The Pennsylvania State University. The Graduate School. Department of Food Science OPTIMIZATION OF AN ALGINATE BASED EDIBLE COATING WITH

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1 The Pennsylvni Stte University The Grdute School Deprtment of Food Science OPTIMIZATION OF AN ALGINATE BASED EDIBLE COATING WITH BEESWAX, NISIN AND EDTA TO MAXIMIZE SHELF LIFE OF FRESH MUSHROOMS (Agricus bisporus) A Thesis in Food Science by Anneliese Luttmnn Submitted in Prtil Fulfillment of the Requirements for the Degree of Mster of Science December 2011

2 ii The thesis of Anneliese Luttmnn ws reviewed nd pproved* by the following: Rmswmy C. Anntheswrn Professor of Food Science Thesis Co-Adviser Deprtment of Food Science Grdute Progrm Coordintor John D. Floros Professor of Food Science Thesis Co-Adviser Hed of the Deprtment of Food Science Robert B. Beelmn Professor of Food Science *Signtures re on file in the Grdute School

3 iii ABSTRACT Fresh white button mushroom (Agricus Bisporus) is highly perishble crop with limited shelf life. Enzymtic browning, bcteril spoilge nd high respirtion rte re the min cuses of qulity deteriortion. Clcium lginte cotings with lipids, ntimicrobil, nd chelting gents cn dely qulity deteriortion nd extend shelf life. The three objectives of this thesis were to evlute the effect of three cotings: lginte (A), lginte contining beeswx (AB), nd lginte contining beeswx, nisin nd N 2 EDTA (ABN) on the qulity of fresh white button mushrooms; to determine the effect of lginte, beeswx, nisin nd N 2 EDTA concentrtion on the qulity of fresh white button mushroom s mesured by color chnge (ΔL*), weight loss (WL), degree of cp opening (MI) nd bcteril growth (BG); nd to optimize the coting composition tht mximizes shelf life using response surfce methodology. As prt of the first objective of this thesis, mushrooms were coted with A, AB nd ABN nd stored t 12 C/60%R.H. for 16 dys. After 8 dys of storge, mushrooms coted with ABN hd significntly (p<0.05) lower ΔL* s compred to uncoted control (C). Cotings AB nd A hd significntly (p<0.05) lower ΔL* s compred to C fter dy 14 nd 16 respectively. No significnt difference (p>0.05) ws found in the rte of WL compred to C. Mushrooms coted with A, AB nd ABN hd significntly (p<0.05) lower MI s compred to C. Mushrooms coted with ABN hd significntly (p<0.05) lower BG during the erly dys of storge. Overll, shelf life of mushrooms coted with ABN ws extended by 6 dys. These results indicte tht edible cotings cn be used to extend shelf life of fresh mushrooms. As prt of the second objective of this thesis, mushrooms were coted with vrious concentrtions of lginte, beeswx, nisin nd N 2 EDTA s determined by centrl composite experimentl design, nd stored t 12 C/60%RH for 16 dys. Alginte significntly (p<0.01) reduced the rte of BG throughout storge. Beeswx significntly decresed ΔL* (p<0.01) nd BG (p<0.05) fter 6 dys in storge, but incresed WL

4 (p<0.10). N 2 EDTA significntly reduced ΔL* (p<0.05) nd BG (p<0.01), while nisin significntly decresed ΔL*(p<0.10) nd MI (p<0.01) during dys 0-6. Nisin lso incresed WL significntly (p<0.05) throughout storge, but significnt (p<0.01) interctions indicted tht t high concentrtions of N 2 EDTA nd lginte, this effect ws reduced. iv As prt of the third objective of this thesis, response surfce methodology (RSM) ws used to find the optiml concentrtion of ech compound to mximize shelf life by reducing ΔL*, MI nd BG. The optiml coting contined 2.49% lginte, 0.82% beeswx, 8.18mg/mL N 2 EDTA nd 4000IU/mL nisin. In vlidtion study, ΔL* nd MI mesurements of mushrooms with the optiml coting were compred to those of the uncoted control. The optiml coting significntly (p<0.05) reduced ΔL* nd MI, extending the shelf life 7 dys pst tht of the control.

5 v TABLE OF CONTENTS LIST OF TABLES... viii LIST OF FIGURES... xi AKNOWLEDGEMENTS... xvii Chpter 1 RATIONALE AND OBJECTIVES Rtionle Hypothesis nd objectives... 2 References... 3 Chpter 2 LITERATURE REVIEW ON SHELF LIFE EXTENSION OF FRESH MUSHROOMS (Agricus bisporus) Introduction Qulity prmeters of fresh white button mushroom (Agricus bisporus) Color Stipe growth Sporophore development Texture nd wter content Respirtion rte Preservtion methods for fresh mushrooms (Agricus bisporus) Mushroom wsh Temperture control Modified tmosphere pckging Irrdition Edible cotings Summry References Chpter 3 LITERATURE REVIEW ON ALGINATE BASED EDIBLE COATINGS Introduction Physicl properties of lginte bsed edible cotings Film thickness Tensile strength Elongtion t brek Brrier properties Wter vpor permebility nd resistnce Wter solubility nd moisture content Oxygen permebility Alginte bsed composite cotings lipid incorportion... 31

6 3.4. Appliction of lginte bsed edible cotings on fresh produce Fruits Vegetbles Mushrooms Appliction of lginte bsed edible cotings on met products Beef Poultry Sefood Appliction of lginte bsed edible cotings on diry products Summry References Chpter 4 SCREENING STUDY TO LOOK AT THE EFFECT OF ALGINATE BASED EDIBLE COATINGS ON FRESH MUSHROOM (Agricus bisporus) QUALITY Introduction Mterils nd methods Mterils Coting preprtion Mushroom coting, pckging nd storge Color mesurement Weight loss determintion Mturity index mesurement Stndrd plte count Shelf life determintion Sttisticl nlysis Results nd discussion Color Weight loss Mturity index Stndrd plte count Shelf life determintion Conclusions References Chpter 5 OPTIMIZATION OF EDIBLE COATING WITH ALGINATE, BEESWAX, NISIN AND N 2 EDTA TO MAXIMIZE SHELF LIFE OF FRESH WHITE BUTTON MUSHROOM (Agricus bisporus) Introduction Mterils nd methods Mterils Coting preprtion Mushroom coting, pckging nd storge Color mesurement Weight gin nd weight loss determintion vi

7 Mturity index mesurement Stndrd plte count Experimentl design Grphic optimiztion Vlidtion study Shelf life determintion Results nd discussion Color ΔL* Vlue ΔE* Vlue Weight gin Weight loss Mturity index Stndrd plte count Interprettion nd visuliztion of the model ΔL* Vlue Mturity index Stndrd plte count Identifiction of optimum conditions Vlidtion study Summry Conclusions References Chpter 6 SUMMARY AND SUGGESTIONS FOR FUTURE RESEARCH Summry Suggestions for future reserch vii

8 viii LIST OF TABLES Tble 2.1. Whiteness ctegories for mushrooms...8 Tble 2.2. Respirtion rtes of perishble commodities t 5 C...11 Tble 2.3. Respirtion rtes of fresh mushrooms...11 Tble 4.1 Composition of lginte bsed cotings...50 Tble 4.2 Rte of chnge in weight loss during storge of mushrooms...66 Tble 5.1. Experimentl design (coded vlues)...87 Tble 5.2. Experimentl design sorted by run order (coded vlues)...88 Tble 5.3. Coded vlues nd ctul concentrtions...89 Tble 5.4. Rte of chnge in qulity prmeters during storge of mushrooms t 12 C...93 Tble 5.5. ANOVA tble for rte of chnge in lightness (dδl*) during dys 0-6 of storge t 12 C...94 Tble 5.6: Regression coefficients of lginte, beeswx, nisin nd N2EDTA concentrtions on rte of chnge in lightness (dδl*) during dys 0-6 of storge t 12 C...95 Tble 5.7. ANOVA tble for rte of chnge in lightness (dδl*) during dys 6-16 of storge t 12 C...98 Tble 5.8. Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of chnge in lightness (dδl*) during dys 6-16 of storge t 12 C...98 Tble 5.9. ANOVA tble for rte of chnge in color (dδe*) during dys 0-6 of storge t 12 C Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of chnge in color (dδe*) during dys 0-6 of storge t 12 C Tble ANOVA tble for rte of chnge in color (dδe*) during dys 6-16 of storge t 12 C...106

9 Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of chnge in color (dδe*) during dys 6-16 of storge t 12 C Tble ANOVA tble for % weight gin fter coting Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on % weight gin fter coting Tble ANOVA tble for bsolute rte of chnge in weight (dw) during storge of mushrooms t 12 C Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on bsolute rte of chnge in weight (dw) during storge of mushrooms t 12 C Tble ANOVA tble for rte of chnge in mturity index (dmi) 2 during dys 0-6 of storge t 12 C Tble Regression coefficients of lginte, nisin nd N 2 EDTA concentrtions on rte of chnge in mturity index during dys 0-6 of storge t 12 C Tble ANOVA tble for rte of chnge in mturity index (dmi) 2 during dys 6-16 of storge t 12 C Tble 5.20: Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of chnge in mturity index (dmi) during dys 6-16 of storge t 12 C Tble ANOVA tble for rte of bcteril growth (dbg) during dys 0-6 of storge t 12 C Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of bcteril growth (dbg) during dys 0-6 of storge t 12 C Tble 5.23: ANOVA tble for rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C Tble Regression models: effect of coting components on qulity prmeters of fresh mushrooms ix

10 Tble Composition of cotings for vlidtion study Tble Model Vlidtion x

11 xi LIST OF FIGURES Figure 2.1. Agricus mushrooms sles in the U.S.A. by type nd percent of totl...5 Figure 2.2. Appernce of sporophore development...9 Figure 2.3 Appernce of sporophore development...10 Figure 3.1. Alginte monomers...22 Figure 3.2. Block rrngement...22 Figure 4.1. Coting preprtion flow digrm...52 Figure 4.2. Smple preprtion flow digrm...54 Figure 4.3. Mushroom coting flow digrm...56 Figure 4.4. Grphicl illustrtion of color mesurement nd relted clcultions...58 Figure 4.5. Appernce of sporophore development...59 Figure 4.6. Effect of lginte bsed cotings on bsolute ΔL* vlue during storge. (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level)...62 Figure 4.7. Glossiness of mushrooms coted with A nd AB...63 Figure 4.8. Effect of lginte bsed cotings on ΔE* vlue during storge. (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level)...64 Figure 4.9. Effect of lginte bsed cotings on % weight loss during storge. (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level)...66 Figure Effect of lginte bsed cotings on mturity index during storge. (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level)...68 Figure Effect of lginte bsed cotings on bcteril growth indicted by stndrd plte count during storge. (Dt represents verge of six

12 mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level)...70 Figure Shelf life determintion of mushrooms coted with ABN nd uncoted control (C) (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level)...71 Figure Mushrooms t the beginning nd end of shelf life: dy 6 for C nd dy 12 for ABN...72 Figure 5.1. Grphicl illustrtion of color mesurement nd relted clcultions...83 Figure 5.2. Appernce of sporophore development...85 Figure 5.3. Exmples of ΔL* vlue chnges during storge time of coted mushrooms with vrious concentrtions of sodium lginte, beeswx, nisin nd N 2 EDTA...92 Figure 5.4. Min effect of N 2 EDTA on dδl* during dys 0-6 of storge t 12 C...95 Figure 5.5. Min effect of nisin on dδl* during dys 0-6 of storge t 12 C...96 Figure 5.6. Interction between lginte nd beeswx on dδl* during dys 0-6 of storge t 12 C...97 Figure 5.7. Min effect of beeswx on dδl* during dys 6-16 of storge t 12 C...99 Figure 5.8. Interction between nisin nd N 2 EDTA on dδl* during dys 6-16 of storge t 12 C Figure 5.9. Min effect of beeswx on dδe* during dys 0-6 of storge t 12 C Figure Min effect of nisin on dδe* during dys 0-6 of storge t 12 C Figure Min effect of N 2 EDTA on dδe* during dys 0-6 of storge t 12 C Figure Interction between lginte nd beeswx on dδe* during dys 0-6 of storge t Figure Min effect of beeswx on dδe* during dys 6-16 of storge t 12 C xii

13 Figure Interction between nisin nd N 2 EDTA on dδe* during dys 6-16 of storge t 12 C Figure Interction between beeswx nd N 2 EDTA on dδe* during dys 6-16 of storge t 12 C Figure Min effect of lginte on weight gin fter coting Figure Min effect of beeswx on weight gin fter coting Figure Interction between N 2 EDTA nd nisin on % weight gin fter coting Figure Min effect of beeswx on bsolute slope of weight during storge t 12 C Figure Min effect of nisin on bsolute slope of weight during storge t 12 C Figure Interction between lginte nd nisin on bsolute slope of weight during storge t 12 C. The remining two fctors kept constnt t center level Figure Interction between N 2 EDTA nd nisin on bsolute slope of weight during storge t 12 C. The remining two fctors kept constnt t center level Figure Min effect of nisin concentrtion on rte of chnge in mturity index during dys 0-6 of storge t 12 C Figure Interction between Nisin nd N 2 EDTA on rte of chnge in mturity index during dys 6-16 of storge t 12 C Figure Interction between beeswx nd N 2 EDTA on rte of chnge in mturity index during dys 6-16 of storge t 12 C Figure Min effect of lginte on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C Figure Min effect of N 2 EDTA on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C Figure Interction between lginte nd nisin on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C Figure Interction between nisin nd lginte on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C xiii

14 Figure Interction between lginte nd beeswx on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C Figure Interction between beeswx nd N 2 EDTA on bcteril growth rte (dbg) during dys 0-6 of storge t Figure Min effect of lginte on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C Figure Min effect of beeswx on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C Figure Interction between lginte nd beeswx on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C Figure Interction between N 2 EDTA nd beeswx on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C Figure Mode of ction of nisin nd N 2 EDTA on grm negtive bcteri Figure Contour plots for bsolute slope of slope of ΔL* vlue (Block 1) during dys 0-6 of storge t 12 C Figure Contour plots for bsolute slope of slope of ΔL* vlue (Block 2) during dys 0-6 of storge t 12 C Figure Contour plots for bsolute slope of slope of ΔL* vlue (Block 1) during dys 6-16 of storge t 12 C Figure Contour plots for bsolute slope of slope of ΔL* vlue (Block 2) during dys 6-16 of storge t 12 C Figure Contour plots for slope of mturity index (Block 1) during dys 0-6 of storge t 12 C Figure Contour plots for slope of mturity index (Block 2) during dys 0-6 of storge t 12 C Figure Contour plots for slope of mturity index (Block 1) during dys 6-16 of storge t 12 C Figure Contour plots for slope of mturity index (Block 2) during dys 6-16 of storge t 12 C Figure Contour plots for slope of bcteril growth rte (dbg) during dys 0-6 of storge t 12 C xiv

15 Figure Contour plots for slope of bcteril growth rte (dbg) (Block 1) during dys 6-16 of storge t 12 C Figure Contour plots for slope of bcteril growth rte (dbg) (Block 2) during dys 6-16 of storge t 12 C Figure Contour lines of slopes of ΔL* vlue (dδl), mturity index (dmi) nd bcteril growth (dbg) t +1 level of lginte nd beeswx for Block 1 dys 0-6. Arrows indicte direction of desired region, shded re indictes overlpping of ll contours Figure Contour lines of slopes of ΔL* vlue (dδl), mturity index (dmi) nd bcteril growth (dbg) t +1 level of Alginte nd Beeswx for Block 2 dys 0-6. Arrows indicte direction of desired region, shded re indictes overlpping of ll contours Figure Contour lines of slopes of ΔL* vlue (dδl), mturity index (dmi) nd bcteril growth (dbg) t +1 level of Alginte nd Beeswx for Block 1 dys Arrows indicte direction of desired region, shded re indictes overlpping of ll contours Figure Contour lines of slopes of ΔL* vlue (dδl), mturity index (dmi) nd bcteril growth (dbg) t +1 level of Alginte nd Beeswx for Block 2 dys Arrows indicte direction of desired region, shded re indictes overlpping of ll contours Figure Superimposed contour lines from dys 0-6 nd 6-16 Block 1 nd 2, gry shding indictes overlpping of contours Figure ΔL* vlue predicted by model nd experimentl vlidtion. Ech dt point is the verge of three replictions, error brs represent stndrd devition. Dotted line represents ±2 stndrd devitions of the model Figure Mturity index predicted by model nd experimentl vlidtion. Ech dt point is the verge of three replictions, error brs represent stndrd devition. Dotted line represents ±2 stndrd devitions of the model Figure Appernce of mushrooms coted with optiml coting, wsheduncoted mushrooms nd unwshed-uncoted mushrooms Figure Effect of cotings on ΔL* vlue during storge. (Dt represents verge of three replictions; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level) xv

16 Figure Effect of cotings on mturity index during storge. (Dt represents verge of three replictions; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level) xvi

17 xvii AKNOWLEDGEMENTS I would like to thnk my dvisor Dr. Rmswmy Anntheswrn for his constnt support throughout my reserch nd grdute studies, nd for mking this experience very fulfilling one. I would lso like to thnk my co-dvisor Dr. John Floros for his guidnce nd for helping me complete my reserch project nd Dr. Robert Beelmn for being in my committee nd for his vluble dvice. I m hertily grteful with the Food Science Deprtment for providing me with reserch ssistntship to fund my grdute studies. I would like to express my deepest pprecition to my fmily for their wholeherted support nd for lwys encourging me to do my best; my friends t Penn Stte nd in Guteml, especilly Julie, Minl, Vnes nd Olg for giving me their support in numerous wys nd ll the people who in one wy or nother helped me with this reserch project. I would like to thnk the stff t the MTDF for providing the mushrooms, Plsgrd for providing the emulsifiers SMS 2008 (sorbitn monosterte) nd Polysorb 7462 (polysorbte) nd Dnisco for providing Nisplin.

18 To my fmily xviii

19 1 Chpter 1 RATIONALE AND OBJECTIVES 1.1. Rtionle White button mushrooms (Agricus bisporus) re highly perishble nd their shelf life is limited when sold s fresh produce. Over 800 million pounds of white button mushrooms were produced in the United Sttes between , from which 85% were sold fresh (USDA 2009). In 2009, mushrooms rnked 13 th mong the 20 most consumed rw vegetbles in the United Sttes ccording to the Food nd Drug Administrtion (CFR 2009). Shelf life extension technologies such s ntimicrobil wsh, modified tmosphere pckging, humidity controlled pckging, irrdition nd edible cotings hve been proposed to extend the shelf life of mushrooms (Burton nd others 1987; Nussinovitch nd Kmpf 1993; Spers nd others 1994; Roy nd others 1995; b; 1996; Beelmn nd Duncn 1997; Gutm nd others 1998; Brron nd others 2002; Beelmn nd Demirci 2007). However, the coting of mushrooms with biopolymers s crriers of ntimicrobil gents hs not been explored. The use of edible cotings hs been considered s potentil pproch in response to the incresing demnd for fresh nd minimlly processed foods in the food retil industry (Lin nd Zho 2007). The crucil qulity ttributes in fresh produce re ppernce, color, texture, flvor, nutritionl vlue, nd microbil sfety; edible cotings hve been proven to preserve these qulity ttributes by the regultion of moisture nd rom trnsfer, nd oxygen nd crbon dioxide equilibrium (Lin nd Zho 2007). The cpbility of edible cotings to crry functionl ingredients, such s ntimicrobils nd

20 ntioxidnts, help to chieve microbil sfety nd color preservtion of fresh produce (Cuppett 1994) Hypothesis nd objectives The hypothesis of this study ws tht n lginte bsed edible coting contining beeswx, lipid, nisin, n ntimicrobil, nd N 2 EDTA, chelting gent, would extend the shelf life of fresh white button mushrooms (Agricus bisporus). The objectives of this study were: To evlute the effect of three cotings: lginte, lginte contining beeswx, nd lginte contining beeswx, nisin nd N 2 EDTA on the qulity of fresh white button mushrooms (Agricus bisporus) s mesured by color chnge, weight loss, degree of cp opening nd microbil growth. To determine the effect of the concentrtion of lginte, beeswx, nisin nd N 2 EDTA on the qulity of fresh white button mushroom (Agricus bisporus) s mesured by color chnge, weight loss, degree of cp opening nd bcteril growth. To optimize the coting composition tht mximizes shelf life using response surfce methodology.

21 3 References Brron C, Vroquux P, Guilbert S, Gontrd N, Gouble B Modified tmosphere pckging of cultivted mushroom (Agricus bisporus L.) with hydrophilic films. Journl of Food Science 67(1): Beelmn RB, Demirci A, inventors; The Penn Stte Reserch Foundtion, ssignee Process for Antimicrobil Tretment of Fresh Produce, Prticulrly Mushrooms. US Ptent A1. Beelmn RB, Duncn EM, inventors; The Penn Stte Reserch Foundtion, ssignee Preservtion compositions nd methods for mushrooms. US Ptent Burton KS, Frost CE, Nichols R A Combintion Plstic Permeble Film System for Controlling Posthrvest Mushroom Qulity. Biotechnology Letters 9(8): CFR Code of Federl Regultions.. In: Services FDADoHH, editor: Food nd Drug Administrtion. Cuppett SL Edible Cotings s Crriers of Food Additives, Fungicides nd Nturl Antgonists. In: Krocht JM, Bldwin EA, Nisperos-Crriedo MO, editors. Edible Cotings nd Films to Improve Food Qulity. 1 ed. Lncster: Technomic Publishing Compny, Inc. p 379. Gutm S, Shrm A, Thoms P Gmm irrdition effect on shelf-life, texture, polyphenol oxidse nd microflor of mushroom (Agricus bisporus). Interntionl Journl of Food Sciences nd Nutrition 49(1):5-10. Lin D, Zho YY Innovtions in the development nd ppliction of edible cotings for fresh nd minimlly processed fruits nd vegetbles. Comprehensive Reviews in Food Science nd Food Sfety 6(3):60-75.

22 4 Nussinovitch A, Kmpf N Shelf-Life Extension nd Conserved Texture of Alginte-Coted Mushrooms (Agricus bisporus). Lebensmittel-Wissenschft und-technologie 26(5): Roy S, Anntheswrn RC, Beelmn RB Fresh Mushroom Qulity s Affected by Modified Atmosphere Pckging. Journl of Food Science 60(2): Roy S, Anntheswrn RC, Beelmn RB. 1995b. Sorbitol increses shelf life of fresh mushrooms stored in conventionl pckges. Journl of Food Science 60(6): Roy S, Anntheswrn RC, Beelmn RB Modified tmosphere nd modified humidity pckging of fresh mushrooms. Journl of Food Science 61(2): Spers GM, Miller RL, Miller FC, Cooke PH, Choi SW Enzymtic Browning Control in Minimlly Processed Mushrooms. Journl of Food Science 59(5): USDA N Mushrooms. In: USDA NASS, editor: Agriculturl Sttistics Bord. p 12.

23 Volume of sles (million lb) 5 Chpter 2 LITERATURE REVIEW ON SHELF LIFE EXTENSION OF FRESH MUSHROOMS (Agricus bisporus) 2.1. Introduction The white button mushroom (Agricus bisporus) is highly perishble crop with limited shelf life when sold s fresh produce. Figure 2.1. shows sles of white button mushrooms in the United Sttes over the pst yers. In period 777 million pounds of white button mushrooms were produced, from which 86% were sold s fresh mushrooms (USDA 2010). Figure 2.1. Agricus mushrooms sles in the U.S.A. by type nd percent of totl Totl Fresh Mrket Processing Source: (USDA 2010) Period

24 6 According to the Food nd Drug Administrtion, mushrooms rnked 13th mong the 20 most consumed rw vegetbles in the United Sttes (CFR 2009). Severl technologies such s modified tmosphere pckging, humidity controlled pckging, irrdition nd ntimicrobil wsh hve been proposed to extend the shelf life of mushrooms (Burton nd others 1987b; Spers nd others 1994; Roy nd others 1995; b; 1996; Beelmn nd Duncn 1997; Gutm nd others 1998; Brron nd others 2002; Beelmn nd Demirci 2007) Qulity prmeters of fresh white button mushroom (Agricus bisporus) White button mushrooms re composed of thred-like cell structure clled hyphe which is compressed together to form fruiting bodies. As living orgnism, the mushroom grows, respires nd senesces nd the rte t which these chnges occur ffects directly it s qulity nd shelf-life (Nichols 1985). As result, one of the min objectives in the reserch of mushroom s post-hrvest physiology is to chnge the rte of these physiologicl processes to improve the mushroom qulity nd extend shelf life (Nichols 1985). The min prmeters tht ffect the qulity of mushrooms re: color, stipe growth, sporophore development, firmness nd wter content. (Guthrie 1984) Color One of the most importnt qulity fctors in Agricus bisporus is the degree of whiteness (Gormley 1975). The hyphe of white button mushrooms is colorless or trnslucent but ppers white. However, s mushrooms ge they suffer enzymtic browning (Nichols 1985). Enzymtic browning is n enzyme ctlyzed oxidtion rection of phenolic substrtes into quinones, which lter undergo oxidtion nd polymeriztion resulting in drk pigments clled melnins (Jolivet nd others 1998). Mushrooms contin copper oxygenses, lso clled polyphenol oxidses (PPOs: lccses

25 7 nd tyrosinses) nd peroxidses (Jolivet nd others 1998). The min enzyme responsible for browning of the sporophore is tyrosinse, given tht lccse hs very low ctivity in the fruiting body (Turner 1974). At the developing stge of the fruiting bodies, tyrosinse nd the substrte re kept prt by membrne boundry lyers in the cells. However, fter hrvest the mushroom processing nd hndling cuses tissue dmge which llows the enzymes nd substrte to come in contct nd strt the rection (Nichols 1985). The color of white button mushrooms cn lso be ltered by bcteril contmintion, Pseudomons tolsii, P. rectns nd P. gingeri hve been identified s responsible for post-hrvest discolortion (Wells nd others 1996). The brown blotch disese cused by P. tolsii, biotype of P. fluorescens is the most studied (Jolivet nd others 1998), this disese mnifests itself s ple yellow spots tht cn redily turn rich brown (Gndy 1985), the spots my be 1-4 mm in dimeter t the beginning, then turn drker nd sunken, colescing to cover the entire cp surfce (Soler-Rivs nd others 1999). P. tolsii produces the lipodepsipeptide tolsin, n extr cellulr toxin tht hs the bility to disrupt the membrnes of fungl, bcteril, plnt nd niml cells by the formtion of n ion chnnel (Brodey nd others 1991). The membrne disruption llows tyrosinse to come in contct with phenolic substrtes cusing the oxidtion rection (Jolivet nd others 1998). Tolsin hs been shown to ctivte tyrosinse, in study where two susceptible strins nd resistnt strin to bcteril blotch were infected with P. tolsii or with tolsin, the mount of tyrosinse level incresed with the increse of bcteril concentrtion or toxin concentrtion in the susceptible strins, nd remined unchnged in the resistnt strin (Soler-Rivs nd others 1997). Six whiteness grdes were defined by pnel discussion in study, relting the pnel description of whiteness with n L vlue s shown in Tble 2.1. In this study the retiler cceptbility nd the consumer cceptbility were defined s L = 80 nd L = respectively (Gormley 1975).

26 8 Tble 2.1. Whiteness ctegories for mushrooms Ctegory Hunter L vlue Pnel description of whiteness 1 >93 Excellent Very good Good Resonble Poor 6 <69 Very Poor Source: (Gormley 1975) Stipe growth The stipe (stem) of the white button mushroom keeps growing fter hrvest. It is believed tht chitin content increse is relted to the post-hrvest stipe elongtion given tht the level of chitin synthse is high during the mximum stipe elongtion stge nd it decreses t the end of elongtion (Goody nd Deroussethll 1975) (Hmmond 1979). It ws erlier believed tht the stipe elongtion ws due only to cell expnsion nd not to cell division. However, study lter proved tht cell division occurs in the upper prt of the stipe, below the veil, which is the most rpidly expnding region (Crig nd others 1977). A study showed tht by trimming the stipe of mushrooms from 35 to 5mm from the cp, enzymtic browning nd cp opening ws reduced nd thus shelf life ws incresed. Mnnitol is the min substrte for fter-hrvest respirtion. Given tht mnnitol content on the stipe is very high (28% of dry weight), trimming of the stipe reduced the respirtory substrte, nd physiologicl chnges were slowed down (Ajlouni nd others 1992).

27 Sporophore development Hmmond nd Nichols described the ppernce of sporophore development in 7 stges ccording to cp growth, opening nd gill exposure, being 1 the pin, 2 the button with no cp opening, nd 7 the cp completely open nd flt gill surfce s shown in Figure 2.2 (Hmmond nd Nichols 1976). Figure 2.2. Appernce of sporophore development Source: (Hmmond nd Nichols 1976) Guthrie described the sporophore development s post hrvest mturity index, where the mushroom ws ssigned score from 1 to 7 ccording to the cp opening nd gill exposure s shown in Figure 2.3. (Guthrie 1984)

28 10 Stge Description of Sporophore Veil intct (tight) Veil intct (stretched) Veil prtilly broken (less thn hlf) Veil prtilly broken (greter thn hlf) Veil completely broken Cp open, gills well exposed Cp open, gill surfce flt Figure 2.3 Appernce of sporophore development Source: (Hmmond nd Nichols 1976; Guthrie 1984) Texture nd wter content Unlike fruits nd vegetbles, mushrooms re not protected by n externl epiderml structure, thus the trnspirtion rte from the fruiting body is very high nd wter loss is comprble to tht of free wter surfce (Sn Antonio nd Flegg 1964). Weight loss s result of desicction is problem for the mushroom retiler becuse it cn cuse toughening nd shriveling of the cp loosing consumer cceptbility. Moreover, weight loss cuses economicl loss when mushrooms re sold by weight (Gormley 1975). Stiffness or resistnce to deformtion is in essence the sme s firmness, property used by consumers to evlute the texture of mushrooms. Stiffness reduction during storge fter hrvest hs been relted to wter loss nd chnges in hyphl density fter cp opening. Stiffness reduction is more rpid t higher tempertures s is wter loss. Overll stiffness is higher in the stipe thn in the pileus, but fter storge stipe presents higher drops in stiffness thn the pileus (McGrry nd Burton 1994).

29 Respirtion rte Live commodities such s fruits, vegetbles nd mushrooms undergo respirtory metbolism, the process where oxygen is combined with crbohydrtes to form vrious compounds tht end up s crbon dioxide nd wter. The rte of respirtion is inversely relted to the shelf life of the product, the higher the respirtion rte, the lower the shelf life; Tble 2.2 shows clssifiction for respirtion rtes of severl commodities, mushrooms rnk mong the extremely high with respirtion rte higher thn 60 mg CO 2 kg -1 h -1 (Sltveit 2004). Tble 2.2. Respirtion rtes of perishble commodities t 5 C Clss mg CO 2 kg -1 h -1 Commodities Very Low < 5 Nuts, dtes Low 5 10 Apple, citrus, grpe, kiwifruit, onion, potto Moderte Apricot, bnn, cherry, pech, nectrine, per, plum, fig, cbbge, crrot, lettuce, pepper, tomto High Strwberry, blckberry, rspberry, culiflower, lim ben, vocdo Very High Artichoke, snp ben, Brussels sprouts, cut Extremely > 60 High Source: (Sltveit 2004) flowers Asprgus, broccoli, mushroom, pe, spinch, sweet corn Respirtion is highly ffected by temperture; n increse in temperture cn cuse exponentil rises in respirtion rtes. Tble 2.3 shows the respirtion rte of mushrooms t severl tempertures of storge (Admicki 2004). Tble 2.3. Respirtion rtes of fresh mushrooms Temperture mg CO 2 kg -1 h -1 0 C C C C 20 C Source: (Admicki 2004)

30 Preservtion methods for fresh mushrooms (Agricus bisporus) Mushroom wsh During the 1980 s sulfite tretments were bnned from use on fresh produce, reserchers hve explored lterntive wshing tretments contining chemicls such s EDTA, hydrogen peroxide, chlorine dioxide, sodium erythorbte nd clcium chloride (Guthrie 1984; McConnell 1991). Mushrooms wshed with tp wter hve shown higher microbil popultions nd n incresed rte of color deteriortion indicted by lower reflectnce vlues. However, when chlorine dioxide ws dded to the wshing tretments t 50ppm bcteril growth ws decresed nd shelf life ws improved. Chlorine dioxide ntimicrobil ction ws incresed by ddition of clcium chloride (10mM) which lso resulted in firmer mushrooms nd reduced the rte of cp opening (Guthrie 1984). Wsh tretment with combintion of 50ppm of chlorine dioxide, 0.1% sodium erythorbte nd 0.05% clcium chloride hs lso shown to be effective in reducing microbil popultion nd incresing shelf life (Guthrie 1984). Mushrooms wshed with solutions contining 1000ppm EDTA did not show significnt difference in whiteness compred to those treted with sodium sulfite fter 1 dy of storge. Moreover, whiteness ws significntly higher in EDTA treted mushrooms fter 3 dys of storge. A combintion wsh of 1000ppm EDTA nd 1% H 2 O 2 resulted in mushrooms with significntly higher whiteness, reduced purple blotch defect nd stndrd plte counts compred to other wsh tretments tested in this study (McConnell 1991). Wshing tretment contining N 2 EDTA in combintion with sodium erythorbte nd cysteine hs shown to reduce browning in mushrooms compred to unwshed control in whole nd sliced mushrooms fter 6 nd 5 dys of storge t 4 C respectively (Spers nd others 1994).

31 13 A two stge wsh process (US Ptent ) lterntive to sulfite hs been developed; where mushrooms re subjected to high ph wsh ( ) to reduce bcteril popultions on mushroom surfce, followed by neutrliztion buffer contining clcium chloride nd enzymtic browning inhibitors such s erythorbic cid nd EDTA. This method hs been shown to increse qulity of mushrooms compred to sulfite treted mushrooms on dys 0, 3, 6 nd 9 of storge, nd lso resulted in better qulity thn mushrooms wshed with combintion of EDTA nd hydrogen peroxide. This tretment ws effective in reducing microbil counts (Beelmn nd Duncn 1997) Temperture control Control of storge temperture by refrigertion is the most common used technology to extend mushroom shelf life (Burton 1987). Studies hve shown tht the longer mushrooms re kept under refrigertion (1 C) the whiter the mushrooms t the point of removl nd during further storge t room temperture (20 C). Delying the time before mushrooms re stored under refrigertion hd negtive effect on subsequent whiteness. After removl from refrigertion the rte of whiteness chnge ws the sme or even less thn unrefrigerted mushrooms (Gormley 1975). Vcuum cooling is technique were cooling is chieved by reduction in pressure which llows wter to vporize t lower temperture, the het required for wter to evporte is supplied by the product, thus het is removed by evportion. The generl rule of thumb is tht 1% w/w of the totl wter vpor removed results in 10 F reduction in the product temperture (Bernrd 1974). Mushrooms treted with vcuum cooling stored t 5 C did not show significnt difference from conventionlly cooled mushrooms in 5 dys of storge. However, when mushrooms were stored t 18 C fter different periods of storge t 5 C less degree of browning ws obtined in vcuum cooled mushrooms compred to conventionlly cooled ones (p<0.05). The rte of weight loss of vcuum cooled mushrooms ws significntly

32 greter thn tht of the conventionlly cooled mushrooms (p<0.02), fter 4.25 dys of storge t 5 C vcuum cooled mushrooms lost 1.7% more wter (Burton nd others 1987). 14 Mushrooms treted with vcuum cooling prior to storge t 4 C hve shown significntly lower polyphenoloxidse ctivity, lipid oxidtion nd superoxide nion genertion which is relted to membrne lipid oxidtion nd senescence (P<0.05). Mushrooms treted with vcuum cooling lso hd significntly lower browning (P<0.05) up to dy 7 of storge (To nd others 2007) Modified tmosphere pckging When fresh produce is wrpped with plstic film, respirtion of produce reduces oxygen nd increses crbon dioxide concentrtion; this cuses the tmosphere within the pckge to be modified retrding ripening nd senescence of fresh commodities (Burton nd others 1987b). The effect of modified tmosphere pckging (MAP) of mushrooms fter vcuum cooling ws compred to hypobric storge (20-30 kp) nd cold storge, ll tretments stored t 4 C. It ws found tht mushrooms stored with MAP hd significntly lower weight loss (p<0.05) compred to hypobric nd cold storge. Mushrooms stored with MAP lso hd significntly lower respirtion rte nd browning (p<0.05) compred to those t cold storge. (To nd others 2006) In nother study, MAP of mushrooms treted with vcuum cooling prior to storge resulted in significntly lower (p<0.05) superoxide nion genertion, polyphenoloxidse ctivity nd browning of the cp. This tretment lso resulted in significntly higher (p<0.05) firmness (To nd others 2007). Development stge of the sporophore hs been relted to crbon dioxide concentrtions in controlled tmosphere pckging, fter 7 dys mushrooms stored t 10 C under 4 different O 2 concentrtions (5-20%) nd 15% CO 2 did not hve broken

33 15 veil whtever the oxygen concentrtion. In this study higher concentrtions of CO 2 resulted in slower opening of the cp (Briones nd others 1992). In study where chitosn coted mushrooms were pckged in different films to chieve modified tmosphere: whole nd sliced coted mushrooms pckged with polyolefin PD-941 film nd sliced coted mushrooms pckged with PVC film hd lower mturity index compred to uncoted control. The polyolefin PD-941 film provided better brrier to moisture nd oxygen resulting in lower weight loss nd mturity index reduction, this film lso improved color of sliced mushrooms without coting (Kim nd others 2006) Irrdition Food is irrdited by exposure to rdint energy such s gmm rys, electron bems nd x-rys. U.S. food regultions llow irrdition of fresh met, poultry, whet powder, white pottoes, mny spices nd sesonings, fresh shell eggs nd fresh produce (USDA 2005). Low irrdition dose (1kGy) ws pproved for fruits nd vegetbles in 1986 for insect control nd shelf life extension (Deprtment of Helth nd Humn Services 2005). Gmm irrdition pplied t rtes of 4.5kGy/h nd 32kGy/h with totl dose of 2kGy ws shown to increse mushroom shelf life by 4 nd 2 dys respectively, s expressed by higher hue ngle vlues obtined on the treted smples, which indicte less color chnge (Beulieu nd others 1999). Irrdited mushrooms nd controls were stored t 15 C nd 90% RH. Better preservtion of cellulr membrnes ws observed by electron microscopy on mushrooms treted with rte of 4.5kGy/h, thus the lower shelf life extension with the rte of 32kGy/h ws ttributed to decomprtmenttion of vcuolr phenols nd entry of oxygen into the cell, which resulted in more browning (Beulieu nd others 1999).

34 16 Mushrooms irrdited with gmm ry dose of 2kGy nd stored t 10 C, hve shown decrese in extent of cp opening, stipe elongtion nd weight loss fter 11 dys of storge. A 6 log reduction in the surfce microbil lod ws chieved right fter irrdition nd fter 10 dys of storge irrdited mushrooms hd 4 log CFU/g compred to 8 log CFU/g in the non irrdited control. Irrdited mushrooms hd higher firmness nd less browning which ws lso confirmed by reduced polyphenol oxidse ctivity (Gutm nd others 1998). Electron bem irrdition hs shown to preserve qulity of sliced mushrooms. Aerobic counts were reduced by 7 Log CFU/g in mushrooms irrdited with 1-5.2kGy. Recovery ws observed fter 9 nd 12 dys of storge t 4 C in mushrooms irrdited with 1kGy nd 3.1kGy respectively, no recovery ws observed fter 21 dys of storge in mushrooms irrdited with 5.2kGy. Irrdited mushrooms hd significntly lower browning indicted by higher L*-vlues (p<0.05); fter 10 dys of storge t 4 C irrdited mushrooms hd n L*vlue between nd non-irrdited control hd n L*vlue of 69 (Koorpti nd others 2004) Edible cotings Chitosn coting hs shown negtive effects on color on sliced nd whole mushrooms indicted by lower L* vlues nd higher ΔE* vlues. However, the coting tretment resulted in lower mturity index on both whole nd sliced coted mushrooms pckged with polyolefin PD-941 film nd sliced mushrooms pckged with PVC film. No significnt differences in weight loss were found between coted nd uncoted mushrooms (Kim nd others 2006). Alginte bsed edible cotings contining lipids nd emulsifiers hve been used to cot white button mushrooms, this will be covered in detil on Chpter 3 (see section 3.4.3).

35 Summry Fresh Agricus mushrooms sles hve incresed by 25% over the lst 14 yers. High perishbility of fresh white button mushroom Agricus bisporus is determined by enzymtic browning cused by mechnicl nd bcteril dmge of the sporophore tissue, opening of the cp, stipe growth, firmness nd wter content. Temperture control during storge is the most common method used to extend mushroom shelf life. However, reserch hs been crried out in this field nd methods such s ntimicrobil wsh, modified tmosphere pckging, modified humidity pckging, irrdition nd edible cotings hve been proposed to preserve qulity prmeters nd extend mushroom shelf life. References Admicki F Mushroom. In: Gross KC, Yi Wng C, Sltveit M, editors. Agriculture Hndbook Number 66 The Commercil Storge of Fruits, Vegetbles, nd Florist nd Nursery Stocks. Beltsville, MD: USDA. Ajlouni SO, Beelmn RB, Thompson DB, Jeng-Leun MAU Stipe Trimming t Hrvest Increses Shelf Life of Fresh Mushrooms (Agricus bisporus). p Brron C, Vroquux P, Guilbert S, Gontrd N, Gouble B Modified tmosphere pckging of cultivted mushroom (Agricus bisporus L.) with hydrophilic films. Journl of Food Science 67(1): Beulieu M, D'Aprn MBlG, Lcroix M Dose Rte Effect of Gmm Irrdition on Phenolic Compounds, Polyphenol Oxidse, nd Browning of Mushrooms (Agricus bisporus). Journl of Agriculturl nd Food Chemistry 47(7): Beelmn RB, Demirci A, inventors; The Penn Stte Reserch Foundtion, ssignee Process for Antimicrobil Tretment of Fresh Produce, Prticulrly Mushrooms. US Ptent A1.

36 18 Beelmn RB, Duncn EM, inventors; The Penn Stte Reserch Foundtion, ssignee Preservtion compositions nd methods for mushrooms. US Ptent Bernrd N Some Experiments in Vcuum Cooling. Mushroom Journl 14: Briones GL, Vroquux P, Chmbroy Y, Bouqunt J, Bureu G, Psct B Storge of common mushroom under controlled tmospheres. Interntionl Journl of Food Science & Technology 27(5): Brodey CL, Riney PB, Tester M, Johnstone K Bcteril Blotch Disese of the Cultivted Mushroom Is Cused by n Ion Chnnel Forming Lipodepsipeptide Toxin. Moleculr Plnt-Microbe Interctions 4(4): Burton KS. The qulity nd storge life of Agricus bisporus; 1987; Brunschweig, Germny. p Burton KS, Frost CE, Atkey PT Effect of vcuum cooling on mushroom browning. Interntionl Journl of Food Science & Technology 22(6): Burton KS, Frost CE, Nichols R. 1987b. A Combintion Plstic Permeble Film System for Controlling Posthrvest Mushroom Qulity. Biotechnology Letters 9(8): CFR Code of Federl Regultions.. In: Services FDADoHH, editor: Food nd Drug Administrtion. Crig GD, Gull K, Wood DA Stipe Elongtion in Agricus-Bisporus. Journl of Generl Microbiology 102(OCT): Deprtment of Helth nd Humn Services CfDCP Food Irrdition. Gndy DG Bcteril nd Fungl Diseses. In: Flegg PB, Spencer DM, Wood DA, editors. The Biology nd Technology of the Cultivted Mushroom. 1 ed. Littlehmpton, U.K.: John Wiley & Sons Ltd. p Gutm S, Shrm A, Thoms P Gmm irrdition effect on shelf-life, texture, polyphenol oxidse nd microflor of mushroom (Agricus bisporus). Interntionl Journl of Food Sciences nd Nutrition 49(1):5-10.

37 19 Goody GW, Deroussethll A Properties of Chitin Synthetse from Coprinus- Cinereus. Journl of Generl Microbiology 89(JUL): Gormley R Chill Storge of Mushrooms. Journl of the Science of Food nd Agriculture 26: Guthrie BD Studies on the Control of Bcteril Deteriortion of Fresh, Wshed Mushrooms (Agricus bisporus/brunescens). Stte College: Pennsylvni Stte University. 158 p. Hmmond JBW Chnges in Composition of Hrvested Mushrooms (Agricus- Bisporus). Phytochemistry 18(3): Hmmond JBW, Nichols R Crbohydrte-Metbolism in Agricus-Bisporus (Lnge) Sing - Chnges in Soluble Crbohydrtes During Growth of Mycelium nd Sporophore. Journl of Generl Microbiology 93(APR): Jolivet S, Arpin N, Wichers HJ, Pellon G Agricus bisporus browning: review. Mycologicl Reserch 102(12): Kim KM, Ko JA, Lee JS, Prk HJ, Hnn MA Effect of modified tmosphere pckging on the shelf-life of coted, whole nd sliced mushrooms. Lwt-Food Science nd Technology 39(4): Koorpti A, Foley D, Pilling R, Prksh A Electron-bem Irrdition Preserves the Qulity of White Button Mushroom (Agricus bisporus) Slices. Journl of Food Science 69(1):SNQ25-SNQ9. McConnell AL Evlution of wsh tretments for the improvement of qulity nd shelf life of fresh mushrooms (Agricus bisporus). University Prk: Pennsylvni Stte University. 111 p. McGrry A, Burton KS Mechnicl properties of the mushroom, Agricus bisporus. Mycologicl Reserch 98(2): Nichols R Post-hrvest Physiology nd Storge. In: Flegg PB, Spencer DM, Wood DA, editors. The Biology nd Technology of the Cultivted Mushroom. 1 ed. Littlehmpton, U.K.: John Wiley & Sons Ltd. p 347. Roy S, Anntheswrn RC, Beelmn RB Fresh Mushroom Qulity s Affected by Modified Atmosphere Pckging. Journl of Food Science 60(2):

38 20 Roy S, Anntheswrn RC, Beelmn RB. 1995b. Sorbitol increses shelf life of fresh mushrooms stored in conventionl pckges. Journl of Food Science 60(6): Roy S, Anntheswrn RC, Beelmn RB Modified tmosphere nd modified humidity pckging of fresh mushrooms. Journl of Food Science 61(2): Sltveit M Respirtory Metbolism. In: Gross KC, Yi Wng C, Sltveit M, editors. Agriculture Hndbook Number 66 The Commercil Storge of Fruits, Vegetbles, nd Florist nd Nursery Stocks. Beltsville, MD: USDA. Sn Antonio JP, Flegg PB Trnspirtion from Sporophore of Agricus bisporus White. Americn Journl of Botny 51(10):1129-&. Spers GM, Miller RL, Miller FC, Cooke PH, Choi SW Enzymtic Browning Control in Minimlly Processed Mushrooms. Journl of Food Science 59(5): Soler-Rivs C, Arpin N, Olivier JM, Wichers HJ Activtion of tyrosinse in Agricus bisporus strins following infection by Pseudomons tolsii or tretment with tolsin-contining preprtion. Mycologicl Reserch 101: Soler-Rivs C, Jolivet S, Arpin N, Olivier JM, Wichers HJ Biochemicl nd physiologicl spects of brown blotch disese of Agricus bisporus. Fems Microbiol Rev 23(5): To F, Zhng M, Hngqing Y, Jinci S Effects of different storge conditions on chemicl nd physicl properties of white mushrooms fter vcuum cooling. Journl of Food Engineering 77(3): To F, Zhng M, Yu H-q Effect of vcuum cooling on physiologicl chnges in the ntioxidnt system of mushroom under different storge conditions. Journl of Food Engineering 79(4): Turner EM Phenoloxidse Activity in Reltion to Substrte nd Development Stge in Mushroom, Agricus-bisporus. Trnsctions of the British Mycologicl Society 63(DEC):541-7.

39 21 USDA Mushrooms. Wshington, D.C.: Ntionl Agriculturl Sttistics Service (NASS), Agriculturl Sttistics Bord. 13 p. USDA FSIS Irrdition nd Food Sfety Answers to Frequently Asked Questions. Fct Sheets. Wells JM, Spers GM, Fett WF, Butterfield JE, Jones JB, Bouzr H, Miller FC Posthrvest discolortion of the cultivted mushroom Agricus bisporus cused by Pseudomons tolsii, P-'rectns', nd P-'gingeri'. Phytopthology 86(10):

40 22 Chpter 3 LITERATURE REVIEW ON ALGINATE BASED EDIBLE COATINGS 3.1. Introduction Algintes re the slts from lginic cid, n cidic polyscchride tht functions s structurl component in brown seweeds (Pheophycee). Alginic cid is composed of (1,4)-β-D-mnnuronic cid (M) nd α-l-guluronic cid (G) residues (see Figure 3.1), which re rrnged in homopolymeric regions of M nd G blocks nd scttered regions of lternting MG blocks (see Figure 3.2) (King 1983; Drget 2009). β-d-mnnuronte (M) α-l-guluronte (G) Figure 3.1. Alginte monomers MMMMMGGGGGMGMGMGGGGGMGMGM M-block G-block MG-block G-block MG-block Figure 3.2. Block rrngement

41 23 Commercil lginte is extrcted from lge, minly Lminri hyperbore, Mcrocystis pyrifer, Lminri digitt, Ascophyllum nodosum, Lminri jponic, Ecloni mxim, Lessoni nigrescens, Durville ntrtic nd Srgssum spp. Production of lginte from bcteri such s Azotobcter vinelndii nd Pseudomons hs been explored but it is not yet economiclly fesible (Drget 2009). Algintes hve been pplied in the food industry s stbilizers, thickeners, films nd gels. A unique property of lgintes is their bility to form n insoluble mtrix when cross-linked with divlent ctions such s C +2 (King 1983). This property llows for lginte solutions to be pplied s liquid cotings to enrobe foods nd lter become wter insoluble by cross-linking (Kester nd Fennem 1986). Alginte cn be cross-linked by diffusion setting or internl setting method: Diffusion setting: is chrcterized by rpid gelling kinetics nd occurs when crosslinking ions such s C +2 diffuse from n outer source into n lginte solution. Commonly used s n immobiliztion technique (Drget 2009). Internl setting: cross-linking ions re dded to n lginte solution by controlled relese, usully chieved by controlling ph, limiting solubility of cross-linking ion source or using chelting gents (Drget 2009). Edible cotings cn ct s supplement of pckging mteril for qulity improvement nd shelf life extension (Kester nd Fennem 1986). The use of edible cotings hs been considered s potentil pproch in response to the incresing demnd of fresh nd minimlly processed foods in the food retil industry (Lin nd Zho 2007). The crucil qulity ttributes in fresh produce re ppernce, color, texture, flvor, nutritionl vlue, nd microbil sfety (Lin nd Zho 2007). Edible cotings cn be used to retrd desicction in fresh fruits. However, the biopolymer must meet

42 certin gs permebility threshold to void nerobic respirtion which would cuse rpid loss of qulity (Kester nd Fennem 1986). 24 Edible cotings hve been shown to preserve qulity ttributes of fresh fruits nd vegetbles by the regultion of moisture nd rom trnsfer, oxygen nd crbon dioxide equilibrium nd oxidtive rection rtes (Nisperos-Crriedo nd others 1991; Lin nd Zho 2007; Rojs-Grü nd others 2009). Edible cotings hve lso been pplied in met nd diry products to prevent moisture loss nd lipid oxidtion (Mehyr nd others 2007; Conte nd others 2009; Song nd others 2010). The cpbility of edible cotings to crry functionl ingredients, such s ntimicrobils nd ntioxidnts, help to chieve the microbil sfety nd color preservtion of fresh produce nd met products (Cuppett 1994) Physicl properties of lginte bsed edible cotings Film thickness Thickness of edible cotings nd films cn be ffected by viscosity, density drining time nd solid concentrtion of the coting solution. Film thickness is relted to other physicl properties such s gs nd wter vpor permebility (Cisneros Zevllos nd Krocht 2003; Lin nd Zho 2007; Vrgs nd others 2008). Thickness of clcium lginte films hs been shown to increse with concentrtion of the clcium chloride solution used to crosslink it. This hs been ttributed to decrese in the degree of lginte solubiliztion s the concentrtion of clcium chloride increses (Pvlth nd others 1999; Rhim 2004). In other words, s the concentrtion of clcium chloride increses the rte of the cross-linking rection is fster thn the rte of solubiliztion of sodium lginte in the cross-linking solution. When

43 cross-linking by immersion ws compred to cross-linking by mixing prior to csting the film, the lter films showed significntly higher thickness (p<0.05) (Rhim 2004) Tensile strength Tensile strength (P) is the mximum stress pplied to film before breking during tensile testing nd is clculted by: TS = F mx /A Eqution 3.1 Where F mx (N) is the mximum lod pplied before brking nd A (m 2 ) is the cross-sectionl re of the film (thickness x width) (Genndios nd others 1994; Rhim 2004b). Tensile strength of sodium lginte films hs been shown to increse with incresing concentrtions of clcium chloride cross-linking solution, nd cross-linking time; ddition of glycerin s plsticizer to the films hs shown to reduce tensile strength (Pvlth nd others 1999). Tensile strength of sodium lginte films hs been shown to increse when films re cross-linked with clcium chloride by immersion compred to films cross-linked by mixing prior to csting. Moreover, no significnt differences hve been found between the lter type of film nd sodium lginte films (p>0.05) (Rhim 2004). Reltive humidity hs been shown to decrese tensile strength of clcium lginte films with nd without plsticizers. At reltive humidities between 76-85% higher tensile strength ws observed in non-plsticized films, followed by films plsticized with glycerol nd the lowest tensile strength ws found in films plsticized with sorbitol nd

44 fructose, this ws ttributed to the size of the plsticizer molecules, higher moleculr weight plsticizer provided lower tensile strength (Olivs nd Brbos-Cánovs 2008). 26 Incresing concentrtions of grlic oil from 0 to 0.4% (v/v) hs shown to significntly decrese tensile strength of 1% clcium lginte films (p<0.05), the uthors ttributed this effect to the interference of grlic oil with the ionic interction between lginte nd clcium in the film network (Prnoto nd others 2005) Elongtion t brek Elongtion t brek represents the bility of the film to stretch before rupture occurs nd is clculted by E % = L f- L o L o 100% Eqution 3.2 Where L f is the finl length right before rupture nd L o is the originl length of the film (Genndios nd others 1994; Rhim 2004b). Reltive humidity hs been shown to increse % elongtion of clcium lginte films with nd without plsticizers. At reltive humidities between 76-85% higher % elongtion ws observed in films plsticized with glycerol, followed by fructose nd sorbitol plsticized films nd the lowest % elongtion ws found in non-plsticized films, this ws ttributed to the size of the plsticizer molecules, higher moleculr weight plsticizer provided lower % elongtion of the films (Olivs nd Brbos-Cánovs 2008). Elongtion t brek of sodium lginte films hs been shown to decrese when films re cross-linked with clcium chloride by immersion compred to films crosslinked by mixing prior to csting. Elongtion t brek lso decreses with incresing clcium chloride concentrtions in both cross-linking methods (Rhim 2004).

45 27 Incresing concentrtions of grlic oil from 0 to 0.3% (v/v) did not show significnt differences in elongtion t brek of clcium lginte films (p>0.05). However, when the concentrtion ws incresed to 0.4% elongtion t brek ws reduced lmost by hlf (p<0.05) (Prnoto nd others 2005) Brrier properties Brrier properties of edible cotings ginst wter vpor nd gses cn be described by permebility, trnsmission rte nd resistnce (Greener Donhowe nd Fennem 1994). Permebility: describes stedy stte diffusion nd solubility of permete through nonporous brrier without significnt imperfections, combining Fick s first lw of diffusion nd Henry s lw of solubility: P = m t e p A Eqution 3.3 where Δm/Δt is the weight of moisture loss per unit time (g/dy), e is film thickness (m), Δp is the prtil pressure difference between the two sides of the film (P), nd A is the film re exposed to moisture trnsfer (m 2 ) (Greener Donhowe nd Fennem 1994; Hmbleton nd others 2009). Trnsmission rte: obtined from the expression for permebility excluding film thickness nd prtil pressure grdient, therefore it should only be used to compre films with the sme composition nd thickness: P = m t A Eqution 3.4 where Δm/Δt is the weight of mss loss per unit time (g/dy), nd A is the film re exposed to mss trnsfer (m 2 ). (Greener Donhowe nd Fennem 1994).

46 28 Resistnce: describes the bility of heterogeneous mteril to work s brrier to permete. Resistnce to wter vpor cn be obtined by: WV = w - % H 100 T P wv A s J Eqution 3.5 Where w is the wter ctivity of the smples, %RH is the reltive humidity, P wv is the sturted vpor pressure (P), A s is the film re exposed to moisture trnsfer (m 2 ), R is the gs constnt (R wter = P m 3 /K kg), T is the bsolute temperture (K) nd J is the slope of weight loss curve in sttionry conditions (kg/dy)(greener Donhowe nd Fennem 1994; Vrgs nd others 2008) Wter vpor permebility nd resistnce Algintes with higher proportion of guluronic:mnuronic cid hve been shown to provide films with higher wter vpor permebility (Olivs nd Brbos-Cánovs 2008). Wter vpor permebility of sodium lginte films hs been shown to decrese when films re cross-linked with clcium chloride by immersion compred to films crosslinked by mixing prior to csting. Moreover, no significnt differences hve been found between the lter type of film nd sodium lginte films (p>0.05) (Rhim 2004). Cross-linking time with clcium chloride hs shown to decrese wter vpor permebility reching minimum fter 3 minutes of rection; further rection time ws shown to increse wter vpor permebility bck. This ws ttributed to the effect of unsolubiliztion of the film by crosslinkge with clcium chloride, which would reduce wter vpor permebility t the beginning of the rection, fter certin period of time tht lginte ws in contct with the queous cross-linking solution, the solubiliztion of lginte would increse wter vpor permebility bck (Olivs nd Brbos-Cánovs 2008).

47 29 Wter vpor permebility of sodium nd clcium lginte films with nd without plsticizer hs been shown to be incresed by incresing reltive humidity (Olivs nd Brbos-Cánovs 2008; Hmbleton nd others 2009), this effect hs been ttributed to the plsticizing effect tht wter molecules confer to hydrophilic films which promotes molecule mobility nd increses permebility (Gontrd nd others 1996; Hmbleton nd others 2009). Addition of severl plsticizers in clcium lginte films hs been studied, no significnt differences in wter vpor permebility were found between films plsticized with glycerol nd films without plsticizer. However, wter vpor permebility ws decresed by dding fructose or sorbitol to the films (Olivs nd Brbos-Cánovs 2008). Incresing concentrtions of grlic oil from 0 to 0.3% (v/v) did not show significnt differences (p>0.05) in wter vpor permebility of clcium lginte films (rnge g mm/m 2 dy kp). However, when the concentrtion ws incresed to 0.4% wter vpor permebility significntly incresed to g mm/m 2 dy kp (p<0.05). The uthors explined this effect with the hydrophobicity of grlic oil which would extend intermoleculr interctions of the lginte mtrix, llowing moisture to pss through the film (Prnoto nd others 2005). Wter vpor resistnce ws mesured in 2% clcium lginte films with severl concentrtions of glycerol (1-3%), plmitic cid ( %), glycerol monosterte ( %) nd β-cyclodextrine ( %). Severl combintions indicted by n orthogonl experimentl design were tested, but none of the ingredients incorported in the coting were shown to hve significnt effect on the wter vpor resistnce of the coting (Fn nd others).

48 Wter solubility nd moisture content Wter solubility of sodium lginte films cross-linked with clcium chloride by dipping hs been shown to increse with temperture. However, no significnt differences hve been found in solubility with incresing concentrtions of clcium chloride from 2-5%. Swelling rtio of films cross-linked by dipping ws shown to decrese with incresing soking temperture (Rhim 2004). No significnt differences hve been found in moisture content of plsticized nd non-plsticized clcium-lginte films when reltive humidity is lower thn 76%. However, when reltive humidity is incresed from 85-98% steep increse in moisture content of plsticized films hs been observed, moisture content of non-plsticized films lso incresed but it ws significntly lower thn tht of plsticized films (p<0.05) (Olivs nd Brbos-Cánovs 2008). Moisture content of sodium lginte films hs been shown to decrese when films re cross-linked with clcium chloride by immersion compred to films cross-linked by mixing prior to csting. Moreover, films cross-linked by both methods hd lower moisture contents compred to sodium lginte films (Rhim 2004) Oxygen permebility Sodium lginte films hve shown to hve significntly lower oxygen permebility (5.66cm 3 µm/m 2 dkp) compred to pectin (19.49cm 3 µm/m 2 dkp) nd methylcellulose (268.43cm 3 µm/m 2 dkp) (Fontes nd others 2011). Alginte-pple puree bsed films hve been shown to be better oxygen brriers compred to pectin-pple puree bsed films, lginte bsed films hd hlf the permebility of the pectin bsed films (10.20 ± 0.91 nd ± 1.28 cm 3 µm/m 2 d kp

49 respectively), this difference ws ttributed to the lower oxygen permebility of lginte compred to tht of pectin (Rojs Grü nd others 2007) Alginte bsed composite cotings lipid incorportion Composite cotings combine lipids nd hydrocolloids to obtin the combined dvntge of wter vpor brrier provided by lipids nd improved mechnicl properties provided by hydrocolloids (polyscchrides or proteins) (Greener Donhowe nd Fennem 1994; Lin nd Zho 2007; Vrgs nd others 2008). Environmentl scnning electron microscopy (ESEM) hs shown incorportion of emulsified beeswx in n lginte coting to be well dispersed on the surfce of the film. Lipid migrtion towrds the surfce of the film hs been observed fter drying nd ttributed to density difference. Lipid ggregtion hs been found in sodium lginte films by lser light scttering grnulometry, this ggregtion ws ttributed to the lipid migrtion to the surfce observed with ESEM (Hmbleton nd others 2009). Addition of emulsified beeswx to sodium lginte cotings did not show significnt difference in wter vpor permebility of films t vrious reltive humidity grdients (p>0.05). However, when the films incorported rom compounds, the ddition of beeswx to the coting decresed wter vpor permebility t reltive humidity grdients of % nd 30-84% (Hmbleton nd others 2009). Addition of sunflower oil to clcium lginte films did not hve significnt effect on wter vpor permebility compred to films without sunflower oil, permebility vlues rnged from x10 9 g m/p s m 2 (Tpi nd others 2007).

50 Appliction of lginte bsed edible cotings on fresh produce Fruits nd vegetbles re often minimlly processed by peeling, cutting or slicing, these process steps cuse wounding of tissue, increse surfce re nd mke fresh produce more prompt to enzymtic browning, texture brekdown nd microbiologicl spoilge (Rojs-Grü nd others 2007; Rojs-Grü nd others 2009). Edible cotings hve been shown to preserve qulity ttributes of fresh fruits nd vegetbles by the regultion of moisture nd rom trnsfer, oxygen nd crbon dioxide equilibrium nd oxidtive rection rtes (Nisperos-Crriedo nd others 1991; Lin nd Zho 2007; Rojs- Grü nd others 2009). The cpbility of edible cotings to crry functionl ingredients, such s ntimicrobils nd ntioxidnts, help to chieve the microbil sfety nd color preservtion of fresh produce (Cuppett 1994) Fruits During storge whole pples cn suffer flvor deteriortion nd loss of texture by softening. Alginte plsticized with glycerol ws used to cot Brvo de Esmolfe whole pples, coted pples resulted in less texture, color nd weight chnge fter 97 dys of storge t 20 C (Moldão-Mrtins nd others 2003). Fresh cut pples undergo enzymtic browning, nd fster weight loss given tht the surfce re is incresed nd they re no longer protected by the peel which functions s brrier to the environment (Olivs nd others 2007). Clcium lginte bsed cotings with 0.25% potssium sorbte hve been shown to preserve texture, color nd reduce weight loss of fresh cut Gl pples for period of 10 dys stored t 3 C, in this study clcium lginte coting contining cetylted monoglyceride significntly reduced weight loss (p<0.05) when compred to clcium lginte coting nd clcium lginte coting contining butter nd linoleic cid. The three cotings significntly reduced color chnge nd mintined hrdness (p<0.05) compred to control, nd no

51 difference ws found between the three cotings bility to improve color nd texture (Olivs nd others 2007b). 33 Clcium lginte coting contining mlic cid nd severl essentil oils or their ctive compounds were shown to reduce counts of E. Coli O157:H7 in fresh cut pples. The most effective effect ws found in cotings contining 0.7% lemongrss oil or 0.5% citrl oil resulting in 4 log CFU/g reduction compred to uncoted control on dy 0. Moreover, these two cotings were ble to mintin popultions of E. Coli O157:H7 below 2 log CFU/g throughout 30 dys of storge t 5 C. Coted smples lso showed significnt reduction in ntive microflor (p<0.05), in this cse the most effective cotings were the ones contining % cinnmon or % clove essentil oils. Coted smples were shown to hve lower oxygen consumption nd lower crbon dioxide nd ethnol production compred to uncoted controls. Cotings contining cinnmon, clove or lemongrss essentil oil t 0.3% nd the coting without essentil oil were shown to mintin desired firmness over 30 dys of storge, incresing concentrtions of these essentil oils or their ctive compounds were shown to significntly decrese firmness (p<0.05) in the coted pple smples (Rybudi-Mssili nd others 2008b). In study were fresh cut Fuji pples were coted with lginte nd nti-browning gents, n increse in N-cetylcysteine in the coting significntly increse resistnce to wter diffusion which ws clculted from the rte of moisture loss indicted by weight loss. In this study the resistnce to wter diffusion ws incresed when sunflower oil ws dded to the coting. However, incresing the oil in the lginte coting from 0.025% to 0.125% did not hve n effect on the wter vpor resistnce (Rojs Grü nd others 2007). Comprble results were obtined when fresh cut ppy ws coted with n lginte contining 2% glycerol, 1% scorbic cid nd sunflower oil ws dded to the coting. In this study incresing concentrtions of glycerol nd scorbic cid were found

52 34 to significntly increse wter vpor resistnce of the coting (p<0.05 nd p<0.001 respectively), nd no significnt differences were obtined in respirtion rte nd ethylene production of coted nd uncoted smples. The lginte coting lso incresed firmness compred to uncoted smples. (Tpi nd others 2008). Probiotic Bifidobcterium lctis Bb-12 ws incorported in clcium lginte cotings plsticized with glycerol contining sunflower oil, scorbic cid, citric cid or N-cetylcysteine, nd used to cot ppy nd pple cylinders. Vible cells of the probiotic were shown to be mintined between Log CFU/g in ppy cylinders nd between Log CFU/g in pple cylinders for 10 dys of storge t 2 C (Tpi nd others 2007). Apple slices treted with commercil browning inhibitor Nturesel nd coted with sodium lginte contining prebiotics inulin, oligofructose or mixture of inulin nd oligofructose were shown to mintin fructn content throughout 14 dys of storge t 4 C. Smples coted with lginte, nd lginte with oligofructose hd less thn 1% weight loss fter 14 dys of storge, uncoted control lost round 2.5%. All coted smples were significntly firmer thn uncoted control (p<0.001). However, uncoted smples treted with Nturesel showed significntly higher firmness thn lginte coted tretments, this ws ttributed to the clcium content of Nturesel, by dding lginte less clcium would be vilble to strengthen the cell wll, becuse of the crosslinking rection between clcium nd lginte. Coted smples lso hd lower titrtble cidity thn uncoted control (p<0.05) which is desirble given tht rise in cidity cn fvor growth of Slmonell nd Shigell. Coted smples mintined their sensory cceptbility throughout 14 dys of storge, cceptbility of uncoted control dropped significntly (p<0.05). No significnt differences in oxygen nd crbon dioxide hedspce concentrtion were obtined throughout storge (p>0.001) (Rößle nd others 2011).

53 35 Fresh cut pers re susceptible to enzymtic browning nd weight loss. Clcium lginte coting contining N-cetylcysteine nd glutthione s nti-browning gents nd sunflower oil hs shown to increse wter vpor resistnce (clculted from the rte of moisture loss indicted by weight loss) in fresh cut pers stored for 14 dys t 4 C. Coted smples lso hd lower ethylene production which ws further decresed when nti-browning gents were dded to the coting; lower ethnol production ws lso observed in coted smples contining N-cetylcysteine nd glutthione fter 11 dys of storge. The coting with nti-browning gents ws ble to id in the retention of vitmin C for up to 11 dys nd slowed down color chnge indicted by hue ngle nd sensory nlysis throughout the 14 dys of storge. Totl erobic psychrotrophic growth ws reduced by the coting with nti-browning gents by 2 Log CFU/g fter 14 dys of storge compred to uncoted control. Coted smples contining ntibrowning gents scored higher in odor nd tste cceptnce evluted by sensory nlysis (Oms-Oliu nd others 2008). Sodium lginte in combintion with wter-soluble chitosn ws used to cot fresh Elliott blue berries, coted fruit resulted in significntly higher firmness (p<0.05) nd lower decy rte indicted by mold growth. No significnt differences were found in weight loss between coted nd uncoted fruit which ws ttributed by the hydrophilic nture of the coting (Dun nd others). Clcium lginte coting contining glycerol (2%), plmitic cid (0.5%), glycerol monosterte (0.5%) nd β-cyclodextrine (0.5%) nd 10 9 CFU/mL culture of Cryptococcus lurentii ws used to cot strwberries which were stored t 20 C nd 70%RH. The coted strwberries showed less decy incidence (indicted by visul mold growth), less weight loss nd more firmness compred to uncoted control. Moreover, the coting significntly reduced mold counts by 2 log CFU/g from dy 2-5 of storge (Fn nd others).

54 36 Clcium lginte hs shown to significntly decrese moisture loss nd respirtion rte in whole peches stored t 15 C nd 40%RH. Coted peches lso hd slower chnge in titrtble cidity ph nd firmness. The coting did not hve n effect on the totl soluble solids of the fruit (Mftoonzd nd others 2008). Sodium lginte contining D-L mlic cid severl essentil oils, or their ctive compounds, cross linked with clcium lctte ws used to cot fresh cut melon. Cotings contining 0.5% eugenol, 0.5% gerniol or lemongrss essentil oil were the most effective in suppressing microbil growth for up to 21 dys t 5 C. Cotings contining cinnmon (0.7%), plmros ( %), lemongrss ( %) essentil oils, eugenol (0.5%), gerniol (0.5%) or citrl (0.5%) extended microbiologicl shelf life for more thn 21 dys (indicted by reching mximum limit of 10 7 CFU/g); uncoted nd coted controls without essentil oils hd shelf life of round 4 nd 10 dys respectively. The cotings with vrious essentil oils lso reduced oxygen consumption, nd crbon dioxide nd ethnol production compred to uncoted nd coted controls without essentil oils. Cotings with low concentrtion of plmros nd lemongrss essentil oils (0.3%) nd ll concentrtions of cinnmon nd eugenol were shown to mintin firmness by more thn 21 dys. Cotings with cinnmon essentil oil, eugenol, gerniol or citrl were shown to mintin color indicted by chrom vlues for more thn 21 dys in fresh cut melons (Rybudi-Mssili nd others 2008). Tomto hs limited posthrvest shelf life limited by trnspirtion, mold infection, ccelertion of ripening nd senescence. Moreover, tomtoes re sensitive to chilling injury nd recommended storge tempertures re bove 11 C (Zpt nd others 2008). Sodium lginte coting plsticized with glycerol ws shown to reduce ethylene production nd respirtion rte compred to uncoted control (p<0.05). Coted smples were lso shown to hve round 2% less weight loss (p<0.05), higher firmness for up to dy 5 of storge t 20 C nd no significnt chnge in color indicted by hue ngle (p>0.05) for up to dy 6 of storge compred to uncoted control, finlly coted smples scored higher thn control in 5 rnked scle sensory nlysis performed by trined

55 pnel evluting crunchiness, juiciness, sweetness, sourness nd overll qulity (Zpt nd others 2008) Vegetbles Clcium lginte coted lettuce hs shown incresed crispness throughout 12 dys of storge t 2 C nd 6 C, no significnt difference ws found in color chnge, Russet spotting, or crbon dioxide levels in the pckge compred to uncoted control (Ty nd Perer 2004). Clcium lginte ws used to cot minimlly processed crrots. Coted crrots nd coted crrots contining 0.1% nd 0.5% citric cid were ble to mintin firmness. The whiteness index or whiteness blush did not increse significntly (p<0.05) for coted crrots without citric cid, but the cotings tht incorported citric cid resulted in significnt increse of the whiteness index (p<0.05). The coting lone did not hve ny effect on the totl vible count. However, the coting contining 0.5% citric cid reduced the vible count by 2 log CFU/g. Coted nd uncoted smples were subjected to severl tmospheres; under ll tmospheres the coted smples hd lower phenolic content. Coted smples under controlled tmospheres of 80%-30%-0%, 70%-30%-0% nd 50%- 30%-20% of O 2 -CO 2 -N 2 hd significntly higher contents of D-sucrose. Coted smples stored t 8 C nd ir hd significntly lower production of ethnol nd ethylene. (Amntidou nd others 2000) Mushrooms Mushrooms coted with 1% nd 2% clcium lginte nd stored t 4 C, mintined significntly higher L* vlues (p<0.05) from dys 2-6 of storge. Mushrooms coted with 2% lginte resulted in significntly lower weight loss (p<0.05) between dys 1-4 of storge t 20 C. Mushrooms coted with 1% lginte nd stored t

56 38 20 C hd significntly higher L* vlues between dys 1-4 of storge. After 48 hours of storge t 4 C nd 20 C coted mushrooms hd lower strin t frcture nd higher stiffness (resistnce to mstiction) chrcteristics desired in fresh mushrooms (Nussinovitch nd Kmpf 1993). Alginte cotings contining lipids hve shown to reduce browning on mushrooms on the lter dys of storge. Mushrooms coted with 2% lginte; 2% lginte + ergosterol; nd 2% lginte + 0.2% ergosterol + polysorbte 80; resulted in significntly higher L* vlues (less browning) between dys 7-19 of storge (2-3 C) compred to uncoted mushrooms. Mushrooms coted with clcium lginte contining ergosterol nd polysorbte 80 resulted in significntly higher L* vlues (p<0.05) compred to mushrooms coted with lginte between dys 7-19 of storge. Mushrooms coted with clcium lginte nd clcium lginte + ergosterol, during 19 dys of storge were found to lose less weight compred to uncoted ones, but no difference ws found between the two cotings (Hershko nd Nussinovitch 1998) Appliction of lginte bsed edible cotings on met products Beef Clcium lginte (1%) hs shown to be n pproprite delivery system for nisin in beef crcsses. Beef tissue coted with clcium lginte contining 100 ppm of nisin ws shown to reduce growth of Brochothrix thermosphct, responsible for spoilge in vcuum-pckged red met, for up to 7 dys (>2.42logCFU/cm 2 ) compred to smples coted with clcium lginte, smples treted with 100µg/mL of nisin solution nd untreted smples (>6logCFU/cm 2 ) (Cutter nd Sirgus 1996). This system ws lso shown to significntly reduce growth of Brochothrix thermosphct (p 0.05) for up to seven dys when pplied to beef prior to grinding; compred to smples treted

57 with100µg/ml of nisin, smples coted with 1% clcium lginte, nd untreted smples (Cutter nd Sirgus 1997). 39 The growth of E. coli O157:H7 in ground beef treted with nisin nd cheltors immobilized in clcium lginte gels ws studied. Nisin immobilized in 1.5% clcium lginte provided significntly higher (P<0.05) inhibition of E. coli O157:H7, s compred to the non-immobilized control when stored t 10 C. The combintion of nisin nd EDTA in the immobilized system lso provided significntly higher inhibition (P<0.05) effect when compred to the non-immobilized combintion system. The highest inhibition ws chieved with the combintion of nisin nd cetic cid immobilized in 1.5% clcium lginte. This immobilized combintion lso provided significntly higher inhibition (P<0.01) when compred to the non-immobilized system (Fng nd Tsi 2003) Poultry Clcium lginte edible cotings hve been used to overcome the high prevlence of Listeri monocytogenes in poched nd deli turkey products. Alginte cotings contining 2.4% sodium lctte nd 0.3% potssium sorbte, 600IU/g nisin nd 2.4% sodium lctte or 500IU/g nisin nd 0.25% sodium dicette significntly reduced growth of L. monocytogenes by 2-3log CFU/g in poched turkey met over 8 weeks of storge t 4 C compred to uncoted control (p<0.05). Cotings contining nisin in combintion with sodium lctte, sodium dicette or potssium sorbte were lso shown to significntly decrese erobic nd nerobic counts of poched turkey throughout 8 weeks of storge t 4 C (p<0.05). For deli turkey the lginte coting contining ntimicrobils only showed significnt differences in inhibition of L. monocytogenes on week 3 of storge, nd no significnt differences were found between the tretments nd control on growth of erobic nd nerobic bcteri (p>0.05) (Juck nd others 2010).

58 40 Crcss wshing with ntimicrobils s tretment of rw poultry to reduce popultions of Slmonell, hs hd limited success in incresed line speeds becuse of reduced contct time with ntimicrobil gents. Clcium lginte cotings contining cidified sodium chlorite (1,200ppm) were shown to significntly reduce Slmonell on inoculted chicken drumettes (p<0.05) compred to control wshed with cidified sodium chlorite (1,200ppm). Drumettes coted with lginte contining cidified sodium chlorite lso showed less weight loss compred to smples coted with lginte, uncoted control, nd control wshed with cidified sodium chlorite. Clcium lginte coting with cidified sodium chlorite ws shown to hve low bsorption rte (0.006g/min) compred to pe strch coting (0.05g/min) nd compred to wter (0.06g/min) (Mehyr nd others 2007) Sefood Cotings with nisin nd EDTA, clcium lginte without ntimicrobil nd dipping solution of nisin nd EDTA significntly inhibited the growth of mesophilic nd psycrophillic bcteri (P<0.05) on northern snkehed fillets (Chnn rgus) t 4 C. The clcium-lginte coting contining nisin nd EDTA nd the clcium-lginte coting without ntimicrobil significntly (P<0.05) reduced the wter loss in the fillets fter 7 dys of storge s compred to the fillets treted with the dipping solution nd the control. A nine-point hedonic sensory nlysis with trined pnel ws performed. Overll, the clcium-lginte coting with incorported nisin nd EDTA nd the clciumlginte coting without ntimicrobil scored significntly higher (P<0.05) thn the control nd the fillets treted by the dipping solution (Lu nd others 2009). Alginte cotings contining sodium lctte (2.4%) nd sodium dicette (0.25%) hve shown to dely growth of Listeri monocytogenes in cold smoked slmon slices nd fillets over storge period of 30 dys t 4 C. Coted slices nd fillets reched 4.1 nd 4.4 CFU/g respectively when uncoted controls reched 7.3 nd 6.8log CFU/g respectively (Neetoo nd others 2009).

59 41 Brem is highly consumed freshwter fish in Chin with high content of unsturted ftty cids (70% of totl ft) which mkes it very prompt to lipid oxidtion. Clcium lginte coting plsticized with glycerol contining vitmin C or te polyphenols hs been shown to significntly reduce totl vible counts, weight loss nd totl voltile bsic nitrogen (relted to spoilge bcteri nd endogenous enzymes) in coted brem. The coted smples were lso shown to hve significntly lower thiobrbituric cid vlue (p<0.05) which is relted to the extent of lipid oxidtion; coted smples were below the threshold of 2 mgmda/kg fter 21 dys of storge, when uncoted smples reched tht limit fter 12 dys of storge. The coting contining vitmin C ws most effective in reducing chnge in ph between dys 4 nd 22 of storge nd scored significntly better (p<0.05) in sensory nlysis thn other cotings nd control throughout 21 dys of storge (Song nd others 2010) Appliction of lginte bsed edible cotings on diry products Edible cotings hve been used on fresh cheese to overcome microbil spoilge nd to substitute the brine in the pckge reducing pcking weight nd trnsporttion cost. Fior di Ltte cheese coted with clcium lginte contining lysozyme nd N 2 EDTA ws shown to mintin weight chnge compred to uncoted smples pckged with brine (p>0.05), coted smples with nd without modified tmosphere pckging nd control with brine hd significntly lower weight loss compred to uncoted control pckged in modified tmosphere without brine (p<0.05). Coted smples with nd without modified tmosphere pckging nd uncoted control pckged in modified tmosphere hd lower counts of Pseudomons sp. (p<0.05), nd did not rech the limit of 10 6 CFU/g, where ltertions to the product strt to pper, fter 8 dys of storge t 10 C; uncoted control pckged in brine rech tht limit on the 3 rd dy of storge. No differences in overll qulity were found in the sensory evlution between coted nd uncoted smples with or without modified tmosphere pckging nd control with brine (Conte nd others 2009).

60 42 Clcium lginte coting ws used to cot semi-hrd cheese, coted smples resulted in 2% less weight loss compred to uncoted control (p<0.05) throughout 25 dys of storge t 4 C. Stress t filure fter 18 dys of storge significntly incresed from 50.3 to 58.2kP (p<0.05) in uncoted control, smples coted with lginte did not hve significnt chnge in stress t filure, the uthors explined this increse s result of drying in the uncoted control. Sensory nlysis indicted tht coted smples crosslinked with clcium lctte resulted in no bitter tste compred to smples crosslinked with clcium chloride (Kmpf nd Nussinovitch 2000) Summry Alginte slts re extrcted from brown seweeds. The property of lginte to form wter insoluble mtrix when cross-linked with divlent ctions such s C +2, llows it to be pplied s liquid coting to enrobe foods nd become insoluble fter cross-linking. Alginte bsed cotings hve been pplied on whole nd cut fresh produce such s fruits, vegetbles nd mushrooms; to retrd moisture loss, enzymtic browning, off-flvor development nd microbil spoilge. Alginte s crrier of ntimicrobil gents hs lso been used to cot met products such s beef, poultry nd sefood; to reduce lipid oxidtion nd counts of spoilge nd pthogenic bcteri. Alginte cotings pplied in fresh cheese hve shown to reduce microbil spoilge nd moisture loss.

61 43 References Amntidou A, Slump RA, Gorris LGM, Smid EJ High Oxygen nd High Crbon Dioxide Modified Atmospheres for Shelf-life Extension of Minimlly Processed Crrots. Journl of Food Science 65(1):61-6. Cisneros Zevllos L, Krocht J Dependence of coting thickness on viscosity of coting solution pplied to fruits nd vegetbles by dipping method. Journl of food science 68(2): Conte A, Gmmriello D, Di Giulio S, Attnsio M, Del Nobile MA Active coting nd modified-tmosphere pckging to extend the shelf life of Fior di Ltte cheese. Journl of Diry Science 92(3): Cuppett SL Edible Cotings s Crriers of Food Additives, Fungicides nd Nturl Antgonists. In: Krocht JM, Bldwin EA, Nisperos-Crriedo MO, editors. Edible Cotings nd Films to Improve Food Qulity. 1 ed. Lncster: Technomic Publishing Compny, Inc. p 379. Cutter CN, Sirgus GR Reduction of Brochothrix thermosphct on beef surfces following immobiliztion of nisin in clcium lginte gels.. Letters in Applied Microbiology 23:9-12. Cutter CN, Sirgus GR Growth of Brochothrix thermosphct in ground beef following tretments with nisin in clcium lginte gels. Food Microbiology 14(5): Drget KI Algintes. In: Phillips GO, Willims PA, editors. Hndbook of hydrocolloids 2nd ed: CRC Press. p Dun J, Wu R, Strik B, Zho Y. Effect of edible cotings on the qulity of fresh blueberries (Duke nd Elliott) under commercil storge conditions. Posthrvest biology nd technology 59(1):71-9. Fn Y, Xu Y, Wng D, Zhng L, Sun J, Sun L, Zhng B. Effect of lginte coting combined with yest ntgonist on strwberry (Frgri nnss) preservtion qulity. Posthrvest Biology nd Technology 53(1-2):84-90.

62 44 Fng TJ, Tsi H-C Growth ptterns of Escherichi coli O157:H7 in ground beef treted with nisin, cheltors, orgnic cids nd their combintions immobilized in clcium lginte gels. Food Microbiology 20(2): Fontes LCB, Rmos KK, Sivi TC, Queiroz FPC Biodegrdble edible films from renewble sources nd potentil for their ppliction in fried foods. Americn journl of food technology 6(7):555. Genndios A, McHugh TH, Weller CL, Krocht JM Edible Cotings nd Films Bsed on Proteins. In: Krocht JM, Bldwin EA, Nisperos-Crriedo MO, editors. Edible Cotings nd Films to Improve Food Qulity. Lncster, Pennsylvni Technomic Publishing Compny, Inc. p Gontrd N, Thibult R, Cuq B, Guilbert S Influence of Reltive Humidity nd Film Composition on Oxygen nd Crbon Dioxide Permebilities of Edible Films. Journl of Agriculturl nd Food Chemistry 44(4): Greener Donhowe I, Fennem OR Edible Films nd Cotings: Chrcteristics, Formtion, Definitions, nd Testing Methods. In: Krocht JM, Bldwin EA, Nisperos-Crriedo MO, editors. Edible Cotings nd Films to Improve Food Qulity. 1st ed. Lncster, PA: Technomic Publishing Compny, Inc. p Hmbleton A, Debeufort F, Bonnotte A, Voilley A Influence of lginte emulsion-bsed films structure on its brrier properties nd on the protection of microencpsulted rom compound. Food Hydrocolloids 23(8): Hershko V, Nussinovitch A Reltionships between Hydrocolloid Coting nd Mushroom Structure. Journl of Agriculturl nd Food Chemistry 46(8): Juck G, Neetoo H, Chen H Appliction of n ctive lginte coting to control the growth of Listeri monocytogenes on poched nd deli turkey products. Interntionl Journl of Food Microbiology 142(3): Kmpf N, Nussinovitch A Hydrocolloid coting of cheeses. Food Hydrocolloids 14(6): Kester JJ, Fennem OR Edible films nd cotings: review. Food Technol 40(12):47-59.

63 45 King A Brown Seweed Extrcts (Algintes). In: Glicksmn M, editor. Food Hydrocolloids. Boc Rton, FL: CRC Press. p Lin D, Zho YY Innovtions in the development nd ppliction of edible cotings for fresh nd minimlly processed fruits nd vegetbles. Comprehensive Reviews in Food Science nd Food Sfety 6(3): Lu F, Liu DH, Ye XQ, Wei YX, Liu F Alginte-clcium coting incorporting nisin nd EDTA mintins the qulity of fresh northern snkehed (Chnn rgus) fillets stored t 4 degrees C. Journl of the Science of Food nd Agriculture 89(5): Mftoonzd N, Rmswmy HS, Mrcotte M Shelf-life extension of peches through sodium lginte nd methyl cellulose edible cotings. Interntionl Journl of Food Science & Technology 43(6): Mehyr GF, Hn JH, Holley RA, Blnk G, Hydmk A Suitbility of Pe Strch nd Clcium Alginte s Antimicrobil Cotings on Chicken Skin. p Moldão-Mrtins M, Beirão-d-Cost SM, Beirão-d-Cost ML The effects of edible cotings on posthrvest qulity of the "Brvo de Esmolfe" pple. Europen Food Reserch nd Technology 217(4): Neetoo H, Ye M, Chen H Bioctive lginte cotings to control Listeri monocytogenes on cold-smoked slmon slices nd fillets. Interntionl journl of food microbiology 136(3): Nisperos-Crriedo MO, Bldwin EA, Shw PE Development of n edible coting for extending posthrvest life of selected fruits nd vegetbles. Proceedings of the Florid Stte Horticulturl Society(104): Nussinovitch A, Kmpf N Shelf-Life Extension nd Conserved Texture of Alginte-Coted Mushrooms (Agricus bisporus). Lebensmittel-Wissenschft und-technologie 26(5): Olivs GI, Brbos-Cánovs GV Alginte-clcium films: Wter vpor permebility nd mechnicl properties s ffected by plsticizer nd reltive humidity. LWT - Food Science nd Technology 41(2):

64 46 Olivs GI, Mttinson DS, Brbos-Cánovs GV Alginte cotings for preservtion of minimlly processed 'Gl' pples. Posthrvest Biology nd Technology 45(1): Olivs GI, Mttinson DS, Brbos-Cánovs GV. 2007b. Alginte cotings for preservtion of minimlly processed 'Gl' pples. Posthrvest Biology nd Technology 45(1): Oms-Oliu G, Soliv-Fortuny R, Mrtín-Belloso O Edible cotings with ntibrowning gents to mintin sensory qulity nd ntioxidnt properties of fresh-cut pers. Posthrvest Biology nd Technology 50(1): Pvlth AE, Gossett C, Cmirnd W, Robertson GH Ionomeric Films of Alginic Acid. Journl of Food Science 64(1):61-3. Prnoto Y, Slokhe V, Rkshit S Physicl nd ntibcteril properties of lgintebsed edible film incorported with grlic oil. Food reserch interntionl 38(3): Rybudi-Mssili RM, Mosqued-Melgr J, Mrtín-Belloso O Edible lgintebsed coting s crrier of ntimicrobils to improve shelf-life nd sfety of freshcut melon. Interntionl Journl of Food Microbiology 121(3): Rybudi-Mssili RM, Rojs Grü MA, Mosqued-Melgr J, Mrtin Belloso O. 2008b. Comprtive Study on Essentil Oils Incorported into n Alginte-Bsed Edible Coting To Assure the Sfety nd Qulity of Fresh-Cut Fuji Apples. Journl of Food Protection 71(6): Rhim J-W Physicl nd mechnicl properties of wter resistnt sodium lginte films. Lebensmittel-Wissenschft und-technologie 37(3): Rhim JW. 2004b. Physicl nd mechnicl properties of wter resistnt sodium lginte films. Lebensmittel-Wissenschft Und-Technologie-Food Science nd Technology 37(3): Rojs-Grü MA, Rybudi-Mssili RM, Soliv-Fortuny RC, Aven-Bustillos RJ, McHugh TH, Mrtín-Belloso O Apple puree-lginte edible coting s crrier of ntimicrobil gents to prolong shelf-life of fresh-cut pples. Posthrvest Biology nd Technology 45(2):

65 47 Rojs-Grü MA, Soliv-Fortuny R, Mrtin-Belloso O Edible cotings to incorporte ctive ingredients to fresh-cut fruits: review. Trends in Food Science & Technology 20(10): Rojs Grü M, Aven Bustillos R, Olsen C, Friedmn M, Henik P, Mrtin Belloso O Effects of plnt essentil oils nd oil compounds on mechnicl, brrier nd ntimicrobil properties of lginte-pple puree edible films. Journl of food engineering 81(3): Rößle C, Brunton N, Gormley RT, Wouters R, Butler F Alginte Coting s Crrier of Oligofructose nd Inulin nd to Mintin the Qulity of Fresh-Cut Apples. Journl of Food Science 76(1):H19-H29. Song Y, Liu L, Shen H, You J, Luo Y Effect of sodium lginte-bsed edible coting contining different nti-oxidnts on qulity nd shelf life of refrigerted brem (Meglobrm mblycephl). Food Control 22(3-4): Tpi M, Rojs Grü M, Crmon A, Rodriguez F, Soliv Fortuny R, Mrtin Belloso O Use of lginte- nd gelln-bsed cotings for improving brrier, texture nd nutritionl properties of fresh-cut ppy. Food hydrocolloids 22(8): Tpi MS, Rojs-Grü MA, Rodríguez FJ, Rmírez J, Crmon A, Mrtin-Belloso O Alginte- nd Gelln-Bsed Edible Films for Probiotic Cotings on Fresh- Cut Fruits. Journl of Food Science 72(4):E190-E6. Ty SL, Perer C Effect of 1-methylcyclopropene tretment nd edible cotings on the qulity of minimlly processed lettuce. Journl of food science 69(2):C131- C5. Vrgs M, Pstor C, Chirlt A, McClements DJ, Gonzlez-Mrtinez C Recent dvnces in edible cotings for fresh nd minimlly processed fruits. Criticl Reviews in Food Science nd Nutrition 48(6): Zpt PJ, Guillén F, Mrtínez-Romero D, Cstillo S, Vlero D, Serrno M Use of lginte or zein s edible cotings to dely posthrvest ripening process nd to mintin tomto (Solnum lycopersicon Mill) qulity. Journl of the Science of Food nd Agriculture 88(7):

66 48 Chpter 4 SCREENING STUDY TO LOOK AT THE EFFECT OF ALGINATE BASED EDIBLE COATINGS ON FRESH MUSHROOM (Agricus bisporus) QUALITY Abstrct Fresh white button mushrooms re highly perishble crop with limited shelf life. Biopolymers bsed cotings cn be used s crriers of ntimicrobil nd chelting gents to increse fresh produce shelf life. The objective of this study ws to evlute the effect of three cotings: lginte (A), lginte contining beeswx (AB), nd lginte contining beeswx, nisin nd N 2 EDTA (ABN) on the qulity of fresh white button mushrooms (Agricus bisporus) s mesured by color chnge, weight loss, degree of cp opening nd microbil growth. Mushrooms were stored t 12 C nd 60% R.H. for 16 dys. Mushrooms coted with ABN hd significntly (p<0.05) lower ΔL* vlue s compred to uncoted control (C) fter dy 8 of storge. Cotings AB nd A hd significntly (p<0.05) lower ΔL* vlue compred to C fter dy 14 nd 16 respectively. No significnt (p>0.05) differences were found in mushroom WL between cotings A, B, ABN nd C. Moreover, no difference ws found in the rte t which coted mushrooms lost weight compred to C. Mushrooms coted with A, AB nd ABN hd significntly (p<0.05) lower MI s compred to C. Mushrooms coted with ABN hd significntly (p<0.05) lower BG during the erly dys of storge. Coting mushrooms with ABN extended the shelf life from 6 to 12 dys when stored t 12 C nd 60% reltive humidity. These results indicte tht edible cotings cn be used to extend shelf life of fresh mushrooms.

67 Introduction The most importnt qulity ttributes in fresh produce re color, texture, flvor, nutritionl vlue nd microbil sfety (Lin nd Zho 2007). The white button mushroom is highly perishble with limited shelf life when sold s fresh retil commodity. As mushrooms ge they suffer enzymtic browning, cused by polyphenol oxidses, which re kept prt from the substrte by intct membrne boundry lyers in the cells. However, fter picking the mushroom processing nd hndling, cuses tissue dmging which llows the enzymes nd substrte to come in contct nd strt the browning rection (Nichols 1985). The color of white button mushrooms cn lso be ltered by bcteril contmintion, Pseudomons tolsii, P. rectns nd P. gingeri hve been identified s responsible for post-hrvest discolortion (Wells nd others 1996). The lck of epiderml structure in mushrooms results in high trnspirtion rtes in mushrooms resulting in incresed weight loss (Sn Antonio nd Flegg 1964). Alginte bsed edible cotings hve been shown to increse fresh produce shelf life (Nisperos-Crriedo nd others 1991; Lin nd Zho 2007; Rojs-Grü nd others 2009). Clcium lginte cotings contining ntimicrobil nd ntibrowning gents hve been shown to chieve microbil sfety nd color preservtion of fresh cut pples nd fresh cut ppy. Moreover, the incorportion of lipids in lginte cotings hs shown n increse in wter vpor resistnce (Rojs Grü nd others 2007; Tpi nd others 2008). Wx emulsions re commonly pplied in fresh citrus fruits to prevent wter loss nd shrinkge, to ct s brrier to gs exchnge nd to crry fungicides nd growth regultors to control decy (Kpln 1986). The min objective of this study ws to evlute the effect of three cotings: lginte (A), lginte contining beeswx (AB), nd lginte contining beeswx, nisin nd N 2 EDTA (ABN) on the qulity of fresh white button mushrooms (Agricus bisporus) s mesured by color chnge, weight loss, degree of cp opening nd microbil growth.

68 Mterils nd methods Mterils White button mushrooms (Agricus bisporus), were obtined from the Pennsylvni Stte University Mushroom Test nd Demonstrtion Fcility (MTDF). Freshly picked mushrooms were obtined in the morning, nd stored t 4 C until used. High viscosity sodium lginte from brown lge ws obtined from Sigm Life Science (Norwy), bleched beeswx ws obtined from Aldrich Chemistry (U.S.A.); nhydrous clcium chloride (CCl 2 ) ws obtined from J.T. Bker (Jpn); Plsgrd SMS 2008 (sorbitn monosterte) nd Plsgrd Polysorb 7462 (polysorbte) were provided by Plsgrd (Denmrk); Nisplin (nisin 20,000 IU/mL) ws provided by Dnisco (Denmrk); disodium etilendimintetrcetic cid dihydrte (N 2 EDTA) ws obtined from Thermo Scientific (IL, U.S.A.) Coting preprtion The coting ws prepred two dys hed of the experiment dy. Three cotings were prepred: sodium lginte coting (A), sodium lginte coting contining beeswx (AB) nd sodium lginte coting contining beeswx, nisin nd N 2 EDTA (ABN), the compositions of the cotings re shown in Tble 4.1. Tretment Tble 4.1 Composition of lginte bsed cotings Sodium Alginte Beeswx Nisin EDTA Polysorbte Sorbitn monosterte (%) (%) (IU/mL) (mg/ml) (%) (% of BW) Uncoted Control (C) A AB ABN

69 51 Disodium EDTA ws dded to deionized (DI) wter nd stirred t 800 rpm until dissolved. Sodium lginte ws dded to the mixture nd stirred t 800 rpm with het until it ws completely dissolved nd it hd reched 70 C. Once dissolved the speed ws reduced to 60 rpm nd stirred for 30 minutes to remove ir bubbles. In seprte flsk beeswx, polysorbte nd sorbitn monosterte were mixed t 60 rpm nd heted to 70 C until melted. Once melted, speed ws incresed to 1000 rpm nd prt of the lginte mixture ws slowly dded to the beeswx mixture nd stirred for 15 minutes. The emulsion ws then dded to the remining lginte mixture nd stirred t 800 rpm for 60 minutes. The het ws turned off, nd the emulsion ws kept stirred t 800 rpm until it reched 25 C. On the experiment dy, Nisplin ws dissolved in DI wter, dded to the coting nd mixed for 30 minutes. A flow digrm of the steps for the coting preprtion is shown in Figure 4.1.

70 Figure 4.1. Coting preprtion flow digrm 52

71 Mushroom coting, pckging nd storge Freshly picked mushrooms were obtined in the morning from the MTDF nd were sorted by size nd ppernce. Selected mushrooms hd cp dimeter between 3 nd 4 cm, nd were free of ny visible signs of disese or physicl dmge. Mushrooms were plced in polystyrene trys: six mushrooms per try for trys used to collect color nd mturity index dt; 6 mushrooms per try with totl weight of 75g for weight loss dt; nd 100g of mushrooms per try for trys used to collect stndrd plte count dt. Mushrooms used for color dt were lbeled 1-6 on the stipe with permnent mrker. Ech try of mushrooms ws wshed by soking in tp wter for 15 seconds to remove pet moss prticles. Mushrooms were then plced bck in trys with folded npkin on the bottom nd llowed to dry for 30 min t 4 C. A flow digrm for the steps involved in smple preprtion is shown in Figure 4.2.

72 Figure 4.2. Smple preprtion flow digrm 54

73 55 Wshed mushrooms were plced in plstic bin with rubber sink protector on the bottom. Mushrooms were coted by sprying with piston pump driven spry gun (Wgner Power Pinter Plus, Chin). Eight psses of coting were pplied t distnce of 30 cm, on the top nd bottom of the mushrooms. The coting ws cross-linked with 4% CCl 2 solution, pplied with n tomizer bottle. Coted mushrooms were plced bck into trys nd llowed to dry for 2 hours t 4 C. Mushroom trys were wrpped with perforted Polyvinylchloride (PVC) film with ntifogging gent, using het seler (Het Seling Equipment LLC., Clevelnd, OH, USA). Ech try ws wrpped mking sure it hd 4 perfortions on the film. Pckged trys were stored t 12 C nd 60% reltive humidity in n environmentl chmber (Lunire Ltd. model CE0932-4, PA, U.S.A.). A flow digrm of the steps for mushroom coting, pckging nd storge is shown in Figure 4.3.

74 Figure 4.3. Mushroom coting flow digrm 56

75 Color mesurement Color of mushrooms ws evluted using the CIE L**b* color spce (Commission Interntionl de l Éclirge or Interntionl Commission on Illumintion) with CR-400 Minolt Chrommeter (Minolt Sensing Inc., Jpn). An observer of 2 (recommended for smll objects i.e., equivlent to circle of 15 mm dimeter observed from distnce of 45 cm) nd illuminnt C (simulted overcst-sky dylight) were used (Billmeyer nd Sltzmn 1981) (McDougll 2002). Dt ws collected with the Spectr Mtch Q.C softwre. The originl projection cone with 10 mm perture ws replced with 25 mm perture projection cone to be ble to cover lrger re of the cp. Color dt ws obtined prior to coting nd on dys 2, 4, 6, 8, 10, 12, 14 nd 16 of storge. The ΔL* nd ΔE* vlues were clculted for ech mushroom using the following equtions: L * * =L time = 0 * - L time = t (Eqution 4.1) E * = ( L * ) 2 +( * ) 2 +( b * ) 2 (Eqution 4.2) Where L*, * nd b* define the loction of ny given color in the uniform color spce: L* is lightness (0=blck, 100=white) nd * nd b* re chrom coordintes (-=green, +=red, -b=blue, +b=yellow) (McDougll 2002). The clculted ΔL* nd ΔE* vlues were verged for ech try (verge of 6 mushrooms), nd the verge of 6 trys, 3 trys per flush, ws used s ech dt point. Figure 4.4 illustrtes the color dt collection procedure.

76 58 Figure 4.4. Grphicl illustrtion of color mesurement nd relted clcultions Weight loss determintion Weight of the mushrooms ws monitored using lbortory scle (model B502-S, Mettler-Toledo GmbH, Greifensee, Switzerlnd). Mushrooms were weighed before wshing, before coting, fter coting, fter pckging nd then on dys 2, 4, 6, 8, 10, 12, 14 nd 16 of shelf life. Three trys per tretment were used to monitor weight throughout storge time. Weight ws recorded for ech try nd the verge of 6 trys, 3 trys per flush, ws one dt point.

77 59 Weight loss throughout the shelf life ws clculted by subtrcting the weight of the mushrooms t time t from the initil weight of the mushrooms dividing by the initil mushroom weight; using the following eqution: % weight loss= weight t - weight initil weight initil 100% (Eqution 4.3) Mturity index mesurement Using the extent of cp opening, the mturity index ws ssigned to ech mushroom bsed on the following seven point scle: Stge Description of Sporophore Veil intct (tight) Veil intct (stretched) Veil prtilly broken (less thn hlf) Veil prtilly broken (greter thn hlf) Veil completely broken Cp open, gills well exposed Cp open, gill surfce flt Figure 4.5. Appernce of sporophore development Source: (Hmmond nd Nichols 1976; Guthrie 1984) The mturity index ws ssigned to ech mushroom on dy 0 nd on dys 2, 4, 6, 8, 10, 12, 14 nd 16 of shelf life. The verge of six mushrooms in one try ws tken, nd the verge of 6 trys, 3 trys per flush, ws one dt point.

78 Stndrd plte count Stndrd plte count ws used to estimte the totl erobic count of mushrooms over storge time. One try ws evluted for bcteril count on dys 0, 3, 6, 10 nd 16. The mushrooms in ech pckge were blended with n equl weight of 2% Difco buffered peptone wter (Becton, Dickinson & Compny, MD, U.S.A.) in sterilized vrible speed lbortory blender (Model LB10, Wring Commercil, CT, U.S.A.). The homogente ws serilly diluted nd spred plted on Difco Eugon Agr (Becton, Dickinson & Compny, MD, U.S.A.) Petri dishes were incubted t 25 C for 48 hours (Guthrie 1984). The men of triplicte pltes ws single mesurement per pckge, bcteril growth ws clculted s follows: BG = Log N f N o (Eqution 4.4) Where N f, is the colony forming units per grm t time t of shelf life; nd N o, is the colony forming units per grm on dy 0 of shelf life Shelf life determintion To evlute the overll shelf life of mushrooms bsed on color chnge, the retiler cceptbility vlue (RAV) of Hunter L = 80 ws used (Gormley 1975). This vlue ws converted from Hunter L vlue to CIE L*vlue using the following equtions (Billmeyer nd Sltzmn 1981): = Hunter L 10 = = (Eqution 4.5)

79 61 L * = 116 n L * = L * = L * (Eqution 4.6) where Y n is the Y tristimulus vlue of the reference white for the selected illuminnt nd observer. Y n = 100 for 2 observer nd C illuminnt (Billmeyer nd Sltzmn 1981). The AV L* of 84 ws converted to ΔL* by subtrcting the AV L* from the verge L* vlue on dy 0 for the mushrooms obtined from the MTDF s follows: AV ΔL* = L* Dy 0 RAV L* (Eqution 4.7) AV ΔL* = AV ΔL* = 11 Shelf life ws determined grphiclly by finding the time (dys) when mushrooms reched the RAV Sttisticl nlysis Mesurements re the verge of six mesurements: three tken on second flush mushrooms nd three tken on third flush mushrooms. Dt nlysis ws conducted using Duncn s Multiple nge Test compring tretments within ech dy of storge using Sttistic (version 6.0 Sttsoft Inc Tuls OK, USA)

80 ΔL* Vlue Results nd discussion Color The ΔL* vlues for fresh mushrooms coted with A, AB nd ABN nd the uncoted control (C) re shown in Figure 4.6. Strting on dy 8, coting ABN showed significntly lower ΔL* vlue (p<0.05) compred to C. Coting A nd AB showed significntly lower ΔL*vlue compred to C between 14 to 16 dys of storge (p<0.05) ABN AB A C b b b Figure 4.6. Effect of lginte bsed cotings on bsolute ΔL* vlue during storge. (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level) b b b Time (D) b b b b b b c b b c Alginte cotings hve been shown in the literture to reduce browning on mushrooms. When mushrooms were coted with 1% nd 2% lginte nd stored t 4 C, coted mushrooms resulted in significntly higher L* vlues (p<0.05) from dys 2-6 of storge (Nussinovitch nd Kmpf 1993). Alginte cotings contining lipids hve been shown to reduce browning on mushrooms between dys 7-19 of storge (Hershko nd

81 63 Nussinovitch 1998). Mushrooms coted with 2% lginte; 2% lginte + ergosterol; nd 2% lginte + 0.2% ergosterol + polysorbte 80; hve shown to result in significntly higher L* vlues (less browning) between dys 7-19 of storge (2-3 C) s compred to uncoted mushrooms. In our study no significnt difference in ΔL* vlue ws found between coting A nd AB. However, coting AB resulted in less visul glossiness on the mushroom surfce nd hd higher resemblnce to uncoted mushrooms (Figure 4.7.) These results re in contrst with n erlier study where mushrooms coted with clcium lginte contining lipids resulted in significntly higher L* vlues (p<0.05) compred to mushrooms coted with lginte between dys 7-19 of storge (Hershko nd Nussinovitch 1998). A AB Figure 4.7. Glossiness of mushrooms coted with A nd AB Other commodities coted with lginte bsed cotings hve shown to reduce chnges in color throughout storge. Fresh cut Gl pples coted with clcium lginte or clcium lginte contining lipids, such s cetylted monoglyceride or linoleic cid with butter, hve been found to significntly reduce browning (p<0.05) s compred to uncoted control (Olivs nd others 2007). Clcium lginte hs shown to prevent increse in whitening index on minimlly processed crrots fter 8 dys of storge t 8 C

82 ΔE* Vlue (Amntidou nd others 2000). However, when the coting contined 0.1% or 0.5% citric cid the whitening index incresed significntly (p<0.05). 64 During preliminry work, between dys 1 to 4 of storge, yellow nd pink discolortions were observed in some mushrooms. However, chnge in L* vlue ws not registered in the chrommeter. Hence, ΔE* vlues were used. ΔE* vlues for fresh mushrooms coted with A, AB nd ABN nd the uncoted control (C) re shown in Figure 4.8. Coting ABN showed significntly lower ΔE* vlue compred to C strting on dy 8. Coting AB hd significntly lower ΔE* vlue compred to C strting on dy 12, nd coting A ws significntly different thn control until dy 14. No significnt difference on ΔE* vlue ws found between the cotings (A, AB nd ABN) nd control on the erly dys of storge indicting tht the color discolortions observed t the beginning of storge did not hve significnt impct on the totl color difference ABN AB A C b b b b b b b b b b 5 b Time (D) Figure 4.8. Effect of lginte bsed cotings on ΔE* vlue during storge. (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level)

83 65 Beeswx nd shellc hve been dded to locust ben gum cotings to obtin different hydrophobic/hydrophilic rtios (Rojs-Argudo nd others 2005). These cotings were shown to reduce totl color difference in coted cherries. In this study the increse in hydrophobicity of the coting significntly reduced the chnge in ΔE vlue (p<0.05) fter 6 dys of storge t 1 C nd 1 dy t 20 C. However, when cherries were stored for 11 dys t 1 C nd 1 dy t 20 C no significnt difference ws found mong the cotings or uncoted control. In our study, no significnt difference in totl color ws found between the three cotings. At 0.55% beeswx the increse in hydrophobicity of the coting does not pper to hve n effect on ΔE* vlue when compred with the clcium lginte coting without beeswx (A) Weight loss The effect of lginte bsed cotings on mushroom weight loss is shown in Figure 4.9. No significnt difference ws found between mushrooms coted with A, AB or ABN nd C throughout the 16 dys of storge (p>0.05). These results re in contrst with study where mushrooms were coted with clcium lginte nd clcium lginte + ergosterol (Hershko nd Nussinovitch 1998). During 19 dys of storge, coted mushrooms lost less weight s compred to uncoted ones. However, no difference ws found between the two cotings. Alginte coted pples contining lipids such s cetylted monoglyceride hve been found to reduce weight loss when compred to lginte coting, or lginte coting with linoleic cid nd butter (Olivs nd others 2007). Moreover, the three cotings significntly reduced weight loss compred to uncoted control.

84 % Weight Loss ABN AB A C Time (D) Figure 4.9. Effect of lginte bsed cotings on % weight loss during storge. (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level) The rte of chnge in weight loss for ech tretment is shown in Tble 4.2. No significnt differences were found in the rte t which mushrooms lost weight during the 16 dys of storge. Tble 4.2 shows tht the overll rte of chnge in weight loss per dy is the sme for the three tretments nd control, this mens tht the coting ws not ble to ct s moisture brrier for the mushrooms. Tble 4.2 Rte of chnge in weight loss during storge of mushrooms Tretment Rte of WL (%WL/dy) Alg+BW+Ni+N 2 EDTA 1.57 ± 0.08 Alg 1.64 ± 0.02 Alg+BW 1.69 ± 0.09 Uncoted Control 1.65 ± 0.03 Note: Averge of six mesurements. Different letters within ech column re significntly different by Duncn's Multiple Rnge Test t 5% level.

85 67 Given tht the clcium lginte coting is insoluble in wter, it ws expected to result in lower wter vpor permebility, nd it ws expected to decrese even further when beeswx ws dded to the coting, resulting in less overll weight loss. The results obtined in this study cn be explined on the bsis of the coting thickness nd the high trnspirtion rte of mushrooms. Other studies hve shown tht lginte cotings contining lipids decrese weight loss on fresh cut pples (Olivs nd others 2007; Rojs- Grü nd others 2007). However, in these studies the coting ws mde by dipping, nd the pples were stored t lower tempertures. Cut pples hve lower trnspirtion rte thn mushrooms; mushrooms loose 21% of their weight fter 6 dys of storge t 4 C (Nussinovitch nd Kmpf 1993) nd the weight loss of fresh cut pple cultivrs rnges between 0.53% % fter 12 dys t 2 C (Kim nd others 1993). Coting by dipping results in more uniform thickness nd less pinholes thn spry coting, becuse solvents evporte t fster rte from spryed films which cuses premture immobiliztion of polymer chins. Cotings with non-uniform surfces nd pinholes hve higher wter vpor permebility (Kester nd Fennem 1986), nd more weight loss cn be obtined with these cotings. Alginte cotings cn ct s scrificing gent where they give up moisture within the polymer before the food strts dehydrting (Kester nd Fennem 1986). Cotings with more uniform thickness hve more moisture to give up before the food strts drying Mturity index The chnge of the mturity index of coted nd uncoted mushrooms throughout storge is shown in Figure Mushrooms coted with A, AB nd ABN hd significntly lower opening of the cp (p<0.05) from dy 6 to dy 16 of storge. No significnt difference ws found between the three coting tretments throughout storge.

86 Mturity Index ABN AB A C b b b b b b 2 1 b Time (D) Figure Effect of lginte bsed cotings on mturity index during storge. (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level) Development stge of the sporophore hs been relted to crbon dioxide concentrtions in controlled tmosphere pckging. After 7 dys, mushrooms stored t 10 C under 4 different O 2 concentrtions (5-20%) nd 15% CO 2 did not hve broken veil whtever the oxygen concentrtion (Briones nd others 1992). In this study higher concentrtions of CO 2 resulted in slower opening of the cp. In nother study where mushrooms were pckged with vrious perforted films to chieve modified tmospheres, development of the cp ws significntly delyed with incresing concentrtions of CO 2 (6-20%) nd decresing concentrtions of O 2 (15-2%) when stored t 18 C for 4 dys (Burton nd others 1987). Extent of cp opening hs been shown to be incresed by controlling oxygen tmosphere concentrtion between 2-5%, the mximum effect ws found t 5% O 2 concentrtion (Murr nd Morris 1975). In this study the extent of cp opening ws reduced by incresing CO 2 concentrtion between 25-50% or by storing mushrooms under 0% O 2.

87 69 The effect of the coting on mturity index could be ttributed to the selective permebility of lginte to oxygen (Conc nd Yng 1993). Low oxygen permebility of lginte films hs been reported (Hmbleton nd others 2009), nd s mentioned before low oxygen concentrtion hs been relted to slower opening of the cp. In different study, chitosn coted mushrooms were pckged in vrious films to chieve modified tmosphere; whole nd sliced coted mushrooms were pckged with polyolefin PD-941 film, nd sliced coted mushrooms were pckged with PVC film (Kim nd others 2006). All tretments exhibited lower mturity index compred to uncoted control. The uthors relted this effect to the limited oxygen concentrtion Stndrd plte count Microbil qulity of mushrooms ws recorded by stndrd plte count. Figure 4.11 shows the bcteril growth throughout storge. Mushrooms coted with ABN showed reduction on totl bcteril growth on dy 3 of storge (p<0.05). No difference ws found between the three cotings or control on dys 6 nd 10 of storge. This is in greement with study where lginte bsed cotings contining potssium sorbte were used to cot pples (Olivs nd others 2007). In this study no significnt difference ws found in mesophilic, psychrotrophic nd yest nd mold counts mong tretments or control. Clcium lginte ws used to cot minimlly processed crrots, the coting lone did not hve ny effect on the totl vible count (Amntidou nd others 2000). However, the coting contining 0.5% citric cid reduced the vible count by 2 log CFU/g. On dy 16, mushrooms coted with ABN hd significntly higher bcteril counts (p<0.05) thn the uncoted control nd the mushrooms coted with lginte.

88 BG ABN AB A C b b b b b Time (D) Figure Effect of lginte bsed cotings on bcteril growth indicted by stndrd plte count during storge. (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level) Shelf life determintion Shelf life ws determined s shown in Figure The uncoted control reched AV ΔL* of 11 between dys 6 nd 8 of storge, nd the mushrooms coted with ABN reched AV ΔL* of 11 between dys 12 nd 14 of storge. ABN coting ws thus ble to extend the shelf life of mushrooms by 6 dys when stored t 12 C nd 60% reltive humidity. Shelf life of mushrooms coted with ABN ws nerly doubled s compred of tht of the uncoted control. Further shelf life extension might be chieved in mushrooms stored t 4 C. Visul ppernce of uncoted mushrooms nd mushrooms coted with ABN t the beginning nd end of shelf life is shown in Figure 4.13.

89 ΔL* Vlue ABN C RAV c c 20 b b 15 b Shelf life end for C Shelf life end for ABN Time (D) Figure Shelf life determintion of mushrooms coted with ABN nd uncoted control (C) (Dt represents verge of six mesurements; error brs represent stndrd devition; different letters within ech dy re significntly different by Duncn's Multiple Rnge Test t 5% probbility level)

90 72 Tretment Time (Dy) ABN C Figure Mushrooms t the beginning nd end of shelf life: dy 6 for C nd dy 12 for ABN

91 Conclusions Mushrooms treted with coting ABN hd significntly (p<0.05) lower browning during storge s compred to uncoted control. This composite coting extended the shelf life of fresh mushrooms from 6 to 12 dys when stored t 12 C nd 60%R.H.. Mushrooms coted with AB nd A hd significntly (p<0.05) lower browning fter dys 14 nd 16 of storge respectively. No significnt (p>0.05) differences were found in mushroom weight loss between cotings A, AB, ABN nd uncoted control throughout storge. Moreover, no difference ws found in the rte t which coted mushrooms lost weight compred to uncoted control. Mushrooms coted with A, AB nd ABN hd significntly (p<0.05) lower extent of cp opening compred to uncoted ones, but no difference ws found mong the three cotings (p>0.05). Mushrooms coted with ABN hd significntly (p<0.05) lower totl bcteril count during the erly dys of storge.

92 74 References Amntidou A, Slump RA, Gorris LGM, Smid EJ High Oxygen nd High Crbon Dioxide Modified Atmospheres for Shelf-life Extension of Minimlly Processed Crrots. Journl of Food Science 65(1):61-6. Billmeyer F, Sltzmn M Principles of Color Technology. Second ed: Wiley- Interscience. 240 p. Briones GL, Vroquux P, Chmbroy Y, Bouqunt J, Bureu G, Psct B Storge of common mushroom under controlled tmospheres. Interntionl Journl of Food Science & Technology 27(5): Burton KS, Frost CE, Nichols R A Combintion Plstic Permeble Film System for Controlling Posthrvest Mushroom Qulity. Biotechnology Letters 9(8): Conc KR, Yng TCS Edible Food Brrier Cotings. In: Ching C, Kpln DL, Thoms EL, editors. Biodegrdble Polymers nd Pckging. Lncster, PA: Technomic Publishing Compny, Inc. p Gormley R Chill Storge of Mushrooms. Journl of the Science of Food nd Agriculture 26: Guthrie BD Studies on the Control of Bcteril Deteriortion of Fresh, Wshed Mushrooms (Agricus bisporus/brunescens). Stte College: Pennsylvni Stte University. 158 p. Hmbleton A, Debeufort F, Bonnotte A, Voilley A Influence of lginte emulsion-bsed films structure on its brrier properties nd on the protection of microencpsulted rom compound. Food Hydrocolloids 23(8): Hmmond JBW, Nichols R Crbohydrte-Metbolism in Agricus-Bisporus (Lnge) Sing - Chnges in Soluble Crbohydrtes During Growth of Mycelium nd Sporophore. Journl of Generl Microbiology 93(APR): Hershko V, Nussinovitch A Reltionships between Hydrocolloid Coting nd Mushroom Structure. Journl of Agriculturl nd Food Chemistry 46(8):

93 75 Kpln HJ Wshing, Wxing, nd Color-Adding. In: Wrdowski WF, Ngy S, Grierson W, editors. Fresh citrus fruits. Mdison, Wisconsin: AVI Pub Co. Kester JJ, Fennem OR Edible films nd cotings: review. Food Technol 40(12): Kim DM, Smith NL, Lee CY Qulity of Minimlly Processed Apple Slices from Selected Cultivrs. Journl of Food Science 58(5): Kim KM, Ko JA, Lee JS, Prk HJ, Hnn MA Effect of modified tmosphere pckging on the shelf-life of coted, whole nd sliced mushrooms. Lwt-Food Science nd Technology 39(4): Lin D, Zho YY Innovtions in the development nd ppliction of edible cotings for fresh nd minimlly processed fruits nd vegetbles. Comprehensive Reviews in Food Science nd Food Sfety 6(3): McDougll DB Colour mesurement of food Principles nd prctice. In: McDougll DB, editor. Colour in food: improving qulity. Boc Rton, Florid: CRC Press. p Murr D, Morris L Effect of storge tmosphere on posthrvest growth of mushrooms. Journl of the Americn Society for Horticulturl Science 100(3): Nichols R Post-hrvest Physiology nd Storge. In: Flegg PB, Spencer DM, Wood DA, editors. The Biology nd Technology of the Cultivted Mushroom. 1 ed. Littlehmpton, U.K.: John Wiley & Sons Ltd. p 347. Nisperos-Crriedo MO, Bldwin EA, Shw PE Development of n edible coting for extending posthrvest life of selected fruits nd vegetbles. Proceedings of the Florid Stte Horticulturl Society(104): Nussinovitch A, Kmpf N Shelf-Life Extension nd Conserved Texture of Alginte-Coted Mushrooms (Agricus bisporus). Lebensmittel-Wissenschft und-technologie 26(5): Olivs GI, Mttinson DS, Brbos-Cánovs GV Alginte cotings for preservtion of minimlly processed [`]Gl' pples. Posthrvest Biology nd Technology 45(1):89-96.

94 76 Rojs-Argudo C, Pérez-Ggo MB, del Río MA Posthrvest Qulity of Coted Cherries cv. 'Burlt' s Affected by Coting Composition nd Solids Content. p Rojs-Grü MA, Soliv-Fortuny R, Mrtin-Belloso O Edible cotings to incorporte ctive ingredients to fresh-cut fruits: review. Trends in Food Science & Technology 20(10): Rojs-Grü MA, Tpi MS, Rodríguez FJ, Crmon AJ, Mrtin-Belloso O Alginte nd gelln-bsed edible cotings s crriers of ntibrowning gents pplied on fresh-cut Fuji pples. Food Hydrocolloids 21(1): Rojs Grü M, Aven Bustillos R, Olsen C, Friedmn M, Henik P, Mrtin Belloso O Effects of plnt essentil oils nd oil compounds on mechnicl, brrier nd ntimicrobil properties of lginte-pple puree edible films. Journl of food engineering 81(3): Sn Antonio JP, Flegg PB Trnspirtion from Sporophore of Agricus bisporus White. Americn Journl of Botny 51(10):1129-&. Tpi M, Rojs Grü M, Crmon A, Rodriguez F, Soliv Fortuny R, Mrtin Belloso O Use of lginte- nd gelln-bsed cotings for improving brrier, texture nd nutritionl properties of fresh-cut ppy. Food hydrocolloids 22(8): Wells JM, Spers GM, Fett WF, Butterfield JE, Jones JB, Bouzr H, Miller FC Posthrvest discolortion of the cultivted mushroom Agricus bisporus cused by Pseudomons tolsii, P-'rectns', nd P-'gingeri'. Phytopthology 86(10):

95 77 Chpter 5 OPTIMIZATION OF EDIBLE COATING WITH ALGINATE, BEESWAX, NISIN AND N 2 EDTA TO MAXIMIZE SHELF LIFE OF FRESH WHITE BUTTON MUSHROOM (Agricus bisporus) Abstrct Enzymtic browning, bcteril spoilge nd high respirtion rte re the min cuses for the short shelf life of fresh mushrooms (Agricus Bisporus). Alginte cotings with lipids, ntimicrobils nd chelting gents cn dely qulity deteriortion nd extend shelf life. The min objectives of this study were to determine the effect of the concentrtion of lginte, beeswx, nisin nd N 2 EDTA on the qulity of fresh white button mushrooms s mesured by lightness (ΔL*), weight loss (WL), mturity index (MI) nd bcteril growth (BG); nd to find the optiml coting composition tht mximizes shelf life using response surfce methodology (RSM). Cotings with vrious concentrtions of lginte, beeswx, nisin nd N 2 EDTA, s determined by centrl composite experimentl design, were used to cot mushrooms. Mushrooms were then stored t 12 C/60%RH for 16 dys nd mesurements were tken throughout storge. Results showed tht lginte significntly (p<0.01) reduced the rte of BG throughout storge. Beeswx significntly decresed ΔL* (p<0.01) nd BG (p<0.05) fter 6 dys in storge, but incresed WL (p<0.10). N 2 EDTA significntly reduced ΔL* (p<0.05) nd BG (p<0.01), while nisin significntly decresed ΔL*(p<0.10) nd MI (p<0.01) during dys 0-6. Nisin lso incresed WL significntly (p<0.05) throughout storge, but significnt (p<0.01) interctions indicted tht t high concentrtions of N 2 EDTA nd lginte, this effect ws miniml. RSM ws used to find the optiml concentrtion of ech compound to mximize shelf life by reducing ΔL*, MI nd BG. The optiml coting (OC) contined 2.49% lginte, 0.82% beeswx, 8.18mg/mL N 2 EDTA nd 4000IU/mL nisin. In vlidtion study, ΔL* nd MI of mushrooms with OC were compred to those

96 of the uncoted control (C). The OC significntly (p<0.05) reduced ΔL* nd MI, extending the shelf life by 7 pst tht of the control. 78

97 Introduction The use of edible cotings hs been considered s potentil pproch to extend shelf life, in response to the incresing consumer demnd for fresh nd minimlly processed foods (Lin nd Zho 2007). The crucil qulity ttributes in fresh produce re ppernce, color, texture, flvor, nutritionl vlue, nd microbil sfety; edible cotings hve been proven to preserve these qulity ttributes by regulting moisture nd rom trnsfer, nd oxygen nd crbon dioxide equilibrium (Lin nd Zho 2007). The cpbility of edible cotings to crry functionl ingredients, such s ntimicrobils nd ntioxidnts, cn improve the microbil sfety nd color preservtion of fresh produce (Cuppett 1994). Edible cotings pplied on minimlly processed fresh produce hve been shown to work better when combintion of bioctive compounds is incorported into the coting rther thn single individul compound (Rojs Grü nd others 2007; Tpi nd others 2008). Enzymtic browning cused by polyphenol oxidses, bcteril spoilge by Pseudomons spp. nd high respirtion rte re the min cuses contributing to the short shelf life of fresh white button mushrooms Agricus Bisporus (Nichols 1985; Wells nd others 1996; Sltveit 2004). Technologies such s modified tmosphere pckging, humidity controlled pckging, irrdition nd ntimicrobil wsh hve been proposed to extend the shelf life of mushrooms (Burton nd others 1987b; Spers nd others 1994; Roy nd others 1995; b; 1996; Gutm nd others 1998; Brron nd others 2002; Beelmn nd Demirci 2007). However, biopolymer cotings with ntimicrobils nd chelting gents hve not been explored extensively to increse the shelf life of fresh mushrooms. Unlike fruits nd vegetbles, mushrooms lck epiderml structure. This cuses high trnspirtion rte from the fruiting body, nd wter loss comprble to tht of free

98 wter surfce (Sn Antonio nd Flegg 1964). Edible cotings cn ct s skin replcement or fortifier of nturl epiderml lyers tht commonly protect fruits nd vegetbles. 80 Anlysis nd modeling of problems tht require the optimiztion of responses ffected by severl vribles cn be chieved by response surfce methodology (Montgomery 2009). This pproch hs been used to optimize the concentrtion of bioctive compounds dded to edible cotings, nd using qulity prmeters s responses (Mtsos 2006; Rojs-Grü nd others 2007; Tpi nd others 2008). In mny cses, severl qulity prmeters or responses re importnt, such s color chnge, bcteril growth nd mturity index, becuse ll of them re involved in qulity deteriortion nd shelf-life. When multiple responses re involved to obtin n optimum coting composition, n dequte pproch is to overly the contour plots for ech response nd find the region where ll contours overlp closest to the desired response rnge (Montgomery 2009). The min objectives of this study were to determine the effect of lginte, beeswx, nisin nd N 2 EDTA on the qulity of fresh white button mushroom (Agricus bisporus) s mesured by color chnge, weight loss, degree of cp opening nd bcteril growth nd to find n optiml coting composition tht mximizes shelf life using response surfce methodology.

99 Mterils nd methods Mterils The mterils used for this experiment re the sme s the ones described in Chpter 4 (see Section 4.2.1) Coting preprtion The procedure used to prepre the coting ws the sme s the one described in Chpter 4 (see Section 4.2.2) Mushroom coting, pckging nd storge Section 4.2.3). Mushrooms were coted pckged nd stored s described in Chpter 4 (see Color mesurement Color of mushrooms ws evluted using the CIE L**b* color spce (Commission Interntionl de l Éclirge or Interntionl Commission on Illumintion) with CR-400 Minolt Chrommeter (Minolt Sensing Inc., Jpn). An observer of 2 (recommended for smll objects i.e., equivlent to circle of 15 mm dimeter observed from distnce of 45 cm) nd illuminnt C (simulted overcst-sky dylight) were used (Billmeyer nd Sltzmn 1981) (McDougll 2002). Dt ws collected with the Spectr Mtch Q.C softwre. The originl projection cone with 10 mm perture ws replced with 25 mm perture projection cone to be ble to cover lrger re of the cp.

100 82 Color dt ws obtined prior to coting nd on dys 2, 4, 6, 8, 10, 12, 14 nd 16 of storge. ΔL* nd ΔE* vlues were clculted for ech mushroom using the following equtions: L * * * =L time = 0 -L time = t (Eqution 5.1) E * = ( L * ) 2 +( * ) 2 +( b * ) 2 (Eqution 5.2) Where L*, * nd b* define the loction of ny given color in the uniform color spce: L* is lightness (0=blck, 100=white) nd * nd b* re chrom coordintes (-=green, +=red, -b=blue, +b=yellow) (McDougll 2002). The clculted ΔL* nd ΔE* vlues were verged for ech try (verge of 6 mushrooms), nd the verge of 3 trys ws used s ech dt point. ΔL* nd ΔE* vlues were plotted ginst storge time for ech tretment (coting). The dt set ws broken into two time periods: dys 0-6 nd dys 6-16 of storge. A liner regression model ws fitted to ech time period. The slope of the regression model represents the rte of chnge in ΔL* nd ΔE* vlue over time. The bsolute vlue of these slopes were designted s dδl* nd dδe* respectively. Figure 5.1 illustrtes the color dt collection procedure nd the clcultions mde to obtin dδl* for dys 0-6 nd 6-16 of storge.

101 Figure 5.1. Grphicl illustrtion of color mesurement nd relted clcultions 83

102 Weight gin nd weight loss determintion The weight of the mushrooms ws monitored using lbortory scle (model B502-S, Mettler-Toledo GmbH, Greifensee, Switzerlnd). Mushrooms were weighed before wshing, before coting, fter coting, fter pckging nd then on dys 2, 4, 6, 8, 10, 12, 14 nd 16 of storge. Three trys per tretment were used to monitor the weight of mushrooms throughout the entire storge time. To estimte the mount of coting retined on the mushroom surfce, the percent weight gin ws clculted from the weight of the mushrooms fter 2 hours of drying nd the initil mushroom weight using the following eqution: %WG= weight dried - weight initil weight initil 100% (Eqution 5.3) The weight loss throughout storge ws lso clculted by monitoring the weight of the mushrooms. A liner regression model ws fitted to the dt, nd the rte of weight chnge over time (dw) ws obtined from the slope.

103 Mturity index mesurement Using the extent of cp opening, the mturity index ws ssigned to ech mushroom bsed on the following seven point scle: Stge Description of Sporophore Veil intct (tight) Veil intct (stretched) Veil prtilly broken (less thn hlf) Veil prtilly broken (greter thn hlf) Veil completely broken Cp open, gills well exposed Cp open, gill surfce flt Figure 5.2. Appernce of sporophore development (Hmmond nd Nichols 1976; Guthrie 1984) The mturity index ws ssigned to ech mushroom on dy 0 nd on dys 2, 4, 6, 8, 10, 12, 14 nd 16 of storge. The dt points were plotted ginst time for ech tretment (coting). To fit liner regression model, the dt set ws broken into two time periods: dys 0-6 nd dys 6-16 of storge. The rte of chnge in mturity index over time ws obtined from the slope (dmi).

104 Stndrd plte count Stndrd plte count ws used to estimte the totl erobic count of mushrooms over storge time. One try ws evluted for bcteril count on dys 0, 3, 6, 10 nd 16. Mushrooms in ech pckge were blended with n equl weight of 2% Difco buffered peptone wter (Becton, Dickinson & Compny, MD, U.S.A.) in sterilized vrible speed lbortory blender (Model LB10, Wring Commercil, CT, U.S.A.). The homogente ws serilly diluted nd spred plted on Difco Eugon Agr (Becton, Dickinson & Compny, MD, U.S.A.). Petri dishes were incubted t 25 C for 48 hours (Guthrie 1984). The men of triplicte pltes ws single mesurement per pckge, bcteril growth ws clculted s follows: BG = Log N f N o (Eqution 5.4) Where N f, is the colony forming units per grm t time t of storge; nd N o, is the colony forming units per grm on dy 0 of storge. Ech mesurement ws plotted ginst time for ech tretment (coting). To fit liner regression model, the dt set ws broken into two time periods: dys 0-6 nd dys 6-16 of storge. The rte of chnge in bcteril growth (dbg) ws obtined from the slope of ech regression line.

105 Experimentl design A centrl composite rottble design with 2 blocks, 4 fctors t 5 levels ech, nd totl of 24 runs including 4 center points per block ws used for this experiment. The str distnce (α) ws The experimentl order ws rndomized. The experimentl design ws constructed using Sttistic (version 6.0 Sttsoft Inc Tuls OK, USA). Tble 5.1, shows the experimentl design with coded vlues sorted by experiment number. Tble 5.1. Experimentl design (coded vlues) Block Block 1 Block 2 Experiment Number Run Order Sodium Alginte Beeswx Nisin N 2 EDTA

106 88 For ech experimentl run, 11 trys with 6 mushrooms ech were prepred nd coted (3 for color nd mturity index, 3 for weight nd 5 for stndrd plte count). The experimentl design ws blocked to differentite two flushes of single mushroom spwn (1 st flush for block 1, nd 2 nd flush for block 2). To be ble to cot the 132 mushroom trys, ech block ws divided in two consecutive dys tht corresponded to the highest picking dys of ech flush, so tht ech dy 66 trys were coted. Tble 5.2 shows the experimentl design with the coded vlues sorted by run order, nd Tble 5.3 shows the ctul concentrtions tht correspond to the coded vlues for ech component of the coting. Tble 5.2. Experimentl design sorted by run order (coded vlues) Block Block 1 Dy 1 of 1 st Flush Block 1 Dy 2 of 1 st Flush Block 2 Dy 1 of 2 nd Flush Block 2 Dy 2 of 2 nd Flush Experiment Number Run Order Sodium Alginte Beeswx Nisin N 2 EDTA

107 89 Tble 5.3. Coded vlues nd ctul concentrtions Coded Vlue Sodium Alginte Beeswx Nisin N 2 EDTA % % IU/mL mg/ml Dt ws nlyzed using multiple liner regression procedures with MODDE sttisticl softwre for design of experiments nd optimiztion (version , Umetrics, Umeå, Sweden). A second order polynomil ws fitted to ech response dt set: R i β β β β β x x x x x x β β β β 22 x x x 2 x β 1 2 x x β 33 x β 14 β x β x 3 1 x β x 3 4 x 44 4 x 4 x 2 4 ε 4 i (Eqution 5.5) Grphic optimiztion The optimiztion procedure ws performed with the dt obtined from the experimentl design described in Section Response surfce methodology ws utilized to find the concentrtion combintion for n optiml nd sub-optiml coting. Identifiction of the two cotings ws done by overlying the contour plots of ech response nd finding the region where ll contours overlp closest to the desired vlue of qulity.

108 Vlidtion study The models nd optimiztion process were vlidted by coting mushrooms with the identified optiml coting, coting tht belonged to the center point of the experimentl design nd combintion identified s sub-optiml coting. The coting performnce predicted by the models ws then compred to tht found by the experimentl vlidtion study. The three different coting tretments were supplemented by two uncoted controls: wshed nd unwshed mushrooms. The vlidtion study ws performed in triplicte within the sme flush of mushrooms to eliminte the vribility of ech flush. For ech coting 9 trys of mushrooms were coted (3 per replicte). Dt nlysis ws conducted using Duncn s Multiple Rnge Test to compre tretments within ech dy of storge. A two smple T-test ws used to compre slopes of qulity prmeters predicted by the models nd the ctul slopes obtined from the vlidtion experiments dt. All nlysis were done using Sttistic (version 6.0 Sttsoft Inc Tuls OK, USA) Shelf life determintion The procedure used to determine shelf life of fresh mushrooms ws the sme s the one described in Chpter 4 (see Section 4.2.8).

109 Results nd discussion To nlyze the dt the slope of ech qulity prmeter vs. time ws obtined for ech tretment. Tble 5.4 shows the rte of chnge in ech qulity prmeter throughout storge Color ΔL* Vlue Browning of mushrooms incresed over storge time, this is expressed by decrese in lightness (L*vlue). To stndrdize the dt, ΔL* vlues where clculted by subtrcting the L* vlue t ny given time during storge from the initil L* vlue: ΔL* = L* t=0 - L* t Delt L* vlues during storge time for mushrooms coted with severl combintions of sodium lginte, beeswx, nisin nd N 2 EDTA re shown in Error! Reference source not found.. To nlyze the dt the slope of ech qulity prmeter vs. time ws obtined for ech tretment. Tble 5.4 shows the rte of browning expressed by rte chnge in ΔL* vlue (dδl*), higher vlues of dδl* indicte fster chnge in lightness or browning. To evlute the effect of coting composition on mushroom lightness the dt set ws broken into two prts: dys 0-6, nd dys 6-16 of storge. For dys 0-6 the regression model fitted to the dt ws significnt (p<0.01) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.74 (Tble 5.5)

110 Figure 5.3. Exmples of ΔL* vlue chnges during storge time of coted mushrooms with vrious concentrtions of sodium lginte, beeswx, nisin nd N 2 EDTA 92

111 93 Exp. No. Alg % BW % Tble 5.4. Rte of chnge in qulity prmeters during storge of mushrooms t 12 C Nisin IU/mL N 2 EDTA mg/ml dδl* 0-6 dδl* 6-16 dδe* * * * * * * * * * * * * 0.26 *Dt point not included in sttisticl nlysis dδe* 6-16 %WG dw dmi 0-6 dmi 6-16 dbg 0-6 dbg 6-16

112 94 Tble 5.5. ANOVA tble for rte of chnge in lightness (dδl*) during dys 0-6 of storge t 12 C dδl* 0-6 DF SS MS (vrince) F p SD Totl Constnt Totl Corrected Regression Residul Lck of Fit (Model Error) Pure Error (Replicte Error) N = 24 Q2 = Cond. no. = DF = 16 R2 = Y-miss = 0 R2 Adj. = RSD = The concentrtion of N 2 EDTA in the coting significntly decresed dδl* t the 5% level (Tble 5.6). Thus, N 2 EDTA cn significntly slow down the rte of mushroom browning during erly dys of storge. The min effect of N 2 EDTA is shown grphiclly in Figure 5.4, the prbolic shpe of this grph explins why the qudrtic effect of N 2 EDTA is significnt in the model (p<0.10). The decrese in dδl* reched plteu when the concentrtion of N 2 EDTA ws 8.18mg/mL (coded vlue 1) nd no further decrese in dδl* ws chieved when the concentrtion ws incresed to 10mg/mL (coded vlue 1.68). The concentrtion of nisin in the coting significntly decresed dδl* t the 10% level (Tble 5.6). Thus, nisin cn significntly slow down the rte of mushroom browning during the beginning of storge time. The min effect of nisin is shown grphiclly in Figure 5.5.

113 Tble 5.6: Regression coefficients of lginte, beeswx, nisin nd N2EDTA concentrtions on rte of chnge in lightness (dδl*) during dys 0-6 of storge t 12 C dδl* 0-6 Coeff. N Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Block DF = 1 Block (B1) e Block (B2) e EDT*EDT Alg*Bee N = 24 Q2 = Cond. no. = DF = 16 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = Figure 5.4. Min effect of N 2 EDTA on dδl* during dys 0-6 of storge t 12 C

114 ABS Slope Delt L Nisin Figure 5.5. Min effect of nisin on dδl* during dys 0-6 of storge t 12 C The concentrtions of sodium lginte nd beeswx did not show significnt effect on the rte of browning (Tble 5.6). The interction between lginte nd beeswx ws significnt t the 5% level nd is shown in Figure 5.6, ccording to the grph when lginte is held t the high level n increse in beeswx decreses browning, but when lginte is held t low level incresing beeswx concentrtion increses browning. The interction effect of lginte nd beeswx is highly confounded with the liner effect of N 2 EDTA (correltion = 0.77). Given tht the min effect of N 2 EDTA is significnt nd the min effect of lginte nd beeswx re not significnt, it is very likely tht this interction turned out to be significnt becuse it ws confounded.

115 97 Figure 5.6. Interction between lginte nd beeswx on dδl* during dys 0-6 of storge t 12 C For dys 6-16 of storge the regression model fitted to the dt ws significnt (p<0.01) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.72 (Tble 5.7). The concentrtion of beeswx in the coting significntly decresed dδl* t the 1% level (Tble 5.8). Thus, in the lter dys of storge beeswx cn significntly slow down the rte of mushroom browning. The min effect of beeswx is shown grphiclly in Figure 5.7. The concentrtions of sodium lginte, nisin nd beeswx did not show significnt effect on the rte of browning between dys 6-16 of storge (Tble 5.8).

116 Tble 5.7. ANOVA tble for rte of chnge in lightness (dδl*) during dys 6-16 of storge t 12 C dδl* 6-16 DF SS MS (vrince) F p SD Totl Constnt Totl Corrected Regression Residul Lck of Fit (Model Error) Pure Error (Replicte Error) N = 23 Q2 = Cond. no. = DF = 14 R2 = Y-miss = 0 R2 Adj. = RSD = Tble 5.8. Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of chnge in lightness (dδl*) during dys 6-16 of storge t 12 C dδl* 6-16 Coeff. SC Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Block(1&2) Alg*Nis Bee*EDT Nis*EDT N = 23 Q2 = Cond. no. = DF = 14 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = 0.90

117 ABS Slope Delt L Beeswx Figure 5.7. Min effect of beeswx on dδl* during dys 6-16 of storge t 12 C The interction between nisin nd N 2 EDTA hd significnt effect on decresing dδl* (p<0.10). However, none of the min effects of this interction re significnt so it is difficult to speculte bout the mening of this interction. According to the grph (see Figure 5.8), when the concentrtion of N 2 EDTA is low, n increse in nisin concentrtion decreses the chnge in ΔL* vlue; but when the concentrtion of N 2 EDTA is high, nisin slightly increses chnge in ΔL* vlue.

118 Figure 5.8. Interction between nisin nd N 2 EDTA on dδl* during dys 6-16 of storge t 12 C 100

119 ΔE* Vlue To evlute the effect of coting composition on mushroom overll color chnge the dt set ws broken in two prts: dys 0-6, nd dys 6-16 of storge. Tble 5.4 shows the rte of browning expressed by rte of chnge in ΔE* vlue (dδe*). Lrger vlues of dδe* indicte fster chnge in color. For dys 0-6 of storge the regression model fitted to the dt ws significnt (p<0.05) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.74 (Tble 5.9). Tble 5.9. ANOVA tble for rte of chnge in color (dδe*) during dys 0-6 of storge t 12 C Slope Delt E 0-6 DF SS MS (vrince) F p SD Totl Constnt Totl Corrected Regression Residul Lck of Fit (Model Error) Pure Error (Replicte Error) N = 22 Q2 = Cond. no. = DF = 12 R2 = Y-miss = 0 R2 Adj. = RSD = The concentrtion of beeswx nd nisin in the coting significntly decresed dδe* t the 5% level (Tble 5.10). Thus, beeswx nd nisin cn significntly slow down the rte of mushroom color chnge during the erly dys of storge. The min effect of beeswx nd nisin re shown grphiclly in Figure 5.9 nd Figure 5.10 respectively.

120 Slope Delt E 0-6 Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of chnge in color (dδe*) during dys 0-6 of storge t 12 C Slope Delt E 0-6 Coeff. SC Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Block(1&2) EDT*EDT Alg*Bee Alg*Nis Bee*Nis N = 22 Q2 = Cond. no. = DF = 12 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = Beeswx Figure 5.9. Min effect of beeswx on dδe* during dys 0-6 of storge t 12 C

121 Slope Delt E Nisin Figure Min effect of nisin on dδe* during dys 0-6 of storge t 12 C The concentrtion of N 2 EDTA in the coting significntly decresed dδe* t the 10% level (Tble 5.10). Thus, N 2 EDTA cn slow down the rte of mushroom color chnge during the erly dys of storge. The min effect of N 2 EDTA is shown grphiclly in Figure 5.11, the decrese in dδe* reched plteu when the concentrtion of N 2 EDTA ws 8.18mg/mL (coded vlue 1) nd no further decrese in dδe* ws chieved when the concentrtion ws further incresed to 10mg/mL (coded vlue 1.68).

122 Slope Delt E N2EDTA Figure Min effect of N 2 EDTA on dδe* during dys 0-6 of storge t 12 C The concentrtion of sodium lginte did not show significnt effect on dδe* (Tble 5.10). The interction between lginte nd beeswx ws significnt t the 5% level nd is shown in Figure 5.12, ccording to the grph when beeswx is held t the high level n increse in lginte decreses chnge in color, but when beeswx is held t low level incresing lginte concentrtion increses color chnge. The interction effect of lginte nd beeswx is highly confounded with the liner effect of N 2 EDTA (correltion = 0.77). Given tht the min effect of N 2 EDTA is significnt nd the min effect of lginte is not significnt, it is likely tht this interction turned out to be significnt becuse it ws confounded.

123 105 Figure Interction between lginte nd beeswx on dδe* during dys 0-6 of storge t 12 For dys 6-16 of storge the regression model fitted to the dt ws significnt (p<0.01) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.72 (Tble 5.11). The concentrtion of beeswx in the coting significntly decresed dδe* t the 1% level (Tble 5.12). Thus, in the lter dys of the storge beeswx cn significntly slow down the rte of color chnge in mushrooms. The min effect of beeswx is shown grphiclly in Figure 5.13.

124 Tble ANOVA tble for rte of chnge in color (dδe*) during dys 6-16 of storge t 12 C Slope Delt E 6-16 DF SS MS (vrince) Totl Constnt F p SD Totl Corrected Regression Residul Lck of Fit (Model Error) Pure Error (Replicte Error) N = 23 Q2 = Cond. no. = DF = 14 R2 = Y-miss = 0 Comp. = 2 R2 Adj. = RSD = Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of chnge in color (dδe*) during dys 6-16 of storge t 12 C Slope Delt E 6-16 Coeff. SC Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Block(1&2) Bee*Nis Bee*EDT Nis*EDT N = 23 Q2 = Cond. no. = DF = 14 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = 0.90

125 Slope Delt E Beeswx Figure Min effect of beeswx on dδe* during dys 6-16 of storge t 12 C The concentrtions of sodium lginte, nisin nd N 2 EDTA did not show significnt effect on the rte of color chnge between dys 6-16 of storge (Tble 5.12). The interction between nisin nd N 2 EDTA ws significnt (p<0.05), but the min effects of this interction were not significnt so it is difficult to speculte bout the mening of this interction. According to Figure 5.14, when the concentrtion of N 2 EDTA is low, n increse in nisin concentrtion decreses the chnge in ΔE* vlue; but when the concentrtion of N 2 EDTA is high, nisin increses chnge in ΔE* vlue. The interction between beeswx nd N 2 EDTA ws significnt (p<0.10). According to Figure 5.15 when the concentrtion of N 2 EDTA ws low, beeswx hd no effect on decresing color chnge. However, when the concentrtion of N 2 EDTA ws high beeswx ws ble to decrese the chnge in color. This interction is highly confounded with the min effect of lginte (correltion = 0.77), but in this cse the min effect of lginte is not significnt nd the min effect for beeswx is highly significnt (p<0.01).

126 108 Figure Interction between nisin nd N 2 EDTA on dδe* during dys 6-16 of storge t 12 C Figure Interction between beeswx nd N 2 EDTA on dδe* during dys 6-16 of storge t 12 C

127 109 N 2 EDTA hd significnt effect on decresing overll color chnge rte (dδe*) in coted mushrooms nd on reducing the rte of mushroom browning (dδl*) on the erly dys of storge. This is in greement with study where cellulose bsed coting contining lipids, CCl 2, clcium disodium EDTA nd scorbic cid ws used to cot mushrooms. Severl combintions of the ctive ingredients were used. The coting by itself, the coting with EDTA nd scorbic cid, nd tretment of EDTA nd scorbic cid lone resulted in higher L* vlues compred to mushrooms wshed with wter, coted mushrooms contining scorbic cid nd CCl 2, nd tretment of scorbic cid nd CCl 2 lone fter 8 hours of storge t 70 F (Nisperos-Crriedo nd others 1991). In nother study, N 2 EDTA in combintion with sodium erythorbte nd cysteine in mushroom wsh hs shown to reduce browning compred to unwshed control on whole nd sliced mushrooms fter 6 nd 5 dys of storge respectively t 4 C (Spers nd others 1994). Mushrooms wshed with solutions contining 1000ppm of EDTA hve shown to preserve whiteness more effectively thn wsh tretments contining combintions of sulfite, ftty cids nd scorbic cid esters (McConnell 1991). Tetrsodium EDTA ws shown to improve color in cnned mushrooms when dded in the brine fter vcuum hydrtion in citric cid (Kilr nd others 1984). Nisin hd significnt effect on decresing overll color chnge nd reducing browning on the erly dys of storge, this effect is ttributed to the inhibition effect of nisin in combintion with N 2 EDTA ginst Pseudomons sp. (Ukuku nd Fett 2002; Economou nd others 2009; Lu nd others 2010). Microbil lod hs been relted to the overll color qulity of fresh mushrooms (Beelmn nd others 1989), if nisin ws ble to inhibit growth of Pseudomons sp. in the erly dys of storge, this would reduce overll color chnge nd browning. Beeswx hd significnt effect on reducing overll color chnge throughout the 16 dys of storge nd reducing chnge in dδl*vlue in the lter dys of storge (6-16). In sections nd it will be shown tht beeswx hd significnt effect in reducing weight gin nd incresing weight loss of coted mushrooms. Other studies

128 110 hve lso shown tht when tretment results in higher weight loss, color is improved. Mushrooms treted with vcuum cooling nd stored t 18 C fter vrious periods of storge t 5 C hve shown less degree of mushrooms browning compred to conventionlly cooled mushrooms. The rte of weight loss of the vcuum cooled mushrooms ws significntly greter thn tht of the conventionlly cooled mushrooms (p<0.02), fter 102 hours of storge t 5 C vcuum cooled mushrooms lost 1.7% more wter; the uthors speculted tht the evported wter ws extrcellulr nd by removing it, bcteril growth ws suppressed resulting in less browning (Burton nd others 1987). Fresh mushrooms pckged with 15 g pouches of sorbitol to control humidity of the pckge hve shown less surfce moisture, higher weight loss nd higher L vlues (p<0.05) (Roy nd others 1995b). Sodium cseinte coting contining beeswx hs shown to preserve green color in fresh cut sprgus spers fter 14 dys of storge t 4 C nd 66% R.H. expressed by significntly lower * vlues (more negtive = more green) (p<0.05), in this study coted smples with chitosn contining beeswx hd significntly higher * vlues (less negtive = less green). However, the uthors ttributed this effect to the low ph of the chitosn coting degrding chlorophyll to pheophytin nd reducing green color (Fuchs nd others 2008). Whey protein concentrte, whey protein isolte nd hydroxypropyl methyl cellulose cotings, ll of them contining beeswx hve shown to significntly reduce browning in fresh cut pple pieces (p<0.05) compred to the sme polymers contining crnub wx. Moreover, both wxes were ble to significntly reduce browning index compred to uncoted control stored for 7 dys t 5 C or 1 dy t 20 C (Perez-Ggo nd others 2005). The interction between nisin nd N 2 EDTA ws significnt during the lter dys of storge. When N 2 EDTA ws held t low level nd nisin ws incresed dδl* nd dδe* were reduced. In sections nd it will be shown tht the interction between nisin nd N 2 EDTA hd significnt effect in reducing weight gin nd incresing weight loss of coted mushrooms. As mentioned before for the min effect of

129 beeswx, other studies hve shown tht when tretment results in higher weight loss, color is improved (Burton nd others 1987; Roy nd others 1995b) Weight gin Tble 5.4 shows % weight gin (%WG) of mushrooms fter one hour of drying t 4 C. The regression model fitted to the dt ws significnt (p<0.01) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.74 (Tble 5.13). Tble ANOVA tble for % weight gin fter coting % Weight Gin DF SS MS (vrince) F p SD Totl Constnt Totl Corrected Regression Residul Lck of Fit (Model Error) Pure Error (Replicte Error) N = 23 Q2 = Cond. no. = DF = 15 R2 = Y-miss = 0 Comp. = 2 R2 Adj. = RSD = The concentrtion of lginte in the coting significntly incresed %WG fter drying t the 5% level (Tble 5.14). The min effect of lginte is shown grphiclly in Figure This effect cn be relted to the viscosity of the coting, the concentrtion of lginte significntly incresed the viscosity of the coting t the 0.001% level (dt not shown), incresing viscosity of the coting results in thicker films (Cisneros Zevllos nd Krocht 2003); thus %WG is incresed. The concentrtion of beeswx in the coting significntly decresed %WG t the 5% level fter drying (Tble 5.14). The min effect of beeswx is shown grphiclly in

130 % Weight Gin 112 Figure Incresing the mount of beeswx increses hydrophobicity of the coting, nd the cpcity of the film to bind wter might be reduced. After the drying step t 4 C more wter would evporte from the film resulting in lower coting weight. Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on % weight gin fter coting % Weight Gin Coeff. SC Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Block(1&2) Nis*Nis Nis*EDT N = 23 Q2 = Cond. no. = DF = 15 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = Alginte Figure Min effect of lginte on weight gin fter coting

131 % Weight Gin Beeswx Figure Min effect of beeswx on weight gin fter coting The concentrtions of nisin nd N 2 EDTA did not show significnt effect on coting weight (Tble 5.14). The interction between N 2 EDTA nd nisin hd significnt effect on incresing coting weight (p<0.01) when the concentrtion of N 2 EDTA ws high nd nisin ws incresed (Figure 5.18). Since the min effect of nisin nd N 2 EDTA re not significnt it is difficult to speculte bout the mening of this interction. Chelting gents such s N 2 EDTA cn be used to control the cross-linking rection between sodium lginte nd clcium chloride (King 1983; Drget 2009). Higher concentrtion of N 2 EDTA would result in less cross-linking, nd less crosslinking results in less rigidity nd incresed moisture content of film (Rhim 2004). This would llow for more spce within the mtrix to hold bulky molecules like nisin preventing nisin from disrupting the lginte mtrix nd improving weight gin.

132 Figure Interction between N 2 EDTA nd nisin on % weight gin fter coting 114

133 Weight loss When the dt set for mushroom weight loss ws evluted the plots were found to be liner, so for this nlysis the dt set ws not split into two time periods. Tble 5.4 shows the rte of mushroom weight loss expressed by the bsolute slope of weight (dw). Lrger bsolute vlues of dw indicte fster chnge in weight. The regression model fitted to the dt ws significnt (p<0.01) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.78 (see Tble 5.15) Tble ANOVA tble for bsolute rte of chnge in weight (dw) during storge of mushrooms t 12 C dw DF SS MS (vrince) F p SD Totl Constnt Totl Corrected Regression Residul Lck of Fit (Model Error) Pure Error (Replicte Error) N = 23 Q2 = Cond. no. = DF = 13 R2 = Y-miss = 0 R2 Adj. = RSD = The concentrtion of beeswx in the coting significntly incresed dw t the 10% level (Tble 5.16). Thus, beeswx cn significntly increse the rte of mushroom weight loss. The min effect of beeswx is shown grphiclly in Figure In study where fresh cut Fuji pples were coted with lginte nd ntibrowning gents, the resistnce to wter diffusion (clculted from the rte of moisture loss indicted by weight loss) ws incresed when sunflower oil ws dded to the coting. However, incresing the oil in the lginte coting from 0.025% to 0.125% did not hve n effect on the wter vpor resistnce (Rojs-Grü nd others 2009). Similr results were obtined when fresh cut

134 dw ppy ws coted with n lginte contining 2% glycerol, 1% scorbic cid nd sunflower oil ws dded to the coting in the sme proportions (Tpi nd others 2008). 116 Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on bsolute rte of chnge in weight (dw) during storge of mushrooms t 12 C dw Coeff. SC Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Block(1&2) Alg*Alg Bee*Bee Alg*Nis Nis*EDT N = 23 Q2 = Cond. no. = DF = 13 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = Beeswx Figure Min effect of beeswx on bsolute slope of weight during storge t 12 C

135 117 In our work incresing the concentrtion of beeswx from 0.1 to 0.5% (coded vlues to 0) did not hve ny effect on the weight loss. However, when beeswx content ws incresed from 0.5% to 1% (coded vlues 0 to 1.68) it significntly incresed weight loss (p<0.1). This is in greement with study were clcium lginte films were incorported with grlic oil, incresing concentrtions of grlic oil from 0 to 0.3% (v/v) did not show significnt differences (p>0.05) in wter vpor permebility in the films (rnge g mm/m 2 dy kp). However, when the concentrtion ws incresed to 0.4% wter vpor permebility significntly incresed to g mm/m 2 dy kp (p<0.05). The uthors explined this effect with the hydrophobicity of grlic oil which would extend intermoleculr interctions of the lginte mtrix, llowing moisture to pss through the film (Prnoto nd others 2005). Cohesive strength in films provides reduction in porosity nd permebility to gses nd vpors, cohesive strength is enhnced by polrity of the polymer (Kester nd Fennem 1986). Incresing concentrtion of beeswx would reduce the polrity of the lginte mtrix decresing cohesive strength nd providing more permeble film which would result in more weight loss. In section it ws observed tht beeswx significntly reduced the weight gin fter coting (p<0.05), incresing mount of beeswx mens less lginte vilble to form the hydrophilic film so the cpcity of the film to bind wter is reduced. Alginte cotings cn ct s scrificing gent which gives up moisture within the polymer before the food strts dehydrting (Kester nd Fennem 1986). A coting with less cpcity to bind wter would hve reduced cpcity to ct s scrificing gent nd weight loss would be incresed. The concentrtion of nisin in the coting significntly incresed dw t the 5% level (Tble 5.16). Thus, nisin cn significntly increse the rte of mushroom weight loss. The min effect of nisin is shown grphiclly in Figure The negtive effect of nisin seemed to be reduced by incresing the concentrtion of lginte or N 2 EDTA, s

136 dw 118 indicted by the significnt interctions of nisin nd N 2 EDTA nd nisin nd lginte (Figure 5.21 nd Figure 5.22). The concentrtions of sodium lginte nd N 2 EDTA did not show significnt effect on the rte of weight loss (Tble 5.16) Nisin Figure Min effect of nisin on bsolute slope of weight during storge t 12 C The interction between lginte nd nisin ws significnt t the 1% level, when lginte ws kept constnt t high level, n increse in nisin concentrtion hd no effect, but when the concentrtion of lginte ws low n increse in nisin concentrtion significntly incresed the rte of weight loss (Figure 5.21). A lower concentrtion of lginte results in thinner film which might be disrupted when nisin concentrtion is incresed. If the film is disrupted the brrier properties of the film re reduced resulting in incresed weight loss. If the film is thick enough (higher concentrtion of lginte) incresing the mount of nisin would not disrupt the integrity of the film. In study where the properties of biodegrdble Poly butylene dipte-co-terephthlte (PBAT) films contining nisin were studied, it ws found tht incresing concentrtions of nisin resulted in smll pores nd holes in the PBAT mtrix s observed by scnning electron

137 119 microscopy, the uthors concluded tht the interction between the polymer nd nisin reduced the bility of the polymer to build bonds within its mtrix, this would cuse reduction in brrier properties of the film (Bstrrche nd others 2010). Figure Interction between lginte nd nisin on bsolute slope of weight during storge t 12 C. The remining two fctors kept constnt t center level. The interction between nisin nd N 2 EDTA ws significnt t the 5% level, when N 2 EDTA ws kept constnt t high level, n increse in nisin concentrtion hd no effect, but when the concentrtion of N 2 EDTA ws low n increse in nisin concentrtion significntly increse the rte of weight loss (Figure 5.22). As mentioned in the previous section, N 2 EDTA is used to control cross-linking (King 1983; Drget 2009). Higher concentrtion of N 2 EDTA would result in less cross-linking, reducing rigidity nd incresing moisture content of the film (Rhim 2004). This would llow for more spce within the mtrix to hold bulky molecules, like nisin, preventing nisin from disrupting the lginte mtrix nd reducing weight loss.

138 Figure Interction between N 2 EDTA nd nisin on bsolute slope of weight during storge t 12 C. The remining two fctors kept constnt t center level 120

139 Mturity index To evlute the effect of coting composition on mturity index the dt set ws broken in two prts: dys 0-6, nd dys 6-16 of storge. Tble 5.4 shows the mturity index expressed by rte chnge in mturity index (dmi). Higher vlues of dmi indicte fster opening of the cp. For dys 0-6 of storge the regression model fitted to the dt ws not significnt (p>0.05), so the response vrible (slope of mturity index) ws trnsformed using the power trnsformtion (dmi 2 ) s indicted by the Box-Cox plot. The regression model fitted to the dt ws significnt (p<0.01) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.72 (Tble 5.17). Tble ANOVA tble for rte of chnge in mturity index (dmi) 2 during dys 0-6 of storge t 12 C dmi DF SS MS (vrince) F p SD Totl Constnt Totl Corrected Regression Residul Lck of Fit (Model Error) Pure Error (Replicte Error) N = 23 Q2 = Cond. no. = DF = 14 R2 = Y-miss = 0 R2 Adj. = RSD =

140 122 The concentrtion of nisin in the coting significntly decresed the slope of mturity index t the 1% level (Tble 5.18). Thus, nisin cn significntly slow down the rte of cp opening during the erly dys of storge. The min effect of nisin is shown grphiclly in Figure 5.23, the prbolic shpe of this grph explins why the qudrtic effect of nisin is significnt in the model (p<0.05). The decrese in the slope of mturity index reched plteu when the concentrtion of nisin ws 6378 mg/ml (coded vlue 1) nd no further decrese in the slope of mturity index ws chieved when the concentrtion ws further incresed to 8000mg/mL (coded vlue 1.68). The concentrtion of sodium lginte, beeswx nd N 2 EDTA did not show significnt effect on the slope of mturity index. Tble Regression coefficients of lginte, nisin nd N 2 EDTA concentrtions on rte of chnge in mturity index during dys 0-6 of storge t 12 C dmi Coeff. SC Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Block(1&2) Nis*Nis Alg*Nis Nis*EDT N = 23 Q2 = Cond. no. = DF = 14 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = 0.90

141 dmi Nisin Figure Min effect of nisin concentrtion on rte of chnge in mturity index during dys 0-6 of storge t 12 C The rte of cp opening hs been relted to microbil growth throughout storge (Doores nd others 1986). Decrese in extent of cp opening together with lower stipe elongtion nd less microbil counts hve been observed when oxine (stbilized chlorine dioxide) ws dded to wtering tretments on mushrooms. The effect ws intensified when combintion tretment of oxine nd CCl 2 ws dded to irrigtion wter reducing not only bcteril counts nd both senescence symptoms but lso browning (Solomon nd others 1991). Both oxine nd nisin re ntimicrobils, high microbil counts might cuse stress to the mushrooms resulting in fster senescence symptoms. If the microbil counts re reduced by n ntimicrobil this would slow down the extent of cp opening resulting in lower mturity index. This is in greement with the min effect of nisin lso being significnt in decresing overll color chnge nd reducing browning on the erly dys of storge (See section )

142 124 For dys 6-16 of storge the regression model fitted to the dt ws not significnt (p>0.05), so the response vrible (slope of mturity index) ws trnsformed using the power trnsformtion (dmi 2 ) s indicted by the Box-Cox plot. The regression model fitted to the dt ws significnt (p<0.01) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.82 (Tble 5.19). Tble ANOVA tble for rte of chnge in mturity index (dmi) 2 during dys 6-16 of storge t 12 C dmi DF SS MS (vrince) Totl Constnt F p SD Totl Corrected Regression Residul e Lck of Fit e (Model Error) Pure Error e (Replicte Error) N = 24 Q2 = Cond. no. = DF = 12 R2 = Y-miss = 0 R2 Adj. = RSD = The concentrtions of sodium lginte, beeswx, nisin nd N 2 EDTA did not show significnt effect on the rte of chnge in mturity index between dys 6-16 of storge time (Tble 5.20). The interction between nisin nd N 2 EDTA hd significnt effect on decresing the slope of mturity index (p<0.05) when the concentrtion of N 2 EDTA ws low nd nisin ws incresed (Figure 5.24). However, the min effects of nisin nd N 2 EDTA were not significnt so it is difficult to speculte bout the mening of this interction. Nonetheless, this interction ws lso significnt for dδl* nd dδe* in the lter dys of storge nd for weight loss throughout storge. As mentioned before for the min effect of nisin, some studies hve shown tht ntimicrobil tretments cn

143 125 reduce chnges in both color nd mturity index (Doores nd others 1986; Solomon nd others 1991). Other studies hve shown tht when tretment results in higher weight loss it reduces color chnge (Burton nd others 1987; Roy nd others 1995b). In our work, incresing nisin concentrtion in the erly dys of storge incresed the rte of weight loss, decresed the rte of color chnge nd mturity index; nd in the lter dys of storge incresing nisin concentrtion t low levels of N 2 EDTA incresed the rte of weight loss, reduced color chnge nd mturity index. It seems tht there might be reltionship between the rte of weight loss nd the rte of cp opening similr to the one found for the rte of chnge in color nd the rte of cp opening. Tble 5.20: Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of chnge in mturity index (dmi) during dys 6-16 of storge t 12 C dmi Coeff. SC Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Block(1&2) Bee*Bee Nis*Nis EDT*EDT Alg*Nis Bee*EDT Nis*EDT N = 24 Q2 = Cond. no. = DF = 12 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = 0.90

144 126 Figure Interction between Nisin nd N 2 EDTA on rte of chnge in mturity index during dys 6-16 of storge t 12 C The interction between beeswx nd N 2 EDTA hd significnt effect on decresing the slope of mturity index (p<0.05) when N 2 EDTA ws t high level nd beeswx concentrtion ws incresed (Figure 5.25). However, this interction is highly confounded with the min effect of lginte (correltion =0.77), which in this cse is lmost significnt (P=0.106), the contour plots (see Figure 5.41 nd Figure 5.42) show tht lginte only decreses dmi when beeswx concentrtion is high nd lginte concentrtion is incresed, t medium nd lower levels of beeswx, lginte does not ffect the slope of mturity index. A higher concentrtion of lginte results in thicker coting which will hve better brrier properties nd lower permebility to gses.

145 127 Figure Interction between beeswx nd N 2 EDTA on rte of chnge in mturity index during dys 6-16 of storge t 12 C Stndrd plte count To evlute the effect of coting composition on the bcteril growth indicted by stndrd plte count the dt set ws broken in two prts: dys 0-6, nd dys 6-16 of storge. Tble 5.4 shows the rte of bcteril growth expressed by rte chnge in Log(Nf/No), (dbg). Lrger vlues of dbg indicte fster bcteril growth. For dys 0-6 of storge the regression model fitted to the dt ws significnt (p<0.05) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.79 (Tble 5.21).

146 128 Tble ANOVA tble for rte of bcteril growth (dbg) during dys 0-6 of storge t 12 C dbg 0-6 DF SS MS (vrince) Totl Constnt F p SD Totl Corrected Regression Residul Lck of Fit (Model Error) Pure Error (Replicte Error) N = 21 Q2 = Cond. no. = DF = 10 R2 = Y-miss = 0 Comp. = 3 R2 Adj. = RSD = The concentrtion of lginte in the coting significntly decresed the rte of bcteril growth t the 1% level (Tble 5.22). Thus, lginte cn significntly slow down the rte of bcteril growth on mushrooms during the erly dys of storge. The min effect of lginte is shown grphiclly in Figure 5.26.

147 dbg 0-6 Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of bcteril growth (dbg) during dys 0-6 of storge t 12 C dbg 0-6 Coeff. SC Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Nis*Nis Alg*Bee Alg*Nis Bee*Nis Bee*EDT Nis*EDT N = 21 Q2 = Cond. no. = DF = 10 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = Alginte Figure Min effect of lginte on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C

148 dbg The concentrtion of N 2 EDTA in the coting significntly decresed the rte of bcteril growth t the 1% level (Tble 5.22). Thus, N 2 EDTA cn significntly slow down the rte of bcteril growth on mushrooms during the erly dys of storge. The min effect of N 2 EDTA is shown grphiclly in Figure The concentrtions of nisin nd beeswx did not show significnt effect on the rte of bcteril growth (Tble 5.22) N2EDTA Figure Min effect of N 2 EDTA on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C The interction between lginte nd nisin hd significnt effect on lowering the rte of bcteril growth (p<0.05) when the concentrtion of lginte ws kept t constnt high level nd the concentrtion of nisin ws incresed (Figure 5.28). Similrly when the concentrtion of nisin ws kept t constnt high level nd the concentrtion of lginte ws incresed, the rte of bcteril growth ws decresed (Figure 5.29).

149 131 Figure Interction between lginte nd nisin on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C Figure Interction between nisin nd lginte on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C

150 132 The interction between lginte nd beeswx hd significnt effect on lowering the rte of bcteril growth (p<0.05) when the concentrtion ws high for lginte nd beeswx concentrtion ws incresed (Figure 5.30), this interction is highly confounded with the liner effect of N 2 EDTA (correltion = 0.77) which is significnt (p<0.01) so it is difficult to conclude if the effect is due to the min effect of N 2 EDTA or this interction. Figure Interction between lginte nd beeswx on bcteril growth rte (dbg) during dys 0-6 of storge t 12 C The interction between beeswx nd N 2 EDTA hd significnt effect on lowering the rte of bcteril growth (p<0.01) when the concentrtion of beeswx ws t the high level nd N 2 EDTA ws incresed (Figure 5.31). However, this interction is highly confounded with the liner effect of lginte (correltion = 0.77) which is significnt in the model (p<0.01) so it is difficult to speculte if the effect is due to the min effect of lginte or this interction.

151 133 Figure Interction between beeswx nd N 2 EDTA on bcteril growth rte (dbg) during dys 0-6 of storge t 12 For dys 6-16 of storge the regression model fitted to the dt ws significnt (p<0.01) with no significnt lck of fit nd coefficient of determintion (R 2 ) of 0.80 (Tble 5.23). The concentrtion of lginte nd beeswx in the coting significntly decresed the rte of bcteril growth t the 1% nd 5% level respectively (Tble 5.24). Thus, in the lter dys of storge time lginte nd beeswx cn significntly slow down the rte of bcteril growth. The min effect of lginte nd beeswx re shown grphiclly in Figure 5.32 nd Figure The concentrtions of nisin nd N 2 EDTA did not show significnt effect on the rte of bcteril growth between dys 6-16 of the storge (Tble 5.24).

152 Tble 5.23: ANOVA tble for rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C dbg 6-16 DF SS MS (vrince) Totl Constnt F p SD 134 Totl Corrected Regression Residul Lck of Fit e (Model Error) Pure Error (Replicte Error) N = 22 Q2 = Cond. no. = DF = 12 R2 = Y-miss = 0 Comp. = 4 R2 Adj. = RSD = Tble Regression coefficients of lginte, beeswx, nisin nd N 2 EDTA concentrtions on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C dbg 6-16 Coeff. SC Std. Err. P Conf. int(±) Constnt e Alg Bee Nis EDT Block(1&2) Bee*Bee Nis*Nis Alg*Bee Bee*EDT N = 22 Q2 = Cond. no. = DF = 12 R2 = Y-miss = 0 R2 Adj. = RSD = Conf. lev. = 0.90

153 dbg 6-16 dbg Alginte Figure Min effect of lginte on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C Beeswx Figure Min effect of beeswx on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C

154 136 The interction between lginte nd beeswx hd significnt effect on decresing the rte of bcteril growth (p<0.05) when the concentrtion ws high for lginte nd beeswx ws incresed (Figure 5.34), this interction is highly confounded with the min effect of N 2 EDTA (correltion = 0.77), but in this cse the min effect of N 2 EDTA is not significnt. Figure Interction between lginte nd beeswx on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C The interction between beeswx nd N 2 EDTA hd significnt effect on decresing the rte of bcteril growth (p<0.01) when the concentrtion ws high for beeswx nd N 2 EDTA ws incresed (Figure 5.35). However, this interction is highly confounded with the min effect of lginte (correltion = 0.77) nd the min effect of lginte is significnt t the 1% level.

155 137 Figure Interction between N 2 EDTA nd beeswx on rte of bcteril growth (dbg) during dys 6-16 of storge t 12 C The concentrtion of lginte in the coting significntly decresed the rte of bcteril growth t the 1% level throughout storge. As explined in section 5.3.2, lginte significntly incresed weight gin fter coting (p<0.05) this is trnslted in thicker coting, which would relese more nisin nd N 2 EDTA onto the mushroom surfce. Thus, lginte cn significntly slow down the rte of bcteril growth on mushrooms during the erly dys of storge. The concentrtion of N 2 EDTA in the coting significntly decresed the rte of bcteril growth t the 1% level in the erly dys of storge. Indigenous microflor of fresh mushrooms includes 54% of fluorescent Pseudomons nd 10% Flvobcterium, these re both grm-negtive bcteri. Nisin is n effective ntimicrobil ginst grmpositive bcteri but hs limited ctivity ginst grm-negtives. However, grm-negtive bcteri cn become vulnerble to nisin by exposure to chelting gents such s N 2 EDTA (Delves-Broughton nd others 1996). N 2 EDTA removes ctions Mg +2 nd C +2, from the cell wll of grm negtives disrupting the phospholipid nd lipoprotein

156 138 structure of the wll nd mking it more permeble to ntimicrobil gents, this mechnism is shown in Figure 5.36 (Delves-Broughton nd others 1996). Incresing the concentrtion of N 2 EDTA would increse the vulnerbility of mushroom microflor to nisin nd tht explins why incresing the concentrtion of N 2 EDTA significntly decreses the rte of bcteril growth on the erly dys of storge.

157 Figure Mode of ction of nisin nd N 2 EDTA on grm negtive bcteri 139

158 140 It is likely tht the min effect of N 2 EDTA ws not significnt in the lter dys of storge becuse the diffusion from the coting might hd lredy reched equilibrium by this time. EDTA incorported t 400ppm in n in vitro study hs shown to increse the ntimicrobil ctivity of hydrogen peroxide, octnoic nd dodecnoic cids, monocrpylin, scorbyl decnote nd scorbyl dodecnote ginst Pseudomons isolted from fresh mushrooms. Moreover, when EDTA ws incorported t 1000ppm together with hydrogen peroxide t 10,000ppm in wshing solution for fresh mushrooms, purple blotch disese ws inhibited nd color ws preserved for up to 6 dys (McConnell 1991). The concentrtion of beeswx did not show significnt effect on the rte of bcteril growth on the erly dys of storge but it ws significnt t the 1% level in the ltter dys of storge. The concentrtion of beeswx in the coting lso reduced the weight gin nd incresed the weight loss of coted mushrooms (see sections nd 5.3.3). Mushrooms with higher weight loss hve lower moisture. Moisture ccumultion in the cp hs been found to fvor growth of Pseudomons tolsii (Brber nd Summerfield 1990), overwtering during growing lso increses bcteril popultions (Chikthimmh nd Beelmn 2006). Mushrooms with lower moisture would hve less wter vilble for bcteril growth. Fresh mushrooms pckged with 15 g pouches of sorbitol to control humidity of the pckge hve shown reduction in totl plte count (p<0.05) (Roy nd others 1995b). The interction between lginte nd nisin hd significnt effect on lowering the rte of bcteril growth in the erly dys of storge. A high concentrtion of lginte provides thicker coting which would hve higher cpcity to hold the ntimicrobil gent, so n increse in the concentrtion of nisin would decrese the rte of bcteril growth.

159 141 The interction between lginte nd beeswx hd significnt effect on lowering the rte of bcteril growth throughout storge, this interction is highly confounded with the liner effect of N 2 EDTA (correltion = 0.77) which is significnt in the erly dys of storge but not in the lter storge time. Reducing polrity of films reduces the cohesive strength nd increses permebility to solutes (Kester nd Fennem 1986), n increse in beeswx concentrtion would reduce polrity of the coting. Alginte is negtively chrged nd nisin is positively chrged, reduction in polrity of the coting would reduce the electrosttic interction between lginte nd nisin llowing for more nisin to be relesed from the coting mtrix which would reduce the rte of bcteril growth. In preliminry work 6.5% increse in inhibition zone ws observed in gr diffusion biossy with Pseudomons fluorescens, when the concentrtion of beeswx ws incresed from 0.2% to 0.4% in the lginte film contining 750 IU/mL nisin nd 5mg/ml of N 2 EDTA. The interction between beeswx nd N 2 EDTA hd significnt effect on lowering the rte of bcteril growth (p<0.01) throughout the storge time, this interction is highly confounded with the liner effect of lginte (correltion = 0.77) which is significnt in the model (p<0.01) in the erly nd lter dys of storge. Nonetheless, this interction could be explined similrly s the previous interction; n increse in beeswx concentrtion would decrese the polrity of the coting nd increse the permebility to solutes (Kester nd Fennem 1986) llowing more N 2 EDTA to be relesed from the coting nd increse vulnerbility of grm-negtive bcteri to nisin (Delves-Broughton nd others 1996) further reducing bcteril growth Interprettion nd visuliztion of the model The models developed in Sections re summrized in Tble 5.25.

160 Tble Regression models: effect of coting components on qulity prmeters of fresh mushrooms Slope ΔL* vlue 0-6 Slope ΔL* vlue 6-16 Slope ΔE* vlue 0-6 Slope ΔE* vlue 6-16 Slope weight loss % WG (Slope MI) (Slope MI) Regression P-vlue Lck of Fit P-vlue R Dropped Runs 11 18, ,11,24 12,18 Coeff sign mening - less chnge - less chnge - less chnge - less chnge - less loss + more gin - less chnge - less chnge - less growth - less growth Block Coeff b c b Alginte Coeff b Beeswx Coeff b c b b Nisin Coeff c b b EDTA Coeff b c Alg*BW Conf EDTA Coeff b b b Alg*Nis Coeff b Alg*EDTA Conf BW Coeff BW*Nis Coeff BW*EDTA Conf Alg Coeff c b Nis*EDTA Coeff b b Alg 2 Coeff c BW 2 Coeff Nis 2 Coeff b b EDTA 2 Coeff c c, b nd c superscripts denote sttisticl significnce t the 1, 5 nd 10% levels respectively. Model would not be significnt if this point ws not tken out. Smple dried out nd lst mesurement ws inccurte BG 0-6 BG 6-16

161 ΔL* Vlue From the models developed in section (see Tble 5.25) the contour plots were obtined for ech block during the erly (0-6) nd lter (6-16) dys of storge. Figure 5.37 nd Figure 5.38 show the effect of the concentrtions of lginte, beeswx, nisin nd N 2 EDTA on d ΔL* vlue on dys 0 to 6 of storge for blocks 1 nd 2 respectively. In these plots the lower contours men less chnge in ΔL* vlue which is desirble. The highly significnt effect of the block cn be observed by compring the vlues of the contours for both figures, this indictes the high vribility between different mushrooms flushes. Even though the vlue of the rte of chnge is different, the sme trend cn be observed in both figures. The significnt effect of N 2 EDTA cn be seen in both plots, s N 2 EDTA is incresed the slope of ΔL* vlue decreses. The effect of nisin brely shows up in the plots given tht nisin is only significnt t the 10% level. Figure 5.39 nd Figure 5.40 show the effect of the concentrtions of lginte, beeswx, nisin nd N 2 EDTA on dδl* vlue on dys 6 to 16 of storge for blocks 1 nd 2 respectively. In these plots the lower contours men less chnge in ΔL* vlue which is desirble. The vribility between mushrooms flushes shows up gin which chnges the vlues of the contours when compring both figures but, gin, the sme trend cn be observed. The significnt effect of beeswx cn be seen in both plots, s beeswx is incresed the slope of ΔL* vlue decreses. The interction between nisin nd N 2 EDTA shows up s the sddle points observed in these plots, s explined in Section , when the concentrtion of N 2 EDTA is low, n increse in nisin concentrtion decreses the chnge in ΔL* vlue. However, when the concentrtion of N 2 EDTA is high, nisin slightly increses chnge in ΔL* vlue.

162 Figure Contour plots for bsolute slope of slope of ΔL* vlue (Block 1) during dys 0-6 of storge t 12 C

163 Figure Contour plots for bsolute slope of slope of ΔL* vlue (Block 2) during dys 0-6 of storge t 12 C

164 Figure Contour plots for bsolute slope of slope of ΔL* vlue (Block 1) during dys 6-16 of storge t 12 C

165 Figure Contour plots for bsolute slope of slope of ΔL* vlue (Block 2) during dys 6-16 of storge t 12 C

166 148 In preliminry experiments s shown in Chpter 4, uncoted control stored t 12 C nd 60%.H. reched the etiler Acceptbility Vlue ( AV) ΔL* of 11 between dys 6 nd 8 of storge. Our gol ws to extend tht to 16 dys. To clculte the predicted shelf life from the contour plots one contour ws chosen for ech storge period. Ech contour is slope which mens specific chnge in ΔL* vlue units per dy. The contour of 0.5 ws chosen for the erly dys of storge nd for the lter dys of storge nd the shelf life ws predicted s follows: 0.5 dy 6 dys = 3 units in L vlue dy 10 dys = 7.25 units in L vlue 3 units units = units in 16 dys

167 Mturity index From the models developed in section the contour plots were obtined for ech block during the erly (0-6) nd lter (6-16) dys of storge. Figure 5.41 nd Figure 5.42 show the effect of the concentrtions of lginte, beeswx, nisin nd N 2 EDTA on the slope of mturity index on dys 0 to 6 of storge for blocks 1 nd 2 respectively. In these plots the lower contours men less chnge in mturity index which is desirble. The effect of the block cn be observed by compring the vlues of the contours for both figures. Even though the vlue of the rte of chnge in mturity index vries between flushes, the sme trend cn be observed in both figures. Figure 5.43 nd Figure 5.44 show the effect of the concentrtions of lginte, beeswx, nisin nd N 2 EDTA on the slope of mturity index on dys 6 to 16 of storge for blocks 1 nd 2 respectively. In these plots the lower contours men less chnge in mturity index which is desirble. The vribility between mushrooms flushes cn be observed gin which chnges the vlues of the contours when compring both figures. However, the sme trend cn be observed. The interction between nisin nd N 2 EDTA nd the interction between beeswx nd N 2 EDTA shows up s sddle points nd chnges in direction of the contours s the concentrtions re incresed. As explined in Section 5.3.4, when the concentrtion of N 2 EDTA is low, n increse in nisin concentrtion decreses the chnge in mturity index. In contrst, when the N 2 EDTA is t high level nd beeswx concentrtion is incresed the slope of mturity index decreses.

168 Figure Contour plots for slope of mturity index (Block 1) during dys 0-6 of storge t 12 C 150

169 Figure Contour plots for slope of mturity index (Block 2) during dys 0-6 of storge t 12 C 151

170 Figure Contour plots for slope of mturity index (Block 1) during dys 6-16 of storge t 12 C 152

171 Figure Contour plots for slope of mturity index (Block 2) during dys 6-16 of storge t 12 C 153

172 154 In preliminry experiments s shown in Chpter 4, uncoted control stored t 12 C nd 60% R.H. reched mturity index of 4 (more thn hlf of the veil prtilly broken) between dys 6 nd 8 of storge. Our gol ws to extend tht to 16 dys. To clculte the predicted shelf life from the contour plots one contour ws chosen for ech storge period. Ech contour is slope which mens specific chnge in mturity index units per dy. The contour of 0.45 ws chosen for the erly dys of storge nd 0.16 for the lter dys of storge nd the shelf life ws predicted s follows: 0.45 dy 6 dys = 2.7 units in MI 0.16 dy 10 dys = 1.6 units in 2.7 units units = 4.3 units in 16 dys

173 Stndrd plte count From the models developed in section the contour plots were obtined for ech block during the erly (0-6) nd lter (6-16) dys of storge. Figure 5.45 shows the effect of the concentrtions of lginte, beeswx, nisin nd N 2 EDTA on the slope of bcteril growth on dys 0 to 6 of storge. In this plot the lower contours men less bcteril growth which is desirble. In the erly dys of storge the vribility between the flushes in bcteril growth ws not high enough for the block to be significnt. In Figure 5.45 the significnt effects of lginte nd N 2 EDTA cn be observed, s the concentrtion of this components is incresed the slope of bcteril growth decreses. The interctions between lginte nd nisin, lginte nd beeswx nd beeswx nd N 2 EDTA, cn be observed s chnges in direction in the contours. As explined in Section 5.3.5, the rte of bcteril growth ws lowered when the concentrtion of lginte ws kept t high level nd the concentrtion of nisin ws incresed, similrly when lginte ws kept t high level nd beeswx ws incresed. Finlly when beeswx ws t high level nd N 2 EDTA ws incresed the slope of bcteril growth ws lso decresed. Figure 5.46 nd Figure 5.47 show the effect of the concentrtions of lginte, beeswx, nisin nd N 2 EDTA on the slope of bcteril growth on dys 6 to 16 of storge for blocks 1 nd 2 respectively. In these plots the lower contours men less bcteril growth which is desirble. The vribility between mushrooms flushes shows up gin which chnges the vlues of the contours when compring both figures. However, the sme trend cn be observed. The significnt effect of lginte nd beeswx cn be seen in both plots, s the concentrtion of these components is incresed the slope of bcteril growth decreses. The interctions between lginte nd beeswx, nd beeswx nd N 2 EDTA, cn be observed s sddle points nd chnges in direction in the contours. As explined in Section 5.3.5, the rte of bcteril growth ws decresed when lginte ws

174 t high concentrtion nd beeswx ws incresed, similrly when the concentrtion of beeswx ws high nd N 2 EDTA ws incresed the bcteril growth ws slowed down. 156

175 Figure Contour plots for slope of bcteril growth rte (dbg) during dys 0-6 of storge t 12 C 157

176 Figure Contour plots for slope of bcteril growth rte (dbg) (Block 1) during dys 6-16 of storge t 12 C 158

177 Figure Contour plots for slope of bcteril growth rte (dbg) (Block 2) during dys 6-16 of storge t 12 C 159

178 160 In preliminry experiments s shown in Chpter 4, uncoted control stored t 12 C nd 60% R.H. hd bcteril growth of 1.75 Log CFU/g in the first 3 dys of storge. Our gol ws to extend tht to 10 dys. To clculte the predicted shelf life from the contour plots one contour ws chosen for ech storge period. Ech contour is slope which mens specific chnge in bcteril growth per dy. The contour of ws chosen for both the erly nd lter dys of storge nd the shelf life ws predicted s follows: Log dy 6 dys = 1.05 Logs Log dy 4 dys = 0.7 Logs 1.05 Logs Logs = 1.75 Logs in 10 dys

179 Identifiction of optimum conditions The contours chosen in the previous section were superimposed to identify the pproprite combintion of lginte, beeswx, nisin nd N 2 EDTA. Figure 5.48, represents the region of high concentrtion of lginte nd beeswx of Figure 5.37, Figure 5.41 nd Figure 5.45, which belong to the first block during dys 0-6 of storge. Figure Contour lines of slopes of ΔL* vlue (dδl), mturity index (dmi) nd bcteril growth (dbg) t +1 level of lginte nd beeswx for Block 1 dys 0-6. Arrows indicte direction of desired region, shded re indictes overlpping of ll contours.

180 162 Figure 5.49, represents the region of high concentrtion of lginte nd beeswx of Figure 5.38, Figure 5.42 nd Figure 5.45, which belong to the second block during dys 0-6 of storge. Figure Contour lines of slopes of ΔL* vlue (dδl), mturity index (dmi) nd bcteril growth (dbg) t +1 level of Alginte nd Beeswx for Block 2 dys 0-6. Arrows indicte direction of desired region, shded re indictes overlpping of ll contours.

181 163 Figure 5.50, represents the region of high concentrtion of lginte nd beeswx of Figure 5.39, Figure 5.43 nd Figure 5.46, which belong to the first block during dys 6-16 of storge. Figure Contour lines of slopes of ΔL* vlue (dδl), mturity index (dmi) nd bcteril growth (dbg) t +1 level of Alginte nd Beeswx for Block 1 dys Arrows indicte direction of desired region, shded re indictes overlpping of ll contours.

182 164 Figure 5.51 represents the region of high concentrtion of lginte nd beeswx of Figure 5.40, Figure 5.44 nd Figure 5.47 which belong to the second block during dys 6-16 of storge. Figure Contour lines of slopes of ΔL* vlue (dδl), mturity index (dmi) nd bcteril growth (dbg) t +1 level of Alginte nd Beeswx for Block 2 dys Arrows indicte direction of desired region, shded re indictes overlpping of ll contours.

183 165 From ech superimposed plot, the region where ll contours overlpped ws tken nd plotted in Figure The region with high level of N 2 EDTA nd medium level of nisin ws identified s the one were ll the desired contours overlpped. Thus, the desired conditions explined in the previous section were met when lginte, beeswx nd N 2 EDTA were t high level (2.49%, 0.82% nd 8.18mg/mL respectively) nd when nisin ws t medium level (4000 IU/mL). Figure Superimposed contour lines from dys 0-6 nd 6-16 Block 1 nd 2, gry shding indictes overlpping of contours.

184 Vlidtion study After identifying the desired concentrtions of ech component of the coting, vlidtion study ws performed where the optiml coting (OC) ws used to cot mushrooms. In this study the combintion tht belonged to the center point of the experimentl design (CC) nd combintion identified s sub-optiml coting (SC) were lso used (see Tble 5.26). Tble Composition of cotings for vlidtion study Sodium Beeswx Nisin N Alginte 2 EDTA Tretment Actul Actul Actul Coded Coded Coded (%) (%) (IU/mL) Coded Actul (mg/ml) Optiml Coting (OC) Sub-optiml Coting (SC) Center Point of Design (CC) Uncoted wshed (UW) Uncoted unwshed (UU) The ΔL* vlue of ech coting tretment ws compred to tht predicted by the model. Figure 5.53 shows tht overll, the OC fell within the re delimited by the model for ΔL* vlue. Figure 5.53 lso compres the OC to uncoted wshed (UW) nd unwshed uncoted (UU) mushrooms. The model predicted tht mushrooms coted with the OC would hve shelf life of 21 dys, defined by retiler cceptbility vlue (RAV) ΔL* of 11 (See Figure 5.53). The lowest shelf life predicted by the model ws 13 dys, nd the highest shelf life ws bove 25 dys (See Figure 5.53). However, the model ws developed using dt of 16 dys of storge. Thus, the shelf life prediction of 21 dys obtined with the model ws n extrpoltion. It is only possible to predict shelf life bove 16 dys, nd not the exct dys of shelf life fter tht period of time.

185 167 Figure ΔL* vlue predicted by model nd experimentl vlidtion. Ech dt point is the verge of three replictions, error brs represent stndrd devition. Dotted line represents ±2 stndrd devitions of the model The mushrooms coted with the OC reched ΔL* vlue of 11 close to dy 14 of storge. Uncoted mushrooms reched this threshold between dys 6 nd 8 of storge. The OC extended shelf life of mushrooms by 7 dys when stored t 12 C, which represented temperture buse (See Figure 5.53). The model ws developed using first nd second flush mushrooms. The high vribility between flushes resulted in model which gve brod rnge of shelf life prediction. The vlidtion study ws performed only on second flush mushrooms to reduce the vribility obtined when dt ws collected from different flushes. This cn explin why the shelf life obtined with the vlidtion study ws closer to the lower limit predicted by the model.

186 168 The mturity index of ech coting tretment ws compred to tht predicted by the model. Figure 5.54 shows tht overll, the OC fell within the re delimited by the model for mturity index. Figure 5.54 lso compres the OC to uncoted wshed (UW) nd unwshed uncoted (UU) mushrooms. Figure Mturity index predicted by model nd experimentl vlidtion. Ech dt point is the verge of three replictions, error brs represent stndrd devition. Dotted line represents ±2 stndrd devitions of the model Shelf life of mushrooms coted with the OC ws nerly doubled compred to tht of the uncoted control. Further shelf life extension might be chieved in mushrooms stored t 4 C. Visul ppernce of uncoted mushrooms nd mushrooms coted with the optiml coting t the beginning nd end of shelf life is shown in Figure 5.55.

187 Tretment Time (Dy) Optiml coting Wshed control Unwshed control Figure Appernce of mushrooms coted with optiml coting, wshed-uncoted mushrooms nd unwshed-uncoted mushrooms