CHAPTER IV EXTENDED SHELF-LIFE STUDY OF A THERMALLY PROCESSED SWEET MILK AND EGG MIX

Transcription

1 CHAPTER IV EXTENDED SHELF-LIFE STUDY OF A THERMALLY PROCESSED SWEET MILK AND EGG MIX A. Abstrct o A milk nd egg mix processed t 92 C with 10 sec hold time ws evluted over seven week refrigerted storge period for nutrient composition nd functionlity. The process ws more thn sufficient to destroy Coxiell burnetti nd Slmonell senftenberg, the most het resistnt orgnisms of concern in processing milk nd egg mix. The spoilge orgnisms received 425 D process which ws more thn dequte for providing reltively sfe product. There were lso no significnt differences in the chemicl nd physicl chnges over the storge period. The microbil counts lso showed no significnt differences over the storge period with colony forming units/ ml of less thn 1 log (p<0.05) The nutritionl profile of the milk nd egg mix ws improved when dried eggs (solids nd liquid proportion equivlent to whole egg) were replced with dried egg white, cholesterol reduced egg yolk, nd skim milk. The ft nd cholesterol were reduced in the sweetened mixes by 22% nd 37.5%, respectively, in the cholesterol reduced formultion (CRF) s compred to the control formultion (CF). The protein content of the milk nd egg mix ws not ltered by the utiliztion of cholesterol- reduced egg yolk in the CRF s compred to the (CF). Addition of -glctosidse decresed the lctose by 96%. The CF ws more yellow thn the CRF in the mixes nd bked gels (p<0.05). There were lso no differences in the gel strength mong the bked gels mde from the mixes. 70

2 B. Introduction Consumer concerns with nutrition, food sfety, nd qulity hve cused mnufcturers to recognize the importnce of shelf-life evlution (Mn nd Jones, 1994). Shelf-life is defined s the number of dys fter production tht product cn be consumed while keeping its qulity stndrds, meeting consumer expecttions, nd most of ll, remining sfe for consumption (Lewis nd Dle, 1994). Fctors which limit the shelf-life of food cuse chemicl, physicl, nd biologicl chnges which result in sensory chnge. Microbil growth is one of the mjor fctors which limit the shelf-life of products (Wlker, 1994). Spoilge orgnisms re neither pthogenic nor toxin producing (Stumbo, 1973). The most importnt gener of orgnisms cusing food spoilge re Pseudomons, Achromobcter, Lctobcillus, Leuconostoc, Proteus, Micrococus, nd Aerobcter. Yests nd molds lso contribute to food spoilge. The mximum het resistnce of these orgnisms my be chrcterized by D 150 vlues between 1 to 3 min nd z vlue in the o rnge of 8 to 10 F. Psteuriztion time nd temperture combintions bsed on these resistnce vlues hve been effective in extending the shelf-life of these products under refrigertion. Temperture fluctution during storge of foods, prticulrly under refrigerted conditions, could hve dmging results on the product qulity nd storge (Wlker, 1994). Thus, the control of temperture to ensure sfety nd qulity of the product is needed. For mny yers, the distribution of diry products ws only fesible to locl nd regionl res becuse of the short shelf life. Tody, modern technologicl dvnces hve creted mss mrket of new products which hve n extended shelf-life. These products re ble to be distributed to vst res becuse of their incresed shelf-life. The objective of this study ws to provide n dequte het tretment to milk nd egg mix tht would reduce the microbil lod nd increse chnces for n extended shelf life product. The gol ws to provide process tht would render seven week extended shelf life under refrigerted conditions. The physicl, chemicl, nd microbil 71

3 properties were monitored over the period for ny chnges. 1. Mix Formultions nd Preprtion C. Mterils nd Methods Formultions. Fluid sweetened milk nd egg mix formultions for dessert ppliction were evluted. In the experiment, two formultions with two levels of lctose were tested in stright fctoril design. All other formultion prmeters were the sme s those described in Chpter III. Preprtion. The formultion ws scled up 24 times the stndrd formultion (Penfield nd Cmpbell, 1990) in order to mke btches of pproximtely 6.5 L for processing. All other preprtion prmeters were completed s previously described in Chpter III. Therml Processing. A lbortory scle psteurizer (Microthermics UHT/ HTST Lb 25 - DH, Microthermics, Inc., Rleigh, NC) with homogenizer (Niro Hudson Model NS2006A, Hudson, Wisconsin) in line ws used in the therml processing. The st nd homogenizer ws operted t totl pressure of 2500 psi ( stge; stge). This type of unit is tubulr het exchnger. The product enters the system through product tube nd goes through two tubulr product heters (Microthermics, 1992). The temperture of the product in these heters is controlled by djusting the stem pressure used to generte the hot wter, nd the quntity of hot wter bypssing the hot wter genertor. The product is cooled within the system by the use of chilled wter. The system ws operted t flow rte of 1000 ml/ min with 10 s hold time. Thermocouples were plced throughout the processing pth (Tble 18) so the different temperture chnges within the heting line could be monitored nd recorded. Tble Unit plcement of thermocouples between sections of the heting line. Thermocouple Unit Plcement 1 Pump outlet - Preheter inlet 2 Preheter outlet - Homogenizer 3 Heter 2 outlet - Hold tube inlet 4 Hold tube outlet - Cooler inlet 5 Cooler outlet - Product outlet 72

4 The tempertures were recorded nd retrieved using computer progrm set up by Microthermics (Rleigh, NC) t their fcility. The product entered the system t ~ 30 o C o before entering the preheter which ws set t of 60 C. Next the product entered the st homogenizer, nd ws homogenized t totl pressure of 2500 psi ( stge; 500- nd o 2 stge). The product continued to flow through heter no.2 which ws set t 102 C o o (215 F) before entering the hold tube nd held t 92 C for 10 s. The product then reched o the cooler set t 23 C for product cool down. Before exiting the product outlet, the o product ws cooled down to ~ 30 C nd collected into sterile continers. The sterile continers were put ice in cooler until lter trnsported bck to the Virgini Tech o fcility (on tht sme dy) nd were plced in 4 C wlk-in cooler. Enzyme Addition. The enzyme ws dded to the sterile continers under filtered positive ir flow hood to rid the ir of contminnts which would reduce the microbiologicl qulity of the mix. The enzyme ws dded one dy fter processing nd trnsporttion bck to the Virgini Tech fcility. The - glctosidse enzyme (1.8 ml; TM Lctozym 3000L, Novo Nordisk, Frnklinton, NC) ws filtered through sterile 0.45 um crodisc (Gelmn Sciences prod. no 4184, Fisher Scientific, Pittsburgh, PA) nd dded to 20 control formultion bottles nd 20 cholesterol-reduced formultion bottles. o The mix ws then stored t 4 C before further nlysis. Bked Gel Preprtion. The preprtion procedures were completed s previously described in Chpter III. 2. Chemicl Anlyses o Lctose nd Glctose Concentrtions. Mix ws stored for 5 dys t 4 C prior to the first nlysis for lctose nd glctose concentrtions. Lctose nd glctose concentrtions were mesured spectrophotometriclly t weeks 1, 3, 5, nd 7 of the storge period. The nlysis procedures for lctose nd glctose concentrtions were completed s previously described in Chpter III. o Protein Concentrtion. Smples were stored t 4 C for pproximtely 4 dys prior to nlysis. The nlysis ws completed on ll 4 custrd formultions the first week of the study to see if they were comprble in protein composition to the sweet milk nd 73

5 egg mix reported in Chpter III. The nlysis procedures for the protein concentrtion ws completed s previously described in Chpter III. o Totl Ft Concentrtion. All smples were stored t 4 C for pproximtely 4 dys prior to nlysis. The nlysis ws completed on ll 4 milk nd egg formultions only on the first week of the study to see if they were comprble in ft composition to the sweet milk nd egg mix reported in Chpter III. The nlysis procedures for the ft concentrtion ws completed s previously described in Chpter III. Cholesterol Concentrtion. Two ml of chloroform contining pproximtely mg lipid obtined by the Bligh nd Dyer (1959) method for lipid extrction ws used to determine the cholesterol concentrtion. Smple vils tht contined lipid mounts closer to the 30 mg mount were divided into 2 liquots so tht their bsorbnce would fll on the stndrd curve. The smples were stored for pproximtely 4 wks t o 0 C in seled vils flushed with nitrogen. The chloroform ws removed by strem of dry nitrogen before continuing with the cholesterol procedure described by Ktes (1986). Cholesterol concentrtions were mesured spectrophotometriclly t 550 nm by uv/vis spectrophotometer (Model lmbd 3B, Perkin- Elmer, Norwlk, CT). Serum cholesterol (99%, ct. no. C7921, Sigm, St. Louis, MO) ws used to mke stndrd stock solution contining l mg/ml of cholesterol. The stock ws used to prepre working stndrds in the rnge of mg. The mount of cholesterol in the smples ws quntified by using the working stndrds nd reding the smples ginst liner regression curve. Ftty Acid Anlysis. Two ml of chloroform contining pproximtely mg lipid obtined by the Bligh nd Dyer (1959) method for lipid extrction ws used to determine the ftty cid profile. The smples were stored for pproximtely 4 wks t 0 o C in seled vils flushed with nitrogen. The nlysis ws done on ll four custrd formultions to see if it ws comprble in ft composition to the sweet milk nd egg mix reported in Chpter III. The nlysis procedures for determining the ftty cid profile ws completed s previously described in Chpter III. Moisture. The nlysis ws completed on ll 4 mix formultions the first week of the study to see if it ws comprble in moisture content to the sweet milk nd egg mix 74

6 reported in Chpter III. The procedure for moisture nlysis ws completed s previously described in Chpter III. Ash. The nlysis ws completed on ll 4 custrd formultions the first week of the study to see if it ws comprble in sh content to the sweet milk nd egg mix reported in Chpter III. The procedure for sh nlysis ws completed s previously described in Chpter III. 3. Physicl nd Microbiologicl Anlyses Color. Color mesurements were obtined on the mix nd bked gel from ech formultion on weeks 1, 3, 5, nd 7. Color mesurements of the top nd bottom surfces of bked gel smples were tken t 3 different loctions (Minolt Chrommeter, Model CR-200, Minolt Cmer Co., Ltd., Osk, Jpn). The gels were removed from the dish to mesure the color of the bottom surfce. The color mesurements were mde on ll 4 formultions of bked custrd, every other week for 7 wk, fter the mix ws prepred. The color mesurements were reported s Hunter L,, nd b vlues. Clibrtion of the instrument ws crried out using white tiled Minolt Reference tile ( L = 97.91, = , b= +2.44). Mens nd stndrd devitions were clculted by the sttistics mode on the chrommeter. The color of the custrd mix before bking ws lso evluted by the sme procedure. The mix ws mesured through cler glss test tube held in the tube holding pprtus (Minolt Cmer Co., Ltd., Osk, Jpn). Gel Strength. Firmness ws mesured on ll 4 formultions of bked custrd, on weeks 1, 3, 5, nd 7 fter the mix ws prepred. Firmness ws mesured s the force (g) needed to penetrte n 80 g bked custrd smple. A Stevens L.F.R.A. Texture Anlyzer (Model TA , Texture Technologies Corp., Scrsdle, NY) with TA - 2 cone ttchment operted t speed of 2 mm/s nd distnce of 5 mm ws used in the nlysis. Mesurements were obtined from 2 different loctions on the top surfce of the custrd nd the vlues verged. The custrds were llowed to sit t room temperture for pproximtely 6 h before mesurements were tken. Syneresis. Syneresis ws mesured on ll 4 formultions of bked custrd every other week for seven weeks fter the bked custrds were prepred. Syneresis, the 75

7 dringe of liquid from bked custrd gel, ws mesured s described by Penfield nd Cmpbell (1990). The gelled custrd smple ws inverted on cheesecloth supported on funnel. The exudte ws collected in 10 ml grduted cylinder over one h period t o C nd the volume recorded. Aerobic Plte Count. A stndrd plte count ws performed on ll 4 mix formultions on weeks 1, 3, 5, nd 7. The procedure for erobic plte count ws completed s previously described in Chpter III. 4. Dt Anlyses Dt Collection. This experiment consisted of 2 replictions of the ctul therml process used on 4 different btches of the sweet milk nd egg mix. The nlysis were completed on weeks 1, 3, 5, nd 7. Some compositionl nlyses (protein, ft, moisture, sh, ftty cid methyl esters, nd cholesterol) were performed on wk 1 to compre formultion compostions to the sweet milk nd egg mix from previous nlyses. All other tests were performed t weeks 1, 3, 5, nd 7 were stndrd plte count (mix), lctose nd glctose concentrtion (mix), color ( mix & gel), texture (gel), nd syneresis (gel). Sttisticl Anlyses. A t- test ssuming equl vrince ws used for the following nlysis: percent totl ft, percent protein, percent cholesterol, percent moisture, percent sh, percent sturted ft, percent monounsturted ft, nd percent polyunsturted ft. A stright fctoril design ws used for nlysis on ll the tests tht were performed over the 7 wk period. The Sttisticl Anlysis System (SAS Institute Inc., 1988) utilizing the PROC ANOVA procedure ws used to nlyze the dt collected from the stright fctoril design. Men difference seprtions were determined using Tukey s HSD. 1. Therml Processing D. Results nd Discussion Therml processing prmeters were modified bsed on ptent for the production of n extended shelf-life sweet milk nd egg bsed product (Dieu nd CuQ, 1989). The therml processing prmeters were similr to those used in Chpter III with 76

8 o the exception of lower hold tube temperture (92 C) with 10 sec hold time. This het tretment gve more thn dequte D vlues for the destruction of Mycobcterium tuberculosis, Coxiell burnetti, Slmonell, nd spoilge orgnisms of concern in this product. As stted before the time nd temperture destruction of n orgnism when deling with public sfety is bsed on the hold tube (Swrtzel, 1986). o o Applying hold tube temperture of 92 C (197 F) t 10 sec still gve more thn dequte process for sfety. A 14,167 D process ws given to Coxiell burnetti, which ws the most het resistnt pthogen in the product; spoilge orgnisms received 425 D process. In spite of the lower temperture pplied in the hold tube, of this study, the product still received more thn dequte het tretment. Aerobic Plte Count. The 4 different formultions of the milk nd egg mix hd microbil counts (cfu/ ml) of less thn l log for the 7 wk storge period. These low counts re ttributed to severl fctors in ddition to the therml process. The use of psteurized milk, commercilly processed dried egg solids, nd processed liquid egg yolk (psteurized prior to cholesterol extrction) ll plyed role in providing lower counts due to their prior het tretments. Additionlly, the use of sterile continers in collecting the mix ided in lowering counts. 2. Chemicl Composition of Sweetened Formultions Proximte Anlysis. The objective of this reserch ws to develop nutritionlly enhnced milk nd egg mixture with shelf life of 6 wk under refrigerted conditions. Proximte nlysis demonstrted decrese in ft in the cholesterol reduced formultion (CRF) s compred to the control formultion(cf)(tble 19). There ws 22% reduction in the CRF. The higher ft content in the CF is ttributed to the type of egg yolk used in the formultion. The clorie contents of the CF nd CRF re very similr with CRF being slightly lower (Tble 19). The CF hd dried egg yolk while the CRF used cholesterol reduced (CR) nd ft reduced (FR) liquid egg yolk. Both tretments were formulted so tht the yolk ws proportionl to egg white solids nd totl moisture level to be equivlent to whole fresh egg. Thus, there ws no difference in the moisture content between the CF nd CRF s would be expected (Tble 19). The milk nd egg 77

9 sources both provided the proteins in the mix. 1 Tble Mens + stndrd devitions for proximte nlysis of sweetened milk nd egg mixture. Formultion Constituent 3 CF 4 CRF (g/100g) b Ft Protein Moisture Ash Crbohydrtes Clories (kcl/ 100g) Mens nd stndrd devitions for 4 replictions Vlue not mesured directly. Clculted s difference bsed on proximte nlysis CF = Control formultion CRF = Cholesterol reduced formultion Vlue bsed on proximte nlysis of three replictions Mens within row with different letters re significntly different ( p< 0.05) , b 78

10 Lctose nd Glctose Concentrtion. As reported in Chpter III, the concentrtions of lctose in the formultion without -glctosidse tretments were not different. The formultions with the -glctosidse enzyme dded hd significnt decreses in the lctose concentrtions nd n increse in the glctose concentrtion (Tble 20). There ws n verge of 96% reduction between the CF nd CRF with - glctosidse ddition. Under refrigerted conditions, the lctose nd glctose concentrtions did not chnge over the 6 wk storge period. These results suggest tht hydrolysis of the lctose ws completed during the first week of storge. Lipid Profile. As reported in Chpter III, the type of egg plyed mjor role in the difference in the type nd proportion of ftty cids found in ech formultion (Tble 21; Tble 22). There ws 8.7% reduction of sturted ftty cids in the CRF tht used the cholesterol nd ft reduced egg yolk.. There ws lso 39% significnt reduction in the monounsturted ftty cids of the CRF. The mount of polyunsturted ftty cids found in the CRF incresed by 61%. Cholesterol ws significntly lowered to 37.5% in the CRF formultion (Tble 22). A difference in the concentrtion of sturted ftty cids in the sweetened formultions, between the study reported in Chpter III nd this study ws observed. The sturted ftty cids reported in Chpter III for the sweetened mix is 57% higher thn in this sweetened formultion. Both studies utilized the sme lot of eggs. This my be ttributed to the smll mount of ft in the product, less thn 1 g, cusing some vrition in the concentrtions ftty cids. 3. Physicl nd Functionl Chrcteristics of Sweetened Formultions Color. There were no differences in color during the 7 wk storge for the mix formultions or the gels bked from those mixes (Figures 1-9). Hunter L,, nd b vlues were mesured for the mix nd top nd bottom surfces of the gel. In this color system, L illustrtes drk to light (0 to100), illustrtes red (+) to green (-), nd b represents yellow (+) to blue (-). The type of egg ws the min effect contributing to the difference in color between the formultions. The L vlues for CRF mix indicted tht it ws lighter thn the CF. There were significnt differences in the L,, nd b 79

11 vlues 1 Tble Interction mens + stndrd devitions for lctose nd glctose of sweetened milk nd egg mixture. Constituent Formultions Lctose (g/l) Glctose (g/l) 2 3 CF - NLR b b CF - LR CRF - NLR b b CRF - LR Mens nd stndrd devitions for 2 replictions CF = Control formultion NLR = No lctose reduction LR = Lctose reduction CRF = Cholesterol reduced formultion Mens within column with different letters re significntly different ( p< 0.05), b 80

12 1 Tble Mens + stndrd devitions for ftty cids in sweetened milk nd egg mixture. Formultion 2 3 Ftty cid (%) CF CRF Butyric - C b Cproic - C6 Trce Trce Cprylic - C Trce Cpric - C Luric - C Myristic - C Plmitic - C b Plmitoleic - C16: b Steric - C Oleic - C18: b Linoleic - C18: b Linolenic - C18: b Archidonic - C20: Mens nd stndrd devitions for 4 replictions CF = Control formultion CRF = Cholesterol reduced formultion Mens within row with different letters re significntly different ( p< 0.05), b 81

13 1 Tble Mens + stndrd devitions for lipid profile of sweetened milk nd egg mixture. Formultion 2 3 Constituent CF CRF b Sturted ft (%) b Monounsturted ft (%) b Polyunsturted ft (%) b Cholesterol (mg/100g) Mens nd stndrd devitions for 4 replictions CF = Control formultion CRF = Cholesterol reduced formultion Mens within row with different letters re significntly different ( p< 0.05), b 82

14 of the mixes for the 4 different formultion (p<0.05) (Tble 23). The L nd vlues of the mix were reltively close within ech color ttribute such tht differences probbly would hve limited significnce with respect to visul observtions. The Hunter b vlues were low enough in the CRF, s compred to s compred to the CF, nd my hve contributed to visul difference. The CF were significntly more yellow thn the CRF. No differences in the L vlues were observed for the top nd bottom surfces of the bked gels. There were significnt differences in the nd b vlues of the top nd bottom gels. The top surfce b vlues were more yellow thn the bottom surfce nd the CF were significntly more yellow thn the CRF. Gel Strength nd Syneresis. There ws no significnt difference in the gel strength between the bked gels utilizing the milk nd egg mix over the 7 wk storge period (Tble 23). The strength of the gels mde with the sweetened milk nd egg mix reported in Chpter III were stronger thn the bked gels in this study. This result could be ttributed to the therml process tht ws pplied. The mix ws homogenized in line in this therml process. There were lso higher preheting tempertures in this study thn in chpter III. These fctors my hve cused denturtion of some of the egg proteins which re vitl in the formtion of gel structure. Syneresis, the dringe of liquid from gel, did not chnge over the 7 wk storge period in the 4 different formultions. 83

15 MIX 100 CF CRF MEAN "L" VALUES STORAGE TIME (WEEKS) Fig. 1 - Men "L" vlues for CF nd CRF for the mix (n= 4) over seven week storge period. 84

16 TOP SURFACE 100 CF CRF MEAN "L" VALUES STORAGE TIME (WEEKS) Fig. 2 - Men "L" vlues for CF nd CRF for the top surfce of the bked gels (n= 4) over seven week storge period. 85

17 BOTTOM SURFACE 100 CF CRF MEAN "L" VALUES STORAGE TIME (WEEKS) Fig. 3 - Men "L" vlues for CF nd CRF for the bottom surfce of the bked gels (n= 4) over seven week storge period. 86

18 MIX 25 CF CRF 20 MEAN "-" VALUES STORAGE TIME WEEKS Fig. 4 - Men "" vlues for CF nd CRF for themix of bked gels (n= 4) over seven week storge period. 87

19 TOP SURFACE 25 CF CRF 20 MEAN "-" VALUES STORAGE TIME (WEEKS) Fig. 5 - Men "" vlues for CF nd CRF for the top surfce of bked gels (n= 4) over seven week storge period. 88

20 BOTTOM SURFACE 25 CF CRF 20 MEAN "-" VALUES STORAGE TIME (WEEKS) Fig. 6 - Men "" vlues for CF nd CRF for the bottom surfce of bked gels (n= 4) over seven week storge period. 89

21 MIX MEAN "b" VALUES CF CRF STORAGE TIME (WEEKS) Fig. 7 - Men " b" vlues for CF nd CRF for the mix (n= 4) over seven week storge period. 90

22 TOP SURFACE MEAN "b" VALUES CF CRF STORAGE TIME (WEEKS) Fig. 8 - Men " b" vlues for CF nd CRF for the top surfce of bked gels (n= 4) over seven week storge period. 91

23 BOTTOM SURFACE MEAN "b" VALUES CF CRF STORAGE TIME (WEEKS) Fig. 9 - Men " b" vlues for CF nd CRF for the bottom surfce of bked gels (n= 4) over seven week storge period. 92

24 1 Tble Interction mens + stndrd devitions for physicl mesurements of sweetened milk nd egg mixture. Formultion Properties CF -NLR CF-LR CRF -NLR CRF-LR Gel strength (g force) Syneresis (ml)* Color Top surfce Hunter L Hunter * Hunter b * Bottom surfce Hunter L Hunter * Hunter b * Mix Hunter L * Hunter * Hunter b * * Mens nd stndrd devitions for 2 replictions CF = Control formultion NLR = No lctose reduction LR = Lctose reduction CRF = Cholesterol reduced formultion Significnt differences t ( p< 0.05) 93

25 E. Conclusion A nutritionlly enhnced milk nd egg mix cn be formulted nd thermlly processed for tody s helth conscious consumers. The product offers lower cholesterol nd ft contents while providing beneficil nutrients such s clcium nd protein. Ft is mjor concern for individuls who re wtching clorie intke, s well s individuls who re monitoring ft nd cholesterol intkes becuse of helth conditions. The product could lso be consumed by the lctose intolernt individuls who shy wy from diry products becuse of gstrointestinl discomfort. Becuse of ll the positive ttributes of this product, the elderly popultion would be good trget mrket. The elderly popultion tends to hve poorer intke of nutrients, nd my be more wre of ft nd cholesterol in their diets due to helth problems. The het tretment pplied to this product ensures reltively sfe product with n extended shelf life. The extended shelf life of this product mkes distribution of this product to food service industries idel. Also, the dded convenience mkes it very mrketble to the consumer with less time to spend in the kitchen prepring mel. 94

26 F. References Bligh, E.G. nd Dyer, W.J A rpid method of totl lipid extrction nd purifiction. Cndin J of Biochem Physiol. 37:911. Dieu, B., nd CuQ, J Process for producing sweet custrd food stuff with long term shelf life bsed on milk nd eggs. U.S. ptent 4,877,625. Ktes, M Techniques of lipidology. Lbortory Techniques in Biochemistry nd rd Moleculr Biology, 3 ed., 122. North- Hollnd Publishing Co., Amsterdm Lewis, M. nd Dle, R.H Chilled yogurt nd other diry desserts. Ch.7, In Shelf Life Evlution of Foods, C.M.D. Mn nd A.A. Jones. ed., Chpmn nd Hll, New York. Mn, C.M.D. nd Jones, A.A Shelf Life Evlution of Foods.P1.Chpmn nd Hll, New York. Microthermics Instruction Mnul for UHT/HTST Lb-25. Microthermics, Rleigh, NC. rd Penfield, M.P. nd Cmpbell, A.M Experimentl Food Science, 3 ed. Acdemic Press, Inc., New York. SAS Institute Inc SAS/STAT User s Guide, 6.03 ed. Cry, NC; SAS Institute, Inc. nd Stumbo, C.R Thermobcteriology in Food Processing. 2 ed. Acdemic Press, New York. Swrtzel, K.R Equivlent-point method for therml evlution of continuous-flow systems. J. Agric. Food Chem. 34: Wlker, S.J The principle nd prctice of shelf life prediction for microorgnisms. Ch. 3, In Shelf Life Evlution of Foods, C.M.D. Mn nd A.A. Jones. ed., Chpmn nd Hll, New York. 95

27 APPENDIX A 96

28 Bligh nd Dyer Lipid Extrction I. Solutions: Methnol:Chloroform (2:1) Methnol: Chloroform: Wter (2:1:0.8) II. Procedure: 1. Add 1 g or ml of smple to 16 x 125 screw cp test tube 2. Add 4 ml of methnol:chloroform mixture. Tighten cp. Vortex 3. Shke or vortex every 15 min for 1-2 hrs. 4. Centrifuge tubes in tble top centrifuge t mximum rpm for 10 min. 5. Trnsfer superntnt to nother 16 x 125 mm screw cp test tube. 6. Add 5 ml of methnol:chloroform:wter to the pellet nd re-extrct. Vortex the pellet nd solvent. Centrifuge gin s bove. 7. Add the second superntnt to the first. 8. Add 3 ml of chloroform nd 3 ml of distilled wter to the combined superntnts. Mix nd centrifuge s bove. 9. Weigh lbeled 10 ml crimp top vil. Record the weight. 10. Withdrw the lower chloroform phse from the centrifuged tube in step 8 nd dd to the weighed vil. 11. Evporte the chloroform under strem of nitrogen. 12. Add 2 ml of chloroform to the lipid, sel nd freeze till methyltion procedure. % Lipid Clcultion: (Wt. empty vil) - (Wt. vil fter chloroform remove) = mg lipid mg lipid/ Wt. smple * 100 = % lipid 97

29 I. Solutions: Cholesterol Anlysis Stock Ferric Chloride Solution: 85% Orthophosphoric Acid Working Ferric Chloride Solution: 4 ml of 85% orthophosphoric cid in 50 ml volumetric flsk nd diluted to totl volume of 50 ml with concentrte sulfuric cid. Stock Stndrd Solution: Add 100 mg of 100% cholesterol to totl volume of l00 ml of glcil cetic cid. Working stndrds: 50, 100, 200, 300, 400, nd 600 ul of stock. (Tke the stndrds nd blnk through the procedure tht follows. II. Procedures: 1. Plce lipid liquot contining pproximtely 0.3 mg of cholesterol in 16 x 150 glss test tube with teflon liner screw cp. 2. Plce liquots of the bove working stndrd in test tubes. 3. If smples were stored in chloroform, evporte solvent under nitrogen. 4. Add 6 ml of glcil cetic cid to the smple tubes. Mix 5. Add pproprite liquots of glcil cetic cid to working stndrds nd blnk to rech totl volume of 6 ml mix. Mix 6. Add 4 ml of working ferric chloride to smples, stndrd, nd blnk tubes. 7. Cool for 10 min. nd red bsorbnce on spectrophotometer set t 550 nm. 8. Clculte the concentrtion of the smples from the stndrd curve. 98

30 I. Solutions: II. Procedure: Boehringer Mnnheim Assy (Lctose nd Glctose Concentrtion) 12% Trichlorcetic cid 1 Norml Sodium Hydroxide solution 1/10 Norml Sodium Hydroxide solution Boehringer Mnnheim Test Kit Regents 1. Two ml of liquid milk nd egg mix were dded to 20 ml of 12% trichlorcetic cid. 2. Smples were mixed nd plced in lrge centrifuge tubes. 3. Tubes plced in Sorvll Refrigerted Superspeed Centrifuge t 6000 rpm for 20 min.. 4. Ten ml of cler superntnt were removed from the tubes nd plced in seprte 50 ml bekers 5. The superntnt from the smples is djusted to ph of using 1 N nd 1/10 N sodium hydroxide solution. 6. After smples were ph, they were brought to totl volume of 25 ml using distilled wter. 7. The smples were further nlyzed ccording to instructions provided in the Boehringer Mnnheim test kit. 99

31 VITAE Trcy D. Sutton ws born in Wilson, NC on November 5, She grduted from Jmes B. Hunt High School in June, 1990, nd entered North Crolin Agriculturl nd Technicl Stte University (NCA&T) in the fll of She grduted from NCA&T Stte University in December, 1994 with Bchelor of Science degree in Food Science. In the Summer of 1995, she strted Virgini Polytechnic Institute nd Stte University (VPI&SU) in the Food Science deprtment under the supervision of Dr. Duncn. She finished her Mster of Science degree in Food Science nd Technology in October,