Improving the quality and safety of unprocessed and fresh-cut vegetables by modifying agents and biopreservation

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1 Improving the quality and safety of unprocessed and fresh-cut vegetables by modifying agents and biopreservation Dr Mala Gamage and Dr Kamaljit Vilkhu Food Science Australia Project Number: VG98106

2 VG98106 This report is published by Horticulture Australia Ltd to pass on information concerning horticultural research and development undertaken for the vegetable industry. The research contained in this report was funded by Horticulture Australia Ltd with the financial support of the vegetable industry. All expressions of opinion are not to be regarded as expressing the opinion of Horticulture Australia Ltd or any authority of the Australian Government. The Company and the Australian Government accept no responsibility for any of the opinions or the accuracy of the information contained in this report and readers should rely upon their own enquiries in making decisions concerning their own interests. ISBN Published and distributed by: Horticultural Australia Ltd Level 1 50 Carrington Street Sydney NSW 2000 Telephone: (02) Fax: (02) norticulture@horticulture.com.au Copyright 2002 Horticulture Australia

3 FOOD SCIENCE AUSTRALIA A JOINT VENTURE OF CSIRO AND AFISC CSIRO I Arise Australian Food Industry Science Centre

4 Horticulture Australia Limited FINAL REPORT IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES BY MODIFYING AGENTS AND BIOPRESERVATION HRDC Project VG (February 2002) Dr Mala Gamage and Dr Kamaljit Vilkhu Food Science Australia Report for Horticulture Australia Limited Food Science Australia

5 Project details VG Project Leaders Dr Mala Gamage Senior Food Technologist Packaging & Coatings section Food Science Australia Private Bag 16, Werribee, Victoria, Australia 3030 Tel ( ) Fax ( ) mala.gamage@foodscience.afisc.csiro.au Dr Kamaljit Vilkhu Senior Research Scientist - Microbiology Food Safety & Quality Food Science Australia Private Bag 16, Werribee, Victoria, Australia 3030 Tel ( ) Fax ( S0) kamaliit.vilkhu@foodscience.afisc.csiro.au Project Aim The aim of this project was to improve the quality and safety of unprocessed and fresh cut vegetables by using modifying agents and bio-preservatives. Project Funding Source National Vegetable Levy Horticulture Australia limited Victorian Department of Natural Resources and Environment Date of Submission The final report for this project was submitted in February 2002 Report for Horticulture Australia Limited Food Science Australia

6 Research Team Dr Mala Gamage, Dr Kamaljit Vilkhu Dr John Coventry Ms Thu Vu Disclaimer Any recommendations contained in this publication do not necessarily represent current Horticulture Australia Policy. No person should act on the basis of the contents of this publication, whether as to matter of fact or opinion or other content, without first obtaining specific, independent professional advice in respect of the matters set out in this report. Report for Horticulture Australia Limited Food Science Australia

7 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES CONTENTS Page LIST OF TABLES 4 LIST OF FIGURES 6 1 MEDIA SUMMARY 9 2 TECHNICAL SUMMARY 10 PART 1- IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES BY MODIFYING AGENTS 11 3 INTRODUCTION 11 4 MATERIALS AND METHODS MATERIALS Lettuce stem discs Sample preparation - Lettuce heads Sample preparation - Broccoli heads Acids Gums METHODS Preliminary experiments on lettuce stem end browning Experiments on cut end of whole lettuce heads Experiments on shredded lettuce Broccoli Sample Evaluation 19 5 RESULTS AND DISCUSSION PRELIMINARY EXPERIMENTS Effect of citric acid solutions atph for seconds Effects of lactic, malic, tartaric and acetic acids and application method Effect of acetic acid treatment and rinsing Effect of acetic acid and application method WHOLE LETTUCE HEADS Effect of low ph and dipping Effect of low ph and blotting Combined effect of heat and low ph on the cut end of whole lettuce heads Effect of low ph and gums I Effect of low ph and gums II Effect of storage temperature on browning prevention, coating applied on the cut end of lettuce heads -I Effect of storage temperature on the browning of coating applied cut end of lettuce heads - II Effect of storage temperature on browning prevention, coating applied on the cut end of lettuce heads -III SHREDDED LETTUCE 63 Report for Horticulture Australia Limited Food Science Australia

8 IMPROVING THE QUALITY AMD SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES The combined effect of heat and low ph on the quality of Shredded lettuce I The combined effect of heat and low ph on the quality of shredded lettuce II BROCCOLI Broccoli I - Effect of moisture retaining agents Broccoli II - Effect of moisture retaining agents and acids 71 6 OVERALL DISCUSSION AND CONCLUSIONS LETTUCE HEADS SHREDDED LETTUCE BROCCOLI HEADS 73 PART 2- IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES BY BIOPRESERVATION 74 3 INTRODUCTION: 74 4 METHODS: CULTURES AND MEDIA MICROBIOLOGICAL AND PH ANALYSIS EVALUATION OF BIOPROTECTIVE L. ALIMENTARRJS CULTURE AGAINST PATHOGENIC AND SPOILAGE BACTERIA IN A LETTUCE EXTRACT TEST SYSTEM EVALUATION OF COATING MATERIAL AND APPLICATION METHOD FOR SHREDDED LETTUCE AND CARROT PIECES EVALUATION OF BIOPROTECTIVE LACTOBACILLUS ALIMENTARRJS AGAINST INDIGENOUS SPOILAGE PSEUDOMONADS ON SHREDDED ICEBERG LETTUCE LEAVES EVALUATION OF BIOPROTECTIVE LACTOBACILLUS ALIMENTARIUS AND CARNOBACTERRJM PISCICOLA AGAINST LISTERIA INNOCUA AND INDIGENOUS PSEUDOMONADS ON LETTUCE LEAVES EVALUATION OF BIOPROTECTIVE AGENTS POLYPHENON 70S, PEDIOCIN AND NISIN ON CARROT PIECES RESULTS AND DISCUSSION: EFFECT OF L ALIMENTARIUS ON L. MONOCYTOGENES, A. HYDROPHILA AND P. FLUORESCENS IN LETTUCE EXTRACT SYSTEM EFFECT OF EDIBLE COATING ON DELIVERY OF BACTERIAL CULTURES TO LETTUCE LEAVES EFFECT OF BIOPROTECTIVE CULTURES ON LISTERIA INNOCUA AND PSEUDOMONAS SPP. ON LETTUCE LEAVES EFFECT OF BIOPROTECTIVE AGENTS POLYPHENON 70S, PEDIOCIN AND NISIN ON FOOD PATHOGENS AND SPOILAGE BACTERIA ON CARROT PIECES WITH EDIBLE COATINGS TECHNOLOGY TRANSFER TECHNOLOGY TRANSFER - PART 1 (IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES BY MODIFYING AGENTS) TECHNOLOGY TRANSFER - PART 2 (IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES BY BIOPRESERVATION) RECOMMENDATIONS 99 Report for Horticulture Australia Limited Food Science Australia 2

9 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES 9 ACKNOWLEDGEMENTS REFERENCES 101 Report for Horticulture Australia Limited Food Science Australia 3

10 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES List of Tables Part 1 Page Table 1 - Treatoent combinations used for evaluation of the combined effects of heat and acid. 14 Table 2 - Treatment solutions used in (f) Effect oflowph and gums - III 15 Table 3 - Treatments used in (g) - Effect oflowph and gums - V 16 Table 4 - Experimental conditions used in (h) Effect oflowph and gums - V16 Table 5 - Solution types and application methods used in (a) - Effect of moisture retaining agents 18 Table 6 - Subjective colour scale for assessing the colour changes in lettuce discs, cut surface of lettuce cores and the cut end of lettuce heads. 19 Table 7 - Subjective colour score for evaluation of browning in shredded lettuce. -20 Table 8 - Summary of experimental findings on the effect of citric, lactic, malic, tartaric and acetic acid treatments on lettuce discs based on number of days Hue value remained > Table 9 - Summary of experimental findings on the effect of acetic, malic and tartaric acid treatments on lettuce cores with or without rinsing with distilled water or calcium hydroxide, based on number of days colour score remained < 4 or marginal acceptability. 26 Table 10 - Summary of experimental findings on the effect of acetic acid treatments on lettuce cores with two application methods, based on number of days colour score remained < 4 (marginal acceptability) and surface ph. 34 Table 11- Summary of the experimental findings on the effect of acetic acid treatments on the cut end of whole lettuce heads with dipping and blotting methods, based on number of days colour score remained < 4 (marginal acceptability). 39 Table 12 - Summary of the experimental findings on the combined effect of acetic acid and heat treatment on the cut end of lettuce heads with dipping method, based on number of days colour score remained < 4 (marginal acceptability).-41 Table 13 - Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads with dipping method, based on number of days colour score remained < 4 (marginal acceptability) (4.2.2 d and e) 45 Table 14- Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage temperatures 4 C, 7 C and 12 C, based on number of days colour score remained < 4 (marginal acceptability). 48 Table 15- Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce head at storage temperatures 4 C, 7 C and 12 G, based on number of days Hue value remained > Table 16 - Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage temperatures 4 C, 7 C and 12 G, based on number of days colour score remained < 4 (marginal acceptability). 53 Table 17 - Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage temperatures 4 C, 7 C and 12 C, based on number of days Hue value remained > Table 18- Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage Report for Horticulture Australia Limited Food Science Australia 4

11 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES temperatures 4 C, 7 C and 12 C, based on number of days colour score remained < 4 (marginal acceptability). 56 Table 19 - Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage temperatures 4 C, 7 C and 12 C, based on number of days Hue value remained > Table 20: Summary of the experimental findings on the combined effect of acetic acid and heat on shredded lettuce at 4 C in polyethylene bags, based on number of days colour score remained < 4 and Hue value remained > 100 [4.2.1 (a)]. -64 Table 20: Summary of the experimental findings on the combined effect of acetic acid and heat on shredded lettuce at 4 C in polyethylene bags, based on number of days colour score remained < 4 and Hue value remained > 100 [4.2.1 (b)] 68 Table 21: Summary of the experimental findings on the combined effect of acetic acid and heat on shredded lettuce at 4 C in polyethylene bags, based on number of days colour score remained < 4 and Hue value remained > 100 [4.2.1 (c)] 68 Part 2 Table 1. Percentage weight gains on shredded lettuce after coating. 85 Table 2. Total plate count on coated and shredded lettuce. 85 Report for Horticulture Australia Limited Food Scmnce Australia

12 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES List of Figures Part 1 Page Figure 1 - Effect of citric acid application on the hue angle of lettuce stem discs stored at 4 C 23 Figure 2 - Effect of lactic acid application on the hue angle of lettuce discs stored at. 23 Figure 3 -Effect of malic acid treatment on the hue angle of lettuce discs stored at-24 Figure 4 - Effect of tartaric acid application on hue angle of lettuce disc stored at-24 Figure 5 - Effect of acetic acid application on the hue value of lettuce discs stored at 27 Figure 6 - Effect of acetic acid treatment (no rinse - HH) on the colour of lettuce core cut end stored at 4 C. 27 Figure 7 - Effect of Malic acid treatment on the colour of lettuce core 28 Figure 8 - Effect of tartaric acid treatment followed by no rinse on the colour of lettuce core cut end stored at 4 C 28 Figure 9 - Effect of acetic acid treatment followed by washing with distilled 2i Figure 10 - Effect of acetic acid treatment followed by washing with Ca(OH)2 on the colour of lettuce core cut end stored at 4 C. 29 Figure 11 - Effect of Tartaric acid treatment followed by washing with distilled water on the colour of lettuce core cut end stored at 4 C 30 Figure 12 - Effect of Tartaric acid treatment followed by washing with Ca(OH)2 on the colour of lettuce core cut end 30 Figure 13 - Effect of Malic acid treatment followed by washing with distilled water on the colour of lettuce core cut end. 31 Figure 14 - Effect of Malic acid acid treatment followed by washing with Ca{OH)2 on the colour of lettuce core cut end stored at 4 C. 31 Figure 15 - Untreated lettuce cores and lettuce cores treated with acetic acid at ph 2.0 for 30 seconds. 32 Figure 16 - Untreated lettuce cores and lettuce cores treated with malic acid at ph 2.0 for 10 min. 32 Figure 17 - Untreated lettuce cores and lettuce cores treated with tartaric acid at ph 2.0 for 10 min. 33 Figure 18 - Visual scores of lettuce cores treated with acetic acid at ph Figure 19 - Visual score of lettuce cores treated with acetic acid at ph Figure 20 - Visual score of lettuce cores treated with acetic acid at ph Figure 21 - Surface ph of lettuce cores treated with acetic acid at ph Figure 22 - Surface ph of lettuce cores treated with acetic acid at ph Figure 23 - Surface ph of lettuce cores treated with acetic acid at ph Figure 24 - Colour change of cut end of lettuce heads dipped in acetic acid 40 Figure 25 - Colour change of cut end of the lettuce heads treated by blotting with acetic acid solutions. 40 Figure 26 - Colour score of cut end of lettuce heads treated with combined heat and acid treatments 42 Figure 27 - Changes in L value of cut surface of whole lettuce heads treated with acid and heat -42 Figure 28 - Changes in hue angle of cut surface of whole lettuce heads treated with acid and heat 43 Figure 29 - Change in browning scores of the cut ends of the lettuce heads treated 46 Report for Horticulture Australia Limited Food Science Australia 6

13 IMPROVING THE QUALITY AN SAFETY OF UNPROCESSED AND FRESH-CUT Figure 30 - Change in browning scores of the cut ends of the lettuce heads treated 46 Figure 31- Colour score of the cut end of lettuce heads treated with gums and acetic acid, and stored at 4 C. 49 Figure 32- Colour score of lettuce butts treated with gums and acetic acid, and stored at7 C. 49 Figure 33 - Colour score of the cut end of lettuce heads treated with gums and acetic acid and stored at 12 C 50 Figure 34 - Hue values of the cut end of lettuce heads treated with AA and gums and stored at 4 C. 50 Figure 35 - Changes in Hue value of lettuce butts treated with acetic acid (AA) and gums and stored at 7 C. 51 Figure 36 - Changes in hue value of untreated lettuce butts stored at 12 C 51 Figure 37 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at 4 C. 54 Figure 38 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at 7 C 54 Figure 39 - Colour score of lettuce butts treated with gum, acetic acid (AA) and 55 Figure 40 - Changes in hue value of lettuce butts treated with acetic acid (AA), CaCI2 & xanthan gum and stored at 4 C 55 Figure 41 - Changes in hue values of lettuce butts with acetic acid (AA), CaCI2 & xanthan gum and stored at 7 C. 57 Figure 42 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at4 C 57 Figure 43 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at 7 C 58 Figure 44 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at 12 C 58 Figure 45 - Changes in hue value of lettuce butts treated with AA, CaCI2 and xanthan gum and stored at 4 C. 59 Figure 46 - Changes in hue value of lettuce butts treated with AA, CaCI2 and xanthan gum and stored at 7 C. 59 Figure 47 - Changes in hue value of lettuce butts treated with AA, CaCI2 and xanthan gum and stored at 12 C. 60 Figure 48 - Colour Score of shredded lettuce treated with heat at different ph levels 65 Figure 49 - Hue values of shredded lettuce treated with heat at different ph levels-65 Figure 50 - Effect of ph and heat treatment on mesophilic and psychrotropic count at dayo 66 Figure 51 - Total mesophilic colony count of shredded lettuce treated with heat at-66 Figure 52 - Combined effect of ph and heat treatment on hue value of shredded lettuce 69 Figure 53 - Combined effect of ph and heat treatment on browning of shredded lettuce 69 Figure 54- Standard plate counts of shredded lettuce subjected to various dipping treatments 70 Figure 55 - Mesophilic counts of shredded lettuce subjected to various dipping treatments 70 Part 2 Page Figure 1. Growth of Lactobacillus alimentarius in presence of target microorganisms in lettuce juice at 4 C. 81 Figure 2. Growth of Pseudomonas fluorescens in lettuce juice at 4 C. 81 Figure 3. Growth of Aeromonas hydrophila in lettuce juice at 4 C. 82 Report for Horticulture Australia Limited Food Science Australia

14 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 4. Growth of Listeria monocytogenes in lettuce juice at 4 C. 82 Figure 5. ph change during the growth of Lactobacillus alimentarius in presence of target microorganisms in lettuce at 4 C. 83 Figure 6. ph change during the growth of Pseudomonas fluorescence in lettuce juice at4 C. 83 Figure 7. ph change during the growth of Aeromonas hydrophila in lettuce juice at 4 C. 84 Figure 8. ph change during the growth of Listeria monocytogenes in lettuce juice at 4 C. 84 Figure 9. Growth of Lactobacillus alimentarius on lettuce leaves at 4 C. 86 Figure 10. Growth of pseudomonads on lettuce leaves at 4 C. 86 Figure 11. Growth of lactic acid bacteria on lettuce leaves at 7 C with Lactobacillus alimentarius. 88 Figure 12. Growth of Listeria innocua on lettuce leaves at 7 C under the influence of Lactobacillus alimentarius. 88 Figure 13. Growth of indigenous pseudomonads on lettuce leaves at 7 C under the influence of Lactobacillus alimentarius. 89 Figure 14. ph changes in lettuce leaves samples with Lactobacillus alimentarius. -89 Figure 15. Growth of lactic acid bacteria on lettuce leaves at 7 C with Carnobacterium piscicola. 90 Figure 16. Growth of indigenous Pseudomonads. on lettuce leaves at 7 C under the influence of Carnobacterium. Piscicola. 90 Figure 17. Growth of Listeria innocua on lettuce leaves at 7 C under the influence of Carnobacterium. Piscicola. 91 Figure 18. ph changes in lettuce leaves samples with Carnobacterium piscicola. -91 Figure 19. Effect of Polyphenon 70S on Listeria innocua inoculated on carrot pieces at4 C. 93 Figure 20. Effect of Polyphenon 70S on Aeromonas hydrophila inoculated on carrot pieces at 4 C. 93 Figure 21. Effect of Polyphenon 70S on indigenous Pseudomonads on carrot pieces at4 C. 94 Figure 22. Effect of Polyphenon 70S on carrot pieces inoculated with E. coli at 8 C. 95 Figure 23. Effect of Polyphenon 70S on carrot pieces inoculated with Salmonella salford at 8 C. 95 Figure 24. Effect of Pediocin and Nisin on Listeria innocua inoculated on carrot pieces at 4 C. 96 Figure 25. Effect of Pediocin and Nisin on indigenous Pseudomonads on carrot pieces at 4 C. 96 Figure 26. ph changes in carrot pieces at 4 C. 97 Report for Horticulture Australia Limited Food Science Australia

15 IMPROVING THE QUALITY AN SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES 1 MEDIA SUMMARY Minimally processed and ready to eat vegetables products have emerged as a major product category in Australia during recent years. While the implementation of strict temperature and hygiene controls throughout the distribution chain and effective farm and processing management practices have contributed to high standards of product safety and quality, new technologies are required to increase product shelf-life and maintain quality. This project investigated the use of food grade acids, gums and antioxidants, and the combined effect of heat treatment and organic acids on the inhibition of browning on whole and fresh-cut vegetables and the effect of food-grade anti-microbial agents and cultures to enhance product safety and quality of fresh cut vegetables. Results on inhibition of enzymatic browning, indicated that acetic acid could inhibit brown colour development on the cut end of the lettuce heads when applied as a dip or a coating. During storage, the acetic acid treated area developed shrinkage, dehydration and softening. However, the application of acetic acid, food grade gum and calcium chloride helped to delay browning by 2-5 days and also minimised dehydration, softening and shrinkage of the treated area. Further commercial trials with known lettuce varieties are anticipated with interested commercial parties. Results on the application of anti-microbial control measures showed small effects with cultures and polyphenol compounds derived from tea but substantial inactivation effects were observed against Listeria with bacteriocin peptides derived from foodgrade cultures. The most promising results with other bioprotective agents have yet to be publicly disclosed, as the technology is the subject of a patent application to protect the interests of the industry. Report for Horticulture Australia Limited Food Science Australia 9

16 IMPROVING THS QUALITY AND SAFETY OF UNPROCiSSIO AN FR1SM-CUT VIGETAfLES 2 TECHNICAL SUMMARY Minimally processed and ready to eat vegetables products have emerged as a major product category in Australia during recent years. A major problem with cut vegetables is the development of brown discoloration on cut surfaces, due to the enzyme activity of polyphenol oxidase. Development of this discoloration as well as drying of cut surfaces and microbial growth limits the acceptability of the produce and time available for marketing of cut vegetable products. The physiological and microbiological quality and safety of these products are generally dependent on the implementation of strict temperature and hygiene controls throughout the distribution chain and appropriate handling, production and harvesting practices. The major microbiological risks for minimally processed vegetable products are bacteria that can grow at refrigerated storage conditions and Listeria monocytogenes is the pathogen of greatest concern, while pseudomonads are of greatest concern with respect to microbial spoilage of products. Additional preservation hurdles would assist in enhancing the microbial safety and quality of these products. Part 1 of this project reports on the use of acidulant, gums, antioxidants and the combined effect of heat treatment and organic acids on the inhibition of browning on fresh-cut vegetables. Part 2 reports on the efficacy of food-grade antimicrobials and bioprotective agents and cultures to provide additional control measures for microbial pathogens and spoilage microorganisms. Results on the inhibition of browning, indicated that acetic acid could inhibit brown colour development on cut end of the lettuce heads when applied as a dip or a coating. During storage, the treated area developed shrinkage, dehydration and softening. The application of acetic acid, gum and calcium chloride helped to delay browning by 2-5 days and also minimised dehydration, softening and shrinkage of the treated area. However, the effectiveness of this coating varied from batch to batch of lettuces. Commercial scale trials with known lettuce varieties are required before commercialising this approach. Results on the application of antimicrobial agents and cultures showed only little effect with tea polyphenols; however, notable inactivation was with anti-listeria bacteriocins and marginal inactivation effects with bacteriocin producing cultures. Suppression of pseudomonads was only not achieved with any treatments other than the undesirable secondary effects of low ph. Results showing the most promising treatments have not been reported here as they comprise the Provisional Patent application PR 3862 and further steps are been taken to facilitate commercialisation. Report for Horticulture Australia Limited Food Science Australia 10

17 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES PART 1- IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES BY MODIFYING AGENTS 3 INTRODUCTION The cut surfaces of freshly harvested vegetables and minimally processed vegetables are prone to physiological responses associated with wounding and wound healing such as browning, increased respiration and ethylene production (Watada et al 1996). Browning of cut vegetables is primarily due to oxidising enzymes that in the presence of oxygen convert phenolic compounds into polymerised, dark coloured pigments (Laurrila et al, 1998). The cutting and shredding inherent in harvesting and/or processing of fresh vegetables exposes cellular content directly to oxygen and enhance the mixing of polyphenol oxidase (PPO) and substrate (McEvily et al, 1992). The activity of phenylalanine ammonialyase and an increase in level of phenolic compounds, also contribute to brown colour development during storage. Browning causes an undesirable effect on the appearance of the cut ends of whole lettuce heads and shredded lettuce, and limits the storage life (Brechet, 1995; Saltveit, 1997). A number of anti-browning agents including reducing agents, enzyme inhibitors, chelating agents, acidulant and complexing agents have been studied in the past for the prevention of browning on fruit and vegetables. The ph optimum for PPO activity was reported to range between ph 4 and 7. However the optimum ph varies with particular substrate (Laurrila et al, 1998, McEvily et al, 1992). Lowering ph below 4 is used to inhibit activity of PPO from many sources. Castner et al (1996) demonstrated the effect of organic acid on the prevention of browning on the lettuce stem cuttings. The effect of temperature on PPO also varies with cultivar and species. It is reported that enzyme activity is completely destroyed at 80 C (Vamos-igayaso, 1981). Inactivation of PPO is feasible at temperatures higher than 50 C (Laurila et al, 1998). Loaiz-Velarde et al (1997) demonstrated that heat-shock treatment could reduce the increase in phenylalanine ammonia lyase activity and subsequent browning of the wounded lettuce tissues. This study was conducted in response to an industry identified need to extend shelf life by reducing the unsightly 'browning' of processed and un-processed vegetables. The results of this study will help to extend the shelf life and reduce waste resulting from regular trimming at retail level. Extended shelf life will lead to an increase in the transport life and allow the products to reach remote Australian and export markets. Overall out come of this project will be to improve profitability of the whole value chain: grower, processor and retailer. In Part 1 of this study the chemical treatments and combinations of natural gums, organic acids, and salts and heat treatment were evaluated to prevent browning on cut surfaces of whole lettuce heads and in shredded lettuce. Report for Horticulture Australia Limited Food Science Australia 11

18 IMPROVING TH1 QUALITY AND SAPITY OF UNPROCESSED AND PRiSH-CUT VEGETAiLfS 4 MATERIALS AND METHODS 4.1 Materials Lettuce stem discs Lettuce stems were obtained from fresh lettuce heads and were sliced (3 mm) using a very sharp knife Sample preparation - Lettuce heads Iceberg lettuce heads were obtained from the retail market and 4-6 outer leaves were removed. The base of the head and the stem was washed with tap water to remove adhering soil. Heads were left to dry on wire mesh trays at room temperature (22 C). A 5 mm thick slice was removed from the stem before treatment Sample preparation - Broccoli heads Broccoli heads were obtained from the retail market. The base f the broccoli heads and the stems were treated as in Acids Food grade glacial acetic acid, lactic acid, citric acid and tartaric acid, were obtained from Consolidated Chemical Co, Australia. Acetic acid solutions with ph 2.0 to 3.5 were made by mixing known weights of glacial acetic acid with distilled water Gums Xanthan and chitosan gums were obtained from Kelco, Germantown, Australia and Primex Ingredients, Norway respectively. Xanthan and chitosan gum powders were dissolved in acetic acid solutions at ph 2.0 and Methods Preliminary experiments on lettuce stem end browning (a) Effect of citric acid solutions at ph for sec Iceberg lettuce stem slices (thickness - 3 mm) were treated with citric acid solutions at ph for sec. Acid treated discs were either rinsed in distilled water or left without rinsing and then stored in seated polyethylene bags at 4 C for 16 days. The colour of the discs was evaluated on 2-day intervals using subjective scores and objective colour measurements as described in (a) Report for Horticulture Australia Limited Food Science Australia 12

19 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES (b) Effect of tactic, malic, and tartaric and acetic acids and application method Iceberg lettuce stem slices with a thickness of 3 mm were treated with lactic, malic, and tartaric and acetic acids at ph level 1.5 and 2 by dipping (1 sec) or blotting. Samples were stored in sealed polyethylene bags at 4 C for 16 days. The colour of the discs was evaluated on 3-day intervals using subjective scores and objective colour measurements as described in (a). (c) Effect of acetic acid treatment and rinsing Lettuce leaves were removed from the head and the core (un-sliced) was used for evaluation of washing treatments in order to retain the firmness of acid treated cut ends of lettuce cores. Trimmed lettuce cores were dipped in tartaric or malic acids at ph 2.0 for 1, 5 and 10 min or in acetic acid for 30 sec, 1 and 5 min. After dipping in acids samples were drained on a mesh for 1 min at room temperature (22 C). Acid treated samples were either rinsed in distilled water (10 min) or Ca(OH) 2 (10 min) or left without rinsing. Eight cores per treatment were stored in sealed polyethylene bags at 4 C. The colour of the discs was evaluated on 2-day intervals using subjective scores as described in (a). (d) Effect of acetic acid and application method Trimmed lettuce cores were either dipped in acetic acid solutions at ph 1.5, 2.0 and 2.5 for 30 sec or dipped in acetic acid solutions and taken out immediately or the acetic acid solution was applied by pressing the core on a sponge in acetic acid solutions. After dipping in acid solutions, samples were drained on a mesh for 1 min at room temperature (22 C). Eight cores per treatment were stored in polyethylene bags at 4 C with a snap seal. The colour of the discs was evaluated using subjective scores as described in (a) Experiments on cut end of whole lettuce heads (a) Effect of low ph and dipping The cut end of the heads was dipped in acetic acid solution either at ph 1.5 or 2.0 for 30 sec. Treated heads were drained on a mesh for 30 sec at room temperature (22 C). A set of 12 heads was kept untreated after removing a 5 mm thick slice of the stem. Treated and untreated lettuce heads were placed in polyethylene bags (49 cm X 32 cm) individually. Six lettuces were placed in a box (29 cm X 44 cm X 15 cm) and lids were placed on the boxes leaving approximately a 1-cm gap. Boxes were stored at 4 C. Samples were evaluated for colour on day 7 and day 10 using subjective scores as described in (a). (b) Effect of low ph and blotting The cut end of the heads was placed for 10 sec on a sponge impregnated with acetic acid solution at either ph1.5 or ph 2.0. Treated heads were drained on a mesh for 30 sec at room temperature (22 C). A set of 12 heads was kept untreated after removing a 5 mm thick slice of the stem. Treated and untreated heads were packed and stored as in (a). Samples were evaluated for colour on day 2 and day 7 using subjective scores as described in 4.2.5(a). Report for Horticulture Australia Limited Food Science Australia 13

20 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FR1SH-CUT VEGETABLES (c) Combined effect of heat and Low ph Ten whole lettuce heads per treatment were treated as follows. Table 1 - Treatment combinations used for evaluation of the combined effects of heat and acid. Treatments ph of acetic acid solution Dipping Time Sec Application method Temperature C T Sponge- blot 20 T Sponge - blot 20 T Dip 55 T Dip 55 T Dip 55 T Dip 55 T Dip 55 T8 Water 90 Dip 55 Treated heads (10 / treatment) were kept on a mesh for 30 sec at room temperature (22 C) and placed in polyethylene bags (49 cm X 32 cm). A set of 10 heads was kept untreated after removing a 5 mm thick slice of the stem. Treated and untreated heads were packed and stored as in (a). Samples were evaluated on day 2, 4, 8 and 17 using the colour scale outlined in (a). (d) Effect of low ph and gums -1 The stem end of the lettuce heads was dipped in acetic acid solution at ph 2.5 or ph 3.5, acetic acid solution +1% chitosan at ph 2.5 or ph 3.5, acetic acid solution* 1% xanthan gum at 2.5 or 3.5 ph for 60 seconds. Treated heads (10 / treatment) were kept on a mesh for 30 sec at room temperature (22 C). A set of 10 heads was kept untreated after removing a 5 mm thick slice of the stem. Treated and untreated heads were packed and stored as in 4.2.2(a). Samples were evaluated on day 3, 7 and 10 using the colour scale outlined in 4.2.5(a). (e) Effect of low ph and gums - II The experiment described in section (d) was repeated with three additional treatments, ph 2.5 with citric acid, ph 2.5 with citric acid + 1% xanthan gum and 1% xanthan gum. (f) Effect of low ph and gums - III Lettuces were obtained from a fresh cut lettuce grower in Werribee and prepared as described in section The cut ends of the lettuce heads were treated by dipping in a treatment solutions outlined in Table 2, for 60 seconds. Treated heads (10 / treatment) were drained on a mesh for 30 sec at room temperature (22 C). Treated and untreated heads were packed and stored as in (a) at 4, 7 and 12 C. Lettuce heads were visually observed for browning on the cut surface and drying on 4, 7, and 12 days of storage using the colour scale outlined in (a). Report for Horticulture Australia Limited Food Science Australia 14

21 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Table 2 - Treatment solutions used in (f) Effect oflowph and gums - III Treatment solution Control Chitosan + ph 2.0 Acetic acid Chitosan + ph 2J Acetic acid Xanthan + pm 2.0 Acetic acid Xanthan + ph 2.5 Acetic acid ph after dissolving gum S (g) Effect of low ph and gums - IV Lettuces were obtained from a grower in Werribee and prepared as described in section The cut ends of the lettuce heads were treated by placing, a sponge impregnated with the treatment solution (Table 3), on the cut end of the lettuce head for 60 seconds. Samples were stored at 4 C, 7 C and 12 C as described in (a) and were evaluated on 2, 5, 7, 9 and 12 days for brown colour development as described in (a). (h) Effect of low ph and gums - V Lettuces were obtained from a grower in Werribee and prepared as described in section The cut ends of the lettuce heads were treated by placing, a sponge impregnated with the treatment solutions outlined in Table 4, on the cut end of the lettuce head for 60 seconds. Samples were stored at 4 C and 12 C as described in (a). A set of samples stored at 4 C for 8 days and were transferred to 7 C and stored for another 4 days. All samples were evaluated on day 2,5,7,9,12 for brown colour development as described in (a). Report for Horticulture Australia Limited Food Science Australia 15

22 IMPROVING THE QUALITY AND SAFETY OF UNPROCISSED AND FRESH-CUT VEGETABLES Table 3 - Treatments used in (g) - Effect oftowph and gums - V Treatment ph Control (No treatment) - O.SM AA 2.48 O.SM AA + O.SM CaCI2 1J9 0.S5M AA + O.SM CaCI % xanthan gum 2.08 Table 4 - Experimental conditions used in (h) Effect oflowph and gums - V Treatment PH Storage condition Control - 4 C 7 C* 12 C O.SM AA + O.SM CaCI2 1J9 4 C 7 C* 0.6M AA + O.SM CaCI C 7 C* O.SM AA + O.SM CaCI % xanthan gum C 7 C* 12 C 0.6M AA + O.SM CaC! % xanthan gum C 7 C* 12 C * Lettuces were kept at 4 C until day 8, and then moved to 7 C. Report for Horticulture Australia Limited Food Science Australia 16

23 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETAiLiS Experiments on shredded lettuce (a) The combined effect of heat and low ph Iceberg lettuces were obtained from the market and 4-6 outer leaves were removed and the heads were washed and left at room temperature to drain. Lettuce heads were shredded with a sharp knife into 2cm wide strips about 10cm long. Shredded lettuce was weighed into 1.5 kg batches and each batch was treated with 6L of solution as described under each treatment. TreajmerriiS C1 - Untreated C2 - Dip in water at 4 C for 30 seconds C3 - Dip in acetic acid solution ph 3.0 at 4 C for 30 sec C4 - Dip in acetic acid solution ph 3.5 at 4 C for 30 sec T1 - Dip in water at 55 C for 90 seconds T4 - Dip in acetic acid solution (ph 3.0) at 55 C for 30 sec T5 - Dip in acetic acid solution (ph 3.5) at 55 C for 30 sec T6 - Dip in Tsunami"(200 ppm) solution at 55 C for 30 sec 77 - Dip in "Tsunami"(200 ppm) solution at 4 C for 30 sec After treatment each batch was de-watered in an Angelo Po spin drier for 2 min. Spin-dried treated lettuce pieces were packed in polyethylene bags (100g / bag) and heat-sealed leaving a minimum amount of headspace. Packages were stored at 4 C. Subjective and objective colour measurements were made as in (b) and (c). Mesophilic and psychotropic microbial counts (4.2.5 (d)) and the internal gas composition (4.2.5 (e)) were evaluated on days 0, 3, 7 and 10. (b) The combined effect of heat and low ph on the quality of shredded lettuce Iceberg lettuces were prepared as in (a) and treated as describe under each treatment. Treatments C1 - Untreated C2 - Dip in water at 4 C for 30 seconds C3 - Dip in acetic acid solution at ph 3.5 at 4 C for 30 seconds C4 - Dip in water at 55 C for 90 seconds T1 - Dip in acetic acid solution (ph 3.5) at 55 C for 15 seconds T4 - Dip in acetic acid solution (ph 3.5) at 55 C for 30 seconds T5 - Dip in acetic acid solution (ph 3.5) at 55 C for 60 seconds After treatment each batch was de-watered, packed and evaluated as described in (a). Report for Horticulture Australia Limited Food Science Australia 17

24 IMPROVING THE QUALITY AW SAFETY OF UNPROCESSED AND FRISH-CUT VEGETABLES Broccoli (a) Effect of moisture retaining agents In our preliminary trials on broccoli heads and florets, drying of the cut end and softening of the cut end were identified as the major changes that occurred during storage. Discolouration of the cut end was not observed after storage. Hence the use of moisture retaining agents was experimented on broccoli florets. The solution types and application methods used in this experiment are outlined in Table 5. Broccoli florets were dipped in solution 1 for 30 seconds and left to dry on a mesh tray at room temperature (22 C). Then samples were sprayed with a second solution and left to dry at room temperature before placing in polyethylene bags. Samples were stored at 4 C for 1 week and visual appearance was observed. Table 5 - Solution types and application methods used in (a) - Effect of moisture retaining agents Solution 1 - dip 30 sec Solution 2 - spray No treatment No treatment 1%CaCI 2 No treatment 1% Alginate No treatment 1% Alginate 1%CaCI 2 1%CaCI 2 1% Alginate 1% Xanthan No treatment 1% Xanthan 1%CaCI 2 1%CaCI 2 1% Xanthan 1% Dextrin No treatment 1% Dextrin 1%CaCI 2 1%CaCI 2 1% Dextrin (b) Effect of low ph and gums The cut end of the broccoli heads was dipped in acetic acid solution + 1% chitosan at 3.5 ph, acetic acid solution+1% xanthan gum at 2.5 or 3.5 ph for 60 seconds. A set of heads was kept untreated after removing a slice off the stem. Treated and untreated heads were kept on a mesh for 30 seconds at room temperature (22 C) and placed in polystyrene boxes box (29 cm X 44 cm X 15 cm). Six heads were placed in each box and lids were placed on the boxes leaving approximately 1-cm gap. Boxes were stored at 4 C for 1 week. Visual appearance of the samples was evaluated on day 3 and 7. Report for Horticulture Australia Limited Food Science Australia 18

25 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Sample Evaluation (a) Sample evaluation of whole lettuce heads The subjective colour measurements of lettuce discs, cut end of the cores and cut end of the lettuce heads were evaluated using a subjective scale described in Table 6. Objective colour readings were obtained using a Minolta Chromo meter (300 series, Japan). Each colour value represents an average of 9 readings. Table 6 - Subjective colour scale for assessing the colour changes in lettuce discs, cut surface of lettuce cores and the cut end of lettuce heads. Score Description 1 FRESH - Look fresh and no browning or pinking 2 GOOD - Slight sign of browning or pinking 3 FAIRLY GOOD - Obvious signs of browning or pinking but < 25% of the circumference 4 MARGINAL - < 50% of the circumference is brown no browning within the center 5 UNACCEPTABLE - > 50% of the circumference is brown 25% browning within the center 6 UNACCEPTABLE - > 50% of the circumference is brown 50% browning within the center 7 UNACCEPTABLE - > 75% of the circumference is brown >75% browning within the center (b) Objective colour measurement of shredded lettuce Minolta Chromo meter (300 series, Japan) was used to measure the colour of samples. Each colour value represents an average of 9 readings. (c) Colour score of shredded lettuce The colour of the shredded lettuce was evaluated using a subjective scale described in Table 7. Report for Horticulture Australia Limited Food Science Australia 19

26 fmproving THE QUALITY ANO SAFETY OF UNPROCESSED AND FRISH-CUT VEGETABLES Table 7 - Subjective colour score for evaluation of browning in shredded lettuce. Score Description 1 FRfSH - Look fresh and no browning or pinking 2 GOOD - Slight sign of browning or pinking 3 FAIRLY GOOD - Slightly pink areas but acceptable (< 10% of the cut surfaces are pink) 4 MARGINAL - Slightly pink areas but still marginally acceptable (< 25% of the cut surfaces are pink) 5 UNACCEPTABLE - Pink < 0% of the cut surfaces are pink 6 Pink % of the cut surfaces are pink 7 Pink % of the cut surfaces are pink (d) Microbiological assessments: Standard microbiological procedures were followed in the enumeration of mesophilic and psychotropic microbial counts (Australian Standard Methods, 1766). (e) Determination of headspace gasses: Carbon dioxide, oxygen and nitrogen contents were analysed by injecting 0.5mlheadspace gas into a gas chromatograph (model GC8A, Shimadzu, Japan) with CTR III packed column (6ft x Vi" SS, Altteeh, Aust.) and a thermal conductivity detector. Argon was the carrier gas. Injector, oven and detector temperatures were 50, 30 and 50 C respectively. Report for Horticulture Australia Limited Food Science Australia

27 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRISH-CUT VEGETABLES 5 RESULTS AND DISCUSSION 5.1 Preliminary experiments Effect of citric acid solutions at ph for seconds Citric acid, at ph 1.5 and ph 2.0 were the most effective ph levels for the control of browning development in lettuce discs. Dipping in ph 1.5 for 60 seconds with or without rinsing showed good inhibition of browning in lettuce stem discs. A maximum shelf life of 16 days was achieved at ph 1.5, 60 seconds dip and with rinsing where as the control was acceptable only for 2 days. However, 60 seconds with no rinse treatment showed loss of texture after 2 days of storage. Further with ph 2.0 and 10-minute dip, rinsed and un-rinsed samples also indicated good browning prevention and shelf life was extended to 15 days Effects of lactic, malic, tartaric and acetic acids and application method Hue value Figures 1-5 show the change in hue value of the acid treated lettuce discs and Table 8, shows the summary of results indicating the number of days lettuce discs remained with acceptable colour. The hue angle of untreated discs decreased with the increase in storage time indicating pinking or browning of the discs. In samples dipped in acid the rate of browning was lower when compared with the untreated controls. The hue values of lettuce discs treated with lactic, malic or tartaric acids were higher than 100 for a longer period than citric acid treated samples (Table 8) that of the discs treated with citric acids. The acetic acid treated samples were not evaluated after 2 days due to excessive softening but brown colour development was not observed. Generally dipping in acids resulted a lighter colour than blotting the acids on lettuce disc. However, citric acid is widely used in the food industry to prevent browning. It is reported that citric acid has dual inhibitory effect on PPO by reducing the ph and by chelating copper ions from enzyme activity sites (McEvily et al., 1992). Effect on firmness The untreated samples were very firm and had a score of 6 until at the end of the experiment. Among the treated samples both malic acid dip and sponge-blot at ph 2.0 and tartaric acid dip at ph 2.0 retained a very firm texture for 10 days. The acetic acid treated samples showed excessive softening after 2 weeks. AH the other treatments showed a gradual loss of firmness during storage. Report for Horticulture Australia Limited Food Science Australia 21

28 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLIS Table 8 - Summary of experimental findings on the effect of citric, lactic, malic, tartaric and acetic acid treatments on lettuce discs based on number of days Hue value remained > 100. Treatment Number of days at Hue value > 100 Control 2 Citric acid ph 1.5 dip 7 Citric acid ph 2.0 dip 2 Citric acid ph 1.5 blot 2 Citric acid ph 2.0 blot 2 Lactic acid ph 1.5 dip 2* Lactic acid ph 2.0 dip 13 Lactic acid ph 1.5 blot 2* Lactic acid ph 2.0 blot 2 Malic acid ph 1.5 dip 7 Malic acid ph 2.0 dip 10 Malic acid ph 1.5 blot 15 Malic acid ph 2.0 blot 2 Tartaric acid ph 1.5 dip 12 Tartaric acid ph 2.0 dip 15 Tartaric acid ph 1.5 blot 15 Tartaric acid ph 2.0 blot 2 Acetic acid ph 1.5 dip 2* Acetic acid ph 2.0 dip 2* Acetic acid ph 1.5 blot 2* Acetic acid ph 2.0 blot 2* * Terminated the evaluations due to excessive softening of the treated lettuce stem discs Report for Horticulture Australia Limited Food Science Australia

29 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 1 - Effect of citric acid application on the hue angle of lettuce stem discs stored at 4 C > a X " " Citric ph 1.5 dip " - Citric ph 2.0 dip Citric ph 1.5 blot -'--Citric ph 2.0 blot -* Control dip - -Control blot Storage time (days) Figure 2 - Effect of lactic acid application on the hue angle of lettuce discs stored at 4 C 120 c CO 0) 3 I Storage time (days) LacfiepHLSdip -*-Lactic ph 2.0 dip Lactic ph 1.5 Wot -*~~ Lactic ph 2.0 blot -*-Control dip - -Control Wot Report for Horticulture Australia Limited Food Science Australia 23

30 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 3 -Effect of malic acid treatment on the hue angle of lettuce discs stored at 4 C "" Malic ph 1.5 dip -*- Malic ph 2.0 dip Malic ph 1.5 blot "-- Malic ph 2.0 Wot ~*~" Control dip - -Control Wot 0 10 Storage time (days) Figure 4 - Effect of tartaric acid apphcation on hue angle of lettuce disc stored at 4 C 0) e CO a) 3 T Tartaric ph 1.5 Dip - -Tartaric ph 2.0 Dip Tartaric ph 1.5 Blot Tartaric ph 2.0 Blot Control dip Control Wot 0 10 Storage time (days) Report for Horticulture Australia Limited Food Science Australia 24

31 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Effect of acetic acid treatment and rinsing Colour score Figures 6,7,8 show the effect of acetic acid treatment with or without rinsing with distilled water or calcium hydroxide. Control samples were unacceptable after two days of storage (Table 10). When comparing the effect of three acids, acetic acid treated samples were whiter in colour for al longer period (12-22 days) than the samples treated with malic (3-11 days) and tartaric (2-8 days) acids. In general Malic acid treated samples retained a whitish appearance longer than the tartaric acid treated samples. Rinsing with either distilled water or calcium hydroxide after acid dip was not effective in controlling browning of the lettuce discs. Acetic acid treated samples retained a acceptable appearance for 22 days when unwashed while samples washed with distilled water or calcium chloride retained acceptable colour only for 15 days (Figures 6,9,10). A similar trend was observed with malic acid (Figures 7,13,14) and tartaric acid (Figures 8,15,16) treated samples. However, washing with calcium hydroxide was evaluated to minimise the effect of acetic acid on the texture of treated area. It is reported that calcium ions can help in retaining the texture of fresh cut vegetables (Picchioni et al 1996). Figures 15,16,17 illustrate the colour difference between treated cores and untreated controls The effect of dipping time was prominent in tartaric acid and malic acid treated samples. Browning developed at a slower rate with the increase in dipping time. Among the acetic acid treated samples the effect of dipping time was prominent when the samples were washed. Hue and L value Hue angle provides an indication of colour change of lettuce cores from white to reddish brown. Hue value of acetic acid treated samples remained stable through the storage period while the hue value of samples treated with tartaric and malic acid decreased rapidly during the first 5 to 10 day period and remained stable at a lower level (Figures not included). L value of the samples indicated the changes in lightness of the samples. L value of the samples followed a similar trend as the hue value (Figures not included). Report for Horticulture Australia Limited Food Science Australia 25

32 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESM-CUT ViGiTASLES Table 9 - Summary of experimental findings on the effect of acetic, malic and tartaric acid treatments on lettuce cores with or without rinsing with distilled water or calcium hydroxide, based on number of days colour score remained < 4 or marginal acceptability. Treatment Number of days at Colour score < 4 Control 2 Acetic acid 30 sec NR 22 Acetic acid 1 min NR 22 Acetic acid 5 min NR 22 Acetic acid 30 sec Dist. water 15 Acetic acid 1 min Dist. water 15 Acetic acid 5 min Dist. water 15 Acetic acid 30 sec Ca(OH) 2 14 Acetic acid 1 min Ca(OH) 2 12 Acetic acid 5 min Ca(OH) 2 19 Malic acid 10minNR 7 Malic acid 20 min NR 9 Malic acid 30 min NR 11 Malic acid 10 min Dist. water 3 Malic acid 20 min Dist. water 5 Malic acid 30 min Dist. water 6 Malic acid 10 min Ca(OH) 2 3 Malic acid 20 min Ca(OH) 2 5 Malic acid 30 min Ca(OH) 2 5 Tartaric acid 10 min NR 4 Tartaric acid 20 min NR 7 Tartaric acid 30 min NR 8 Tartaric acid 10 min Dist. water 2 Tartaric acid 20 min Dist. water 4 Tartaric acid 30 min Dist. water 5 Tartaric acid 10 min Ca(OH) 2 2 Tartaric acid 20 min Ca(OH) 2 3 Tartaric acid 30 min Ca(OH) 2 5 Report for Horticulture Australia Limited Food Science Australia

33 IMPROVING THE QUALITY AND SAFITY OF UNPROCESSED AND FRISH-CUT VEGETABLES Figure 5 - Effect of acetic acid application on the hue value of lettuce discs stored at 4 C 120 H c CD % I Acetic ph 1.5 Dip - B -AceticpH2.0Dip Acetic ph 1.5 Blot - -Acetic ph 2.0 Blot -*~ Control dip - -Control blot Storage time (days) Figure 6 - Effect of acettc acid treatment (norinse- NR) on the colour of lettuce core cut end stored at 4 G. - A. A ~~ Control to 3 2 o O I. -"" -n n -H ~* *?\ Acettc 30secNR Acetic 1 rrin NR - Acetlic5minNR Storage time (days) Report for Horticulture Australia Limited Food Science Australia 27

34 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT ViGETAiLES Figure 7 - Effect of Malic acid treatment on the colour of lettuce core cut end stored at 4 C. s Control Malic 10 min NR Malic 20 min NR Malic 30 min NR Storage time (days) 25 Figure 8 - Effect of tartaric acid treatment foltowed by no rinse on the colour of lettuce core cut end stored at 4 C 8 CO SI.o o O 6 4 is n - 'pi / // / / J *SEp==- QB^fr- H " "Control " "Tartaric 10 min NR Tartaric 20 min NR -x- Tartaric 30 min NR I I i Storage time (days) 25 Report for Horticulture Australia Limited Food Science Australia 28

35 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSiD AND FRESH-CUT VEGlTAiLES Figure 9 - Effect of acetic acid treatmentfollowedby washing wifrt distied water on the colour of lettuce core cut end stored at 4 C. Control Acetic 30sec dist. Water Acetic 1 rrin dist. Wrier Acetic 5 rrin dist. Water Storage time (days) 25 Figure 10 - Effect of acetic acid treatment followed by washing with Ca(OH)2 on the colour of lettuce core cut end stored at 4 C. "" "" Control - -Acetic 30s c Ca (OH)2 Acetic 1 nrinca(oh)2 " Acetic 5 rrin Ca (OH) Storage time (days) 25 Report for Horticulture Australia Limited Food Science Australia 29

36 IMP-ROVING THE QUALITY ANB SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 11 - Effect of Tartaric acid treatment followed by washing with distilled water on the colour of lettuce core cut end stored at 4 C -«n 8 ta L. 3 O "5 O 4 R--X ^rs K " if * if m * / z 1 Control B Tartaric 10 rnin dist. Water Tartaric 20 min dist. Water ' '"' Tartaric 30 min dist. Water 1 15 Storagetime (days) Figure 12 - Effect of Tartaric acid treatment followed by washing with Ca(OH)2 on the colour of lettuce core cut end "Control Tartaric 10 min Ca (OH)2 Tartaric 20 rrwi Ca (OH)2 Tartaric 30 min Ca (OH) Storage time (days) Report for Horticulture Australia Limited Food Science Australia 30

37 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRiSH-CUT VEGETASLES Figure 13 - Effect of Malie acid treatmentfollowedby washing with distilled water on the colour of lettuce core cut end. o "5 o -«Wr ~Wr -X- Cor#d Malic 10 min dist. Water Malic 20 min dist. Water Malic 30 min dist. Water Storage time (days) Figure 14 - Effect of Malic acid treatment followed by washing with Ca(OH)2 on the colour of lettuce core cut end stored at 4 C. ~JK** n " Confrol Storage time (days) 25 Malic 10 min Ca (OH)2 Malic 20 min Ca (OH)2 Malic 30 min Ca (OH)2 Report for Horticulture Australia Limited Food Science Australia 31

38 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED Ah FRESH-CUT VEGETABLES Figure 15 - Untreated lettuce cores and lettuce cores treated with acetic acid at ph 2.0 for 30 seconds.. ai CONTROL S BAYS Figure 16 - Untreated lettuce cores and lettuce cores treated with malic acid at ph 2.0for10min. t - CONTROL S DAYS T7 SDA*S Report for Horticulture Australia Limited i. *.&;«??* Food Science Australia

39 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 17 - Untreated tettuce cores and lettuce cores treated with tartaric acid at ph2.0for10min..,«««s CtWtROL 8 DAYS Tl BDAYS i r Report for Horticulture Australia Limited Food Science Australia 33

40 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Table 10 - Summary of experimental findings on the effect of acetic acid treatments on lettuce cores with two application methods, based on number of days colour score remained < 4 (marginal acceptability) and surface ph. Treatment Number of days at Colour score < 4 Number of days at ph <5.0 Control 2 0 ph sec ph 1.5 dip in out PH 1.5 blot ph sec ph 2.0 dip in out 16 4 PH2.0 blot 13 4 ph sec 5 9 ph 2.5 dip in out 4 7 PH2.5 blot 4 7 Report for Horticulture Australia Limited Food Science Australia 34

41 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 18 - Visual scores of lettuce cores treated with acetic acid at ph s O fi n n- p^~- ^i Control AA-pH sec AA-pH 1.5 dip in & out AA-pH 1.5 blotting Storage time (days) AA: Acetic acid Figure 19 - Visual score of lettuce cores treated with acetic acid at ph 2.0 'Control AA-pH sec AA-pH 2.0 dip in & out AA-pH 2.0 blotting Storage time (days) 20 AA: Acetic acid Report for Horticulture Australia Limited Food Science Australia 35

42 IMPROVING THE QUALITY AND SAFITY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 20 - Visual score of lettuce cores treated with acetic acid at ph g 6 O % 5 4 o O o " "" Control - -AA-pH see AA-pH 2.5 dip in & out -x-aa-ph 2.5 blotting Storage time (days) AA: Acetic acid Figure 21 - Surface ph of lettuce cores treated with acetic acid at ph 1.5 x control AA-pH sec AA-pH 1.5 dip AA-pH 1.5 blot Storage time (days) 20 AA: acetic acid Report for Horticulture Australia Limited Food Science Australia 36

43 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRiSH-CUT VEGETA&LiS Figure 22 - Surface ph of lettuce cores treated with acetic acid at ph control z ex -«AA-pH sec AA-pH 2.0 dip -;*-AA-pH 2.0 blot Storage time (days) AA: acetic acid Figure 23 - Surface ph of lettuce cores treated with acetic acid at ph 2.5 control AA-pH sec AA-pH 2.5 dip AA-pH 2.5 blot Storage time (days) 20 AA: acetic acid Report for Horticulture Australia Limited Food Science Australia 37

44 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Effect of acetic acid and application method Visual colour score Figures 18,19 and 20 show the visual score of lettuce cores treated with acetic acid at ph 1.5, 2.0 and 2.5 respectively and summary of results are shown in Table 10. Visual score of lettuce cores treated with acetic acid solution at ph 1.5 remained at score 1 indicating the inhibition of browning during the 18 day storage period. A tangible difference was not observed in samples treated by the three different methods of application. Lettuce cores dipped in acetic acid solution at ph 2.0 for 30 seconds did not developed brown colour and were acceptable through the 18 day storage period where as the dip in out treatment remained acceptable for 16 days and blotting treatment for 13 days. The cores treated by dipping in and out and blotting at ph 2.0 developed brown colour gradually. These samples reached a score of 4 by the end of the 18th day storage period. All the samples treated at ph 2.5 gradually developed browning and reached a score of 4 (acceptable) within 4-5 days of storage. L and Hue values - L values of the samples treated at ph 1.5 remained between 65 and 75 while the samples treated at ph 2.0 and 2.5 gradually decreased below the above range indicating darkening of the samples during storage. The difference in L values among the methods of application was not very prominent at ph 1.5 but it was obvious at ph 2.0 and 2.5. In general dipping for 30 seconds seems to be more effective than the dip in and out, and blotting (Figures not included). Hue values followed a similar trend as L values. ph- Figures 21, 22 and 23 show the surface ph change in lettuce cores treated with acetic acid at ph 1.5, 2.0 and 2.5. Initial ph of the samples treated at 1.5 ph ranged around 3.5 while samples treated at 2.0 and 2.5 were between 3.5 to > 4.0. The surface ph of control samples were higher than 5.5 and were stable during the storage period. The final ph of lettuce cores dipped for 30 seconds at ph 2.0 had a ph less than 5.0 by 15 days while the blotting and instantly dipping led to ph above 5.0 after 4 days. AH the samples treated at ph 2.5 reached a ph value higher than 6.0 by the 15 th day of storage. When considering the browning trends and surface ph changes, it was evident that to prevent browning by ph modification, surface ph has to be below ph 5.0. To retain the pm at or below ph 5.0 through the storage period, it is essential to treat the lettuce stem with acetic acid at ph 1.5 by any of the application methods (dipping for 30 sec, dipping in and out or blotting), or by dipping for 30 seconds in acetic acid solutions at ph 2.0. Report for Horticulture Australia Limited Food Science Australia 38

45 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES 5.2 Whole lettuce heads Effect of low* ph and dipping Visual Colour Score Figure 24 shows the effect of dipping the cut end of whole lettuce heads in acetic acid solution with ph 1.5 and 2.0. The colour of the untreated controls deteriorated rapidly (2 days) while the colour of the acid treated heads remained unchanged for 10 days (Table 10). General appearance The outer leaves of the heads subjected to dipping treatment were damaged with acid burn. The treated end of the stem was shrivelled during storage Effect of low ph and blotting Visual Colour Figure 25 shows the effect of acid application by sponge blotting at ph 1.5 and 2.0 Colour of the cut surface changed in control samples and was dark pink after 7 days storage. Samples treated with acid solution at ph 1.5 by blotting did not show a colour change during the 7-day storage period. The colour of the samples treated with ph 2.0 solution developed a slight pink tinge on the cut surface after 7 days. General appearance Outer leaves were not acid damaged. The treated stem area was not shrivelled but the outer edge had a dry appearance. Acid application with a sponge impregnated with acid solution, was more effective in controlling acid damage of the outer leaves. Table 11- Summary of the experimental findings on the effect of acetic acid treatments on the cut end of whole lettuce heads with dipping and blotting methods, based on number of days colour score remained < 4 (marginal acceptability). Treatment Number of days at Colour score < 4 Experiment Control 2 AApH1.5dip 10 AA ph 2.0 dip 10 Experiment Control 2 AApH 1.5 blot 7 AA ph 2.0 blot 7 Report for Horticulture Australia Limited Food Science Australia

46 IMPROVING THI QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 24 - Colour change of cut end of lettuce heads dipped in acetic acid solutions s> 8 5 CO o Storage time (days) B co r trol ^^* f\ r\ ' ph 1. dp BAA ph 2.9 dp (AA: Acetic acid, dp: dipping) Figure 25 - Colour change of cut end of the lettuce heads treated by blotting with acetic acid solutions. 1 co 3 O Storage time (days) control AA ph 1.5 bl DAA -ph 2.0 bl (AA: acetic acid, bl: blotting) Report for Horticulture Australia Limited Food Science Australia 40

47 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Combined effect of heat and low ph on the cut end of whole lettuce heads Colour score Figure 26 shows the colour scores of cut end of lettuce heads with combined heat and acid treatment and table 12 shows the summary of results. Colour scores revealed that blotting at ph 1.5 and 2.0 were better than any of the other treatments in the prevention of pink colour development in cut end of lettuce heads. Colour of the treated lettuce heads remained acceptable for 17 days where as the control was acceptable for 3 days. However, the combined effect of heat (55 C, 30 sec) and dipping at ph 2.5 seemed to be more effective than the other combined treatments of heat (55 C) and dipping at ph , which reached an unacceptable level of browning on day 3. L Value Figure 27 shows the changes in L value also followed a similar trend as colour scores. L value is a measure of lightness of the product, which decreased, with pinking of the cut surface. Samples treated by dipping in ph 2.5 solution at 55 C for 30 sec had L values (higher than 60) comparable to blotting with acid at ph 1.5 and ph 2.0. Hue angle Hue angle also followed a simitar trend as L values, showing the benefit of dipping in ph 2.5 solution at 55 C for 30 sec (Figure 28 and Table 12). General appearance Drying and shrivelling of stems was observed in samples treated with acids at ph 1.5 and 2.0 on day 4 of storage. By day 8 the samples treated with acids at ph 1.5 and 2.0 had shrivelled intensely. Samples treated by dipping in ph 2.5 solution at 55 C for 30 sec developed signs of drying in the outer surface of the stem and outer leaves on the day 8, and the outer leaves were yellowish on the day 17. Samples treated at ph 3.0 and 3.5 were not damaged due to acid burn during the storage period. Spoilage was observed in two out of five control samples on day 8. Table 12 - Summary of the experimental findings on the combined effect of acetic acid and heat treatment on the cut end of lettuce heads with dipping method, based on number of days colour score remained < 4 (marginal acceptability), Treatment Number of days at Colour score <4 Number of days at "L value" >60 Number of days at "a value" >100 Control 3 <2 <2 Dip water 55 C 90 sec 3 <2 <2 Blot ph Blot ph Dip ph C 30 sec Dip ph C 30 sec 3 <2 <2 Dip ph C 60 sec 3 <2 <2 Dip ph C 90 sec 3 <2 <2 Report for Horticulture Australia Limited Food Science Australia 41

48 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 26 - Colour score of cut end of lettuce heads treated with combined heat and acid treatments * control "" dp water 55* C 90 sec *-W-pH1.5 *-M-pH2.0 dp-ph C 30 sec "dp-ph C 30 sec (bl - blotting, dp - dipping) Storage time (days) 20 < dp-ph C 60 sec dp-ph C fo see Figure 27 - Changes in L value of cut surface of whole lettuce heads treated with add and heat control dp water 55 C 90 sec bl-ph1.5 bl-ph2.0 dp ph C 30 sec dp ph C 30 sec (bl - blotting, dp - dipping) Storage time (days) 20 dp ph C 60 sec dp ph C 90 sec Report for Horticulture Australia Limited Food Science Australia 42

49 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VIGETASLES Figure 28 - Changes in hue angle of cut surface of whole lettuce heads treated with acid and heat (bl - blotting, dp - dipping) Report for Horticulture Australia Limited Food Science Australia 43

50 IMPROVING THI QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Effect of low ph and gums I Colour Score Figure 29 shows the changes in visual colour (browning) score of the treated cut end area of the lettuce heads. Cut ends of the lettuce heads turned pink gradually with storage time. The samples treated with acetic acid at ph 2.5 with or without gum had a better effect on the browning prevention than at ph 3 (Table 13). Acetic acid at ph % xanthan gum or 1% chitosan the cut end colour remained acceptable up to 5-6 days, in comparison, to the control samples, which were acceptable for 3 days. However, by the seventh day of storage all treatments were unacceptable and had a score above 5. General appearance Drying of the treated area resulted in shrinkage of the stem. Drying and shrinking of the treated stems progressed within the 10-day storage period. Shrinkage was more prominent at ph 2.5 than at ph 3.0. It was observed that application of gums with acids helped to reduce the drying and shrinkage effect of acids especially at ph 2.5. Gums act as moisture retaining agents and thereby prevented drying of the treated area. 5.2.S Effect of low ph and gums II Colour Score Browning of the second set of experiments followed a similar trend as in the previous experiment. Figure 30 shows the browning trend of lettuce heads treated with acid and gum combinations. At ph 2.5 use of either xanthan or chitosan was effective in retaining the acceptable colour of the cut end of lettuce for 9 days (Table 13). The colour of acetic acid only samples at ph 2.5 developed browning at a higher rate than the combined gum and acid treatments. The effect of citric acid at ph 2.5 on browning prevention was similar to the effect of acetic acid at ph 3.0. However, it was noted that there was a batch-to-batch variation in the magnitude of browning and results of the treatment. Citric acid or citric acid and gum combination were not effective in controlling browning. General appearance General appearance results followed a similar trend as in the previous experiment. Application of gums together with acids helped to reduce ttie drying effect of the acids on the stem. Drying effect was more prominent in samples treated with acid at ph 2.5. The applications of acid and gum formulations were more practical than the combined heat and acid treatments. Combined heat and acid treatment generated acid fumes that could lead to occupational health and safety related problems in a commercial situation. The required amount of treatment formulation was less in gum and acid application when compared to the heat and acid treatment. However, it is necessary to understand the batch-to-batch variation in order to standardise the treatments to get consistent results. Report for Horticulture Australia Limited Food Science Australia

51 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Table 13 - Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads with dipping method, based on number of days colour score remained < 4 (marginal acceptability) (4.2.2 d ande) Treatment Number of days at Colour score <4 Experiment (d) Control 3 ph 2.5 AA 4 ph 2.5 AA chitosan 5 ph 2.5 AA xanthan 6 ph 3.0 AA 2 ph 3.0 AA chitosan 2 ph 3.0 AA xanthan 2 Experiment (e) Control 2 ph 2.5 AA 9 ph 2.5 AA chitosan 9 ph 2.5 AA xanthan 9 ph 3.0 AA 3 ph 3.0 AA chitosan 2 ph 3.0 AA xanthan 3 1% xanthan <2 ph 2.5 citric acid 7 ph 2.5 citric acid xanthan 3 Report for Horticulture Australia Limited Food Science Australia 45

52 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 29 - Change in browning scores of the cut ends of the lettuce heads treated with acetic acid and gum formulations -I S control ph2.5aa ph 2.5 AA chitosan ph 2.S AA xanthan pm3.0aa AA: acetic acid Storage time (days) ph 3.0 AA chitosan ph 3.0 AA xanthan Figure 30 - Change in browning scores of the cut ends of the lettuce heads treated with acids and gum formulations -II control score 0 S - o o f 1 //50 V /r m -~~~~. 0* 1^ ft! I 1 i -~~~ Storage time (days) V ph 2.5 AA ph 2.5 AA chitosan ph 2.5 AA xanthan ph3.0aa ph 3.0 AA chitosan ph 3.0 AA xanthan 1% xanthan ph 2.5 CA ph 2.5 CA xanthan AA: acetic acid; CA: citric acid Report for Horticulture Australia Limited Food Science Australia

53 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETASi.ES Effect of storage temperature on browning prevention, coating applied on the cut end of lettuce heads -I In this experiment three storage temperatures, 4, 7 and 12 C, were selected to simulate the ideal and most probable temperature abused conditions. Samples stored at 4 and 7 C were stored for 14 and 8 days respectively. Samples stored at 12 C were discarded after 4 days due to spoilage. Colour score Figures 31, 32 and 33 show the Colour score of the cut end of lettuce heads treated with gums and acetic acid, and stored at 4 C, 7 C and 12 C and Table 14 show the summary of results. The colour of the untreated control samples remained at marginal value of 4 for 3 days at 4 C and for 2 days and 3 days at 7 and 12 C respectively. Samples treated with chitosan at ph 2.5 increased in pink colour formation gradually during storage at all 3 temperatures. Samples treated with xanthan at ph 2.5, xanthan at ph 2.0 and chitosan at ph 2.0 retained the acceptable colour for 12 days at 4 C. At 7 C colour retained acceptable for 8 days when treated with ph2.0 xanthan, ph 2.5 xanthan and ph2.0 chitosan. However, the rate of change in colour was slower at 4 C than at 7 C. The highest rate of colour change was observed at 12 C. Hue Value Hue value of freshly cut lettuce stem is normally around 100 and the hue value decreases with the development of pink colour. Hue value of untreated control samples decreased from an initial value of 100 to 70 during an 8-day storage period at 4 C (Figure 34 and Table 15). Hue values of samples treated with chitosan and xanthan at ph 2.0 and ph 2.5 were higher than 100 for 4-8 days where as the controls were unacceptable after 2days of storage at 4 C. Hue value changes in samples stored at 7 and 12 C are shown in Figures 35 and 36 respectively. However, the samples stored at 12 C were not evaluated throughout the storage time due to spoilage of samples. Appearance Application of acidic gum solution led to dehydration of the cut end of the lettuce. The degree of dehydration varied with the ph value of the solution and the type of gum used. On the 8 th day of storage the shrinkage of the cut end was in the following order in samples stored at 4 C: chitosan ph2.5 < chitosan ph2.0 < xanthan ph2.5 < xanthan ph 2.0. This trend was also observed at 7 C and 12 C. Chitosan treated samples developed a yellowish tinge on the cut surface at the end of the storage period especially at 7 C and 12 C. The application of solutions by dipping led to damage to the outer leaves of the lettuce head. Leaves had acid damaged patches and this effect was severe at 7 C and 12 C. Report for Horticulture Australia Limited Food Science Australia

54 IMPROVING THE QUALITY AMD SAFETY OF UNPROCESSED AND FR1SH-CUT VEGETABLES Table 14- Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage temperatures 4 C, 7 C and 12 C, based on number of days colour score remained < 4 (marginal acceptability). Treatment Number of days at colour score < 4 4 C 7*C 12 C Control phi 2.5 xanthan * ph 2.0 xanthan * ph 2.5 chitosan ph 2.0 chitosan * Table 15- Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce head at storage temperatures 4 C, 7 C and 12 C, based on number of days Hue value remained > 100. Treatment Number of days at Hue value > C 7 C 12 C Control <2 <1 <1 ph 2.5 xanthan 4* 4 ph 2.0 xanthan 4* 3 * ph 2.5 chitosan 6 3 * ph 2.0 chitosan 8 4 Colour was not evaluated due to spoilage. Report for Horticulture Australia Limited Food Science Australia

55 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 31- Colour score of the cut end of lettuce heads treated with gums and acetic acid, and stored at 4 C. - -Control - -Xanthan ph 2.5 Xanthan ph 2.0 )e. QhjjQggn ph 2.5 -*- Chitosan ph Storage time (days) Figure 32- Colour score of lettuce butts treated with gums and acetic acid, and stored at 7 C. o m u. 3 O O O Control Xanthan ph 2.5 Xanthan ph 2.0 Chitosan ph 2.5 Chitosan ph Storage time (days) Report for Horticulture Australia Limited Food Science Australia

56 IMPROVING THE QUALITY AND SAFETY OF UNPROCISSED AND FRESH-CUT VEGETABLES Figure 33 - Colour score of the cut end of lettuce heads treated with gums and acetic acid and stored at 12 C Control Xanthan ph 2.5 Xanthan ph 2.0 Chitosan ph 2.5 Chitosan ph S 10 Storage time (days) Figure 34 - Hue values of the cut end of lettuce heads treated with AA and gums and stored at 4 C. 120 Control Xanthan ph 2.5 Xanthan ph 2.0 Chitosan ph 2.5 Chitosan ph 2.0 AA: acetic acid Storage time (days) Report for Horticulture Australia Limited Food Science Australia 50

57 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 35 - Changes in Hue value of lettuce butts treated with acetie acid (AA) and gums and stored at 7 C. Control Xanthan ph 2.5 Xanthan ph 2.0 Chitosan ph 2.5 Chitosan ph Storage time (days) Figure 36 - Changes in hue value of untreated lettuce butts stored at 12 C Report for Horticulture Australia Limited Food Science Australia 51

58 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Effect of storage temperature on the browning of coating applied cut end of lettuce heads - II Visual score Figure 37 shows the colour score of lettuce butts treated with xanthan gam, acetic acid and CaCI2 stored at 4 C. At 4 C, the browning rate was very high in control samples during 0-4 day storage period. The browning rate was low between 4-18 days. A lower rate of browning was observed in treated samples but the initial rate of browning was lowest with the 0.5M Acetic acid treated samples. The initial rate of browning in samples treated with 0.5M Acetic acid M CaCI2 was similar to the 0.5M Acetic acid treated samples. At 4 C the highest rate of browning among treated samples was observed in 0.5M Acetic acid M CaCI % xanthan gum treated samples (Figure 37 and Table 16). Although the controls stored at 7 and 12 C followed the same trend as at 4 C, for the treated samples the trends were different (Figures 38 & 39). At 7 C the lowest browning rate was observed in samples treated with 0.5M Acetic acid M CaCI % xanthan gum, where as the highest rate was observed in samples treated with 0.5M Acetic acid. Similar behaviour was observed in samples stored at 12 C on day 2. When considering, the effect of different treatments at various temperatures, the control samples, showed a similar browning pattern and rate at 4 and 7 C. However, with the 0.5M Acetic acid or 0.5M Acetic acid M CaGI2 treated samples, browning rate was lower at 4 than at 7 C. Browning rate at 7 C was tower than at 12 C. Samples treated with 0.5M Acetic acid M CaCI % xanthan gum did not show as much difference in browning pattern between 4 and 7 C. Hue value The initial hue value of samples ranged between Samples stored at 4 C and treated with 0.5M acetic acid, had slightly higher hue values than the other treatments. At 7 C all treated samples had similar hue values that were higher than the hue values of control samples for 5-7 days at 7, and 12 days at 4 C. However, all treated samples and controls retained a hue value higher than 100 only for 2 days (Table 17). Appearance Treated lettuce samples had a drier appearance than the untreated controls during the storage period. In some samples treated with acetic acid M CaCI % xanthan gum, fungal colonies were observed at 12 days at 7 C. Report for Horticulture Australia Limited Food Science Australia

59 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETAiLES Table 16 - Summary of tie experimentalfindingson the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage temperatures 4 C, 7 C and 12 C, based on number of days colour score remained < 4 (marginal acceptability). Treatment Number of days at Colour score < C 7 C n a c Control MAA M AA CaCI MAA+ 0.5 CaC% % xanthan Table 17 - Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage temperatures 4 C, 7 C and 12 C, based on number of days Hue value remained > 100. Treatment Number of days at Hue value > C 7 C 12 C Control <2 <2 * 0.5 MAA <2 <2 * 0.5 M AA + <2 <2 * 0.5 CaCl M AA CaCI % xanthan * Not detected <2 <2 * Report for Horticulture Australia Limited Food Science Australia 53

60 IMPROVING THE QUALITY ANO SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 37 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at 4 C. Control 0.SMAA 0.5M AA + 0.5M CaCI Storage time (days) 1S MAA + 0.5M CaCI2+0.25% xanthan Figure 38 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at 7 C Control 0. MAA 0.5M AA + 0.5M CaCI Storage time (days) MAA + 0.5M CaCI2+0.25% xanthan Report for Horticulture Australia Limited Food Science Australia 54

61 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 39 - Colour score of lettuce butts treated with gum, acetic acid (AA) and CaCI2 stored at 12 C I o 6 ' Control 0.5MAA 0.5M AA + 0.5M CaCI2 0.5MAA + 0.5M CaCI2+0.25% xanthan o Storage time (days) Figure 40 - Changes in hue value of lettuce butts treated with acetic acid (AA), CaCI2 & xanthan gum and stored at 4 C 120 Control 100 <D _D ce > 80 d) 3 I Storage time (days) 0.5MAA 0.5M AA + 0.5M CaCI2 0.55MAA + 0.5M CaCI % xanthan Report for Horticulture Australia Limited Food Science Australia 55

62 improving THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT V1GETA1LES Table 18- Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage temperatures 4 C, 7 C and 12 C, based on number of days colour score remained < 4 (marginal acceptability). Treatment Number of days at colour score < 4 4*C 7*C 12 C Control 2 3 <2 : 0.5 M AA CaCI % xanthan 0.6 M AA <2 0.5 CaCI % xanthan 0.5 M AA M CaCi * 0.6MAA * 0.5 M CaCI 2 * Not detectec Table 19 - Summary of the experimental findings on the combined effect of acetic acid and food grade gums on the cut end of lettuce heads at storage temperatures 4 C, 7 C and 12 C, based on number of days Hue value remained > 100. Treatment Number of days at Hue value > C 7 C 12 C Control <2 2 <2 0.5 M AA <2 0.5 CaCI % xanthan 0.6MAA <2 0.5 CaCI % xanthan 0.5 M AA M CaCI * 0.6 M AA * 0.5 M CaCI 2 * Not detected Report for Horticulture Australia Limited Food Science Australia

63 IMF-ROVING THE QUALITY ANO SAFETY OF UNPROCESSID ANO FRESH-CUT ViGlTASLES Figure 41 - Changes in hue values of lettuce butts with acetic acid (AA), CaCI2 & xanthan gum and stored at 7 C " "- Control MAA 1 80 a> 3 I 60 ~*-0.5MAA + 0.5M CaCI Storage time (days) »^~0.55MAA + 0.5M CaCI % xanthan Figure 42 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at 4 C 8 - Confrol o u CO 3 o -*-0.5MAA + 0.5M CaCI2+0.25% xanthan 0.6M AA + 0.5M CaCI2+0.25% xanthan ^<-0.5MAA + 0.5M CaCI Storage time (days) *-0.6MAA + 0.5M CaCI2 Report for Horticulture Australia Limited Food Science Australia 57

64 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 43 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at 7 C 3 Control to» 4 o "o O 2 0.5MAA + 0.5M CaCI2+0.25% xanthan 0.6MAA + 0.5M CaCI2+0.25% xanthan 0.5MAA + 0.5M CaCI Storage time (days) M AA + 0.5M CaCI2 Figure 44 - Colour score of lettuce butts treated with xanthan gum, acetic acid (AA) and CaCI2 stored at 12 C I 6 o m o "o O Storage time (days) -n 10 Control MAA + 0.5M CaCI2+0.25% xanthan -** 0.6MAA + 0.5M CaCI2+0.25% xanthan Report for Horticulture Australia Limited Food Science Australia 58

65 JMPROVtNG THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 45 - Changes in hue value of lettuce butts treated with AA, CaCI2 and xanthan gum and stored at 4 C ) Control 0.5M AA + 0.5M CaCI2 0.6M AA + 0.5M CaCI Storage time (days) M AA + 0.5M CaCI % xanthan -0. MAA + 0.5MCaCI % xanthan AA: Acetic acid Figure 46 - Changes in hue value of lettuce butts treated with AA, CaCI2 and xanthan gum and stored at 7 C. Control CO 3 0.5M AA + 0.5M CaCI2 0.6M AA + 0.5M CaCI Storage time (days) M AA + 0.5M CaCI % xanthan 0.6MAA + 0.5M CaCI % xanthan AA: Acetic acid Report for Horticulture Australia Limited Food Science Australia 59

66 IMPROVING THE QUALITY ANO SAFITY OF UNPROCESSED ANO FRESM-CUT VEGETABLES Figure 47 - Changes in hue value of lettuce butts treated with AA, CaCI2 and xanthan gum and stored at 12 C. 120 Control I X MAA + 0.5M CaCI % xanthan 0.6M AA + 0.5M CaCI % xanthan 4 6 Storage time (days) 10 AA: Acetic acid Report for Horticulture Australia Limited Food Science Australia 60

67 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED ANP FRESH-CUT VEGETABLES Effect of storage temperature on browning prevention, coating applied on the cut end of lettuce heads -III The samples stored at 4 C and 12 C, were stored for todays at a constant temperature. Another set of samples were stored at 4 C for 7 days and then were transferred to 7 C for 3 days to simulate storage and handling at retail conditions. Visual score According to visual scores the control samples were acceptable for 2 days where as the treated samples stored at 4 C retained acceptability for 4 days (Figure 42 and Table 18). However, the effects of treatments were not very prominent when compared with the previous two experiments (Tables 14,16 and 18). In this experiment the colour score of treated samples increased above the marginal score of 4 after 4 days of storage. At 7 C and 12 C the same trend was observed but the treatments with xanthan gum had a slightly slower browning rate than the treatment without gum or the control (Figures 43 & 44). When studying the effect of temperature on different treatments, in control samples browning was faster at 12 C than 7 C and 4 C. Hue value Hue value changes in control sample followed a similar trend at 4 C and after moving samples from 4 to 7 C. At 12 C the hue value of controls remained unchanged during storage. However, the hue value of treated samples was higher than the control samples and hue values of treated samples remained constant when stored at 4 C (Figure 45, Table 19). A decrease in hue value was observed in all treated samples at both 7 C and 12 C but the hue values of treated samples were higher than that of the controls at 7 C. At 12 C a significant difference in hue value was not observed between the treated and the control at the end of the storage period. When considering the temperature effect on different treatments control samples were similar in hue value after 5 days storage at 4 C, 7 C and 12 C (Figures 45,46 and 47). The treatments were more effective at lower temperature in retaining the hue value closer to 100 (4 C than at 7 C or 12 C). At 12 C the effectiveness of treatment decreased with storage time. Appearance The treated samples were drier in appearance than the untreated control samples. Samples stored at 12 C had fungal colonies appearing on day 5. Fungi were observed on treated samples stored at 7 Cand 12 C on day 12 but not on the controls. In all three experiments conducted at 4 C, 7 C and 12 C, the browning rate was higher in control samples than in the treated samples. Browning rate was higher at temperatures greater than at 4 C. The hue value of samples changed from an initial value of >100 to <100 when the colour reached a score of 4 which is the marginal value but still acceptable. Application of acids led to the development of a slight yellow colour and dryness of the cut surface. However, the application of the treatment solution by pressing a sponge impregnated with treatment solution helped to prevent leaf damage caused by acid burn. Report for Horticulture Australia Limited Food Science Australia 61

68 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES In allttweeexperiments tie change in hue value was lower in samples treated with treatment solutions. In tie third experiment moving samples from 4 C to 7 C after S days did not make a tangibte difference in ftm browning score, but a difference in hue value was observed. The effect of treatment solutions on browning prevention varied with the 3 different experiments conducted on lettuce heads. The difference in raw material between the 3 experiments was also seen, through the pattern of browning in control samples. The browning rate of control samples was highest in the second experiment followed by the third and thefirstexperiments. Report for Horticulture Australia Limited Food Science Australia

69 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FR1SM-CUT VEGETABLES 5.3 Shredded lettuce The combined effect of heat and low ph on the quality of Shredded lettuce I Colour Score Figure 48 shows the colour score of shredded lettuce treated with heat at different ph levels. Colour scores increased with time reflecting pink colour development with storage time. The rate of pink colour development was slow in shredded lettuce treated with water at 55 C for 90 sec, tsunami at 55 C and acetic acid (ph 3.5) at 55 C. Lettuce treated with the above three treatments had acceptable quality for 8-10 days. The increase in colour score was very rapid with untreated control, which was unacceptable after 3 days (Table 20). Samples of shredded lettuce treated with acetic acid at ph 3 showed yellowish brown colour development due to acid burn. Hue Angle Figure 49 shows the hue values of shredded lettuce treated with heat at different ph levels. Hue angle is an objective measurement of colour. Hue angle between represents the characteristic light green colour of shredded lettuce. Change of colour towards 90 degrees indicates the yellowing of the green areas and pinking of the cut surfaces. Unwashed lettuce and lettuce treated with acetic acid (ph 3.5) at 55 C had the highest hue value at the beginning of the experiment. Lettuce pieces treated with acetic acid (ph 3.0) at 55 C was yellowing at the start of the experiment indicating acid burn of the shredded leaves at ph 3.0. The yellowish brown colour development was visible even at 0 days. Change in hue value was minimal in lettuce treated with acetic acid (ph 3.5) at 55 C followed by lettuce treated with water at 55 C and Tsunami (200 ppm) at 55 C. All other samples showed a rapid decrease in hue value with storage. Hue angle and visual scores indicated similar results while the changes in L value were difficult to explain. Microbiological assessments Figure 50 shows the effect of ph and heat treatment on mesophilic and psychrotrophic count at day 0. In general, mesophilic counts were higher than the psychrotrophic counts in all treatments. Initial mesophilic and psychrotrophic counts were lowest in lettuce treated with acetic acid (ph 3.0) at 55 C for 30 sec and 2 log cycles lower than unwashed and washing treatments given at 4 C. Heat was more effective on lowering the psychrotrophic counts than on the mesopnittc counts. The low initial counts in samples which received acid and heat combined treatments may have been due to the synergistic detrimental effects of heat and acids on microorganisms. Delaquis et al (1999) reported that washing shredded lettuce in 100 ^g/ml chlorine solution at 47 C for 3 min reduced the initial counts by 3 log cycles. In the present study, the initial counts of the Tsunami (55 C) treated shredded lettuce were lower than the initial counts of untreated samples, but the difference was less than 1 log cycle. Figure 51 shows the total mesophilic colony count of shredded lettuce treated with heat at different ph levels. During storage, the mesophilic counts of samples treated Report for Horticulture Australia Limited Food Science Australia 63

70 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES with combined treatments increased and were in the same range as the unwashed, 4 C water and 4 C acetic acid at ph 3.5 treated samples, after 7 days of storage. The increase of counts in heat-treated samples, during storage may have been due to less competition and selection of the organisms during heat treatment. Table 20: Summary of the experimental findings on the combined effect of acetic acid and heat on shredded lettuce at 4 C in polyethylene bags, based on number of days colour score remained < 4 and Hue value remained > 100 [4.2.1 (a)]. Treatment Number of days at colour score <4 Number of days at Hue value >114 Unwashed C Water 30 sec C Acetic acid (ph 3.0) 30 sec 3 <1 4 C Acetic acid (ph 3.5) 30 sec C Water 90 sec C Acetic acid (ph 3.0) 30 sec 2 <1 55 C Acetic acid (ph 3.5) 30 sec C Tsunami (20ppm) 30 sec C Tsunami (20ppm) 30 sec 4 1 Report for Horticulture Australia Limited Food Science Australia

71 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETAiLES Figure 48 - Colour Score of shredded lettuce treated with heat at different ph levels ~ Unwashed *-4 C Water 30 sec o CO k. 3 O o o o Storage time (days) C Acetic acid (ph 3.0) 30 sec : -4 C Acetic acid (ph 3.5) 30 sec *-55 C Water 90 sec ~55 C Acetic acid (ph 3.0) 30 sec - 55 C Acetic acid (ph 3.5) 30 sec "~55 C Tsunami (20ppm) 30 sec "* "4 C Tsunami (20ppm) 30 sec Figure 49 - Hue values of shredded lettuce treated with heat at different ph levels " Unwashed -4 C Water 30 sec 4 C Acetic acid (ph 3.0) 30 sec 4 C Acetic acid (ph 3.5) 30 sec 55 C Water 90 sec Storage time (days) 12 * 55 C Acetic acid (ph 3.0) 30 see C Acetic acid (ph 3.5) 30 sec 55 C Tsunami (20ppm) 30 sec *"~4 C Tsunami (20ppm) 30 sec Report for Horticulture Australia Limited Food Science Australia 65

72 IMPROVING THE QUALITY AND SAFITY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 50 - Effect of pm and heat treatment on mesophilic and psychrotrophic count at day 0 Unwashed 4 C Water 4 C 4 C 55 C Water 55 C Acetic acid Tsunami Acetic acid (ph 3.0) (POAA 20 (ph 3.0) ppm) Total mesophillic count Total psychrotrophic count 5S C tsunami (POAA 20 ppm) graph 44 Figure 51 - Total mesophilic colony count of shredded lettuce treated with heat at different ph levels * Unwashed I o U) o Storage time (days) 4 C Water 30 sec 4 C Acetic acid (ph 3.5) 30 sec 55 C Water 90 sec "*~~55 C Acetic acid (ph 3.5) 30 sec Report for Horticulture Australia Limited Food Science Australia 66

73 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Oxygen content Headspace oxygen content was comparatively low in lettuce pieces treated with hot water at 55 C. AH the other treatments had oxygen levels below 12 % on day 3 and decreased with storage time. Carbon dioxide content In general the carbon dioxide levels were stable. Headspace carbon dioxide content was high and ranged between 6-7% in lettuce pieces treated with water at 55 C and acetic acid (ph 3.5) at 55 C. Unwashed, 4 C water and 4 C acetic acid (ph 3.5) washed samples recorded a carbon dioxide level around 4-5% The combined effect of heat and low ph en the quality of shredded lettuce II In this experiment it was noted that the water bath temperature dropped from an initial 55 G to 45 C. There fore the treatment temperature of the shredded lettuce was considered as 45 C. Colour score Figure 53 shows the combined effect of ph and heat treatment on browning of shredded lettuce. The shredded lettuce treated with combined acid and heat treatment at ph 3.5 and 45 C retained a visual score at an acceptable level for 7 days. The rate of cobur change of the combined acid and heat-treated samples was also lower than for the samples that were not subjected to heat treatment. The time duration of dipping in ph 3.5 acid solution at 45 C did not have a significant effect on the visual scores of shredded lettuce. Hue angle Figure 52 shows the combined effect of ph and heat treatment on hue value of shredded lettuce. The hue value of shredded lettuce treated with acid at ph 3.5 and 45 C for 15 sec ranged between and was stable during the 10-day storage period. The samples treated with acid at ph3.5 and 45 C for 30 or 60 sec had hue values lower than the samples treated for 15 sec. In all the control samples hue value decreased with storage time. Microbiological Assessments Figure 54 shows the standard plate counts of shredded lettuce subjected to various dipping treatments. The standard plate count was lowest during the 7 day storage period in shredded lettuce treated with acid solution at ph 3.5 and 45 C for 60 sec. Higher counts were observed with untreated samples, water at 4 C for 30 sec and water at 45 C for 90 sec. Mesophilic Count Figure 55 shows the mesophilic counts of shredded lettuce subjected to various dipping treatments. Mesophilic counts were determined only on day 0 and day 4 of the experiment. Mesophilic counts followed a similar pattern as the psychrotrophic counts. The dipping treatment at ph3.5 and 45 C for 60 sec seemed to reduce the initial microbial load and to maintain it throughout the storage period. Report for Horticulture Australia Limited Food Science Australia 67

74 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Table 20: Summary of the experimental findings on the combined effect of acetic acid and heat on shredded lettuce at 4 C in polyethylene bags, based on number of days colour score remained < 4 and Hue value remained > 100 [4.2.1 (b)] Treatment Number of days at Hue value > 110 Control <1 Control water 55 C, 90 sec 7 Control water 4 C, 30 sec 4 ph 3.5, 4 C, 30 sec 4 ph 3.5,45 C, 15 sec 10 ph 3.5, 45 C, 30 sec 8 ph 3.5,45 C, 60 sec <1 Table 21: Summary of the experimental findings on the combined effect of acetic acid and heat on shredded lettuce at 4 C in polyethylene bags, based on number of days colour score remained < 4 and Hue value remained > 100 [4.2.1 (c)] Treatment Number of days at colour score «? 4 Control 2 Water 4 C, 30 sec 4 Water 45 C, 90 sec 9 ph 3.5, 4 C, 30 sec 4 ph3.5,45 C, 15 sec 10 ph 3.5, 45 C, 30 sec 10 ph 3.5, 45 C, 60 sec 10 Report for Horticulture Australia Limited Food Science Australia

75 IMPROVING THE QUALITY ANO SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 52 - Combined effect of ph and heat treatment on hue value of shredded lettuce control , 4 8 Storage time (days) 12 ctrl water 55 C, 90sec ctrl water 4 C, 30sec ph 3.5, 4 C, 30sec ~*K ph 3.5, 45 C,15sec - ph 3.5, 45 C, 30sec H ph 3.5, 45 C, 60sec Figure 53 - Combined effect of ph and heat treatment on browning of shredded lettuce control - water 4 C 30 sec water 45 C 90 sec ph C 30 sec -* ph C 15 sec 4 8 Storage time (days) 12 ph C 30 sec -I ph C 60 sec Report for Horticulture Australia Limited Food Science Australia

76 IMPROVING THl QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 54- Standard plate counts of shredded lettuce subjected to various dipping treatments 5* c >» c.o 8 O 8> Storage time (days) 8 -*- control water 4 C, 30 sec water 45 C, 90 sec ph 3.5, 4 C, 30 sec ph 3.5,45 C, 15 sec ph 3.5, 45 C, 30 sec ph 3.5, 45 C, 60 sec Figure 55 - Mesophilic counts of shredded lettuce subjected to various dipping treatments 0. m 8.00 i 1 o i Storage time (days) -*- "control water 4 C 30 sec water 45 C 90 sec ph C 30 sec ph C 15 sec ph C 30 sec -ph C 60 sec Report for Horticulture Australia Limited Food Science Australia

77 IMPROVING THE QUALITY AND SAFITY OF UNPROCESSED AND FRf SH-CUT VEGETABLES General appearance The crispiness of the lettuce samples decreased while the translucent appearance increased as the increase of heat treatment time was increased. Slight acid burn symptoms were observed in very young leaves subjected to 60-sec treatment time. Further experiments are needed with very close monitoring of the temperature and ph. 5.4 Broccoli Broccoli I - Effect of moisture retaining agents Preliminary studies on broccoli heads revealed that the cut end of the head undergoes softening due to loss of water. Broccoli florets also under go softening on the stem end becomes whitish during storage. Application of gums and calcium chloride was studied in order to reduce moisture loss and retain a firm texture. Treated samples and the control samples were similar in appearance and firmness Broccoli II - Effect of moisture retaining agents and adds Drying and softening of the stems were observed in control samples by day 3 which increased with storage time. Xanthan gum at ph 2.5 resulted in surface drying but was firm in texture. Xanthan gum and chitosan at ph 3.5 resulted in a slightly dry appearance but the stems were very firm in texture. Slight yellowing and white layer formation was observed in the control samples on the day 7 but this was not observed in samples treated with acid and gum combination. Report for Horticulture Australia Limited Food Science Australia 71

78 IMPROVING THi QUALITY AND SAFETY OF UNPROCESSEfJ AND FRESH-CUT VEGETABLES 6 OVERALL DISCUSSION AND CONCLUSIONS 6.1 Lettuce heads Acetic acid provided the highest protection against browning on the cut surface of lettuce cores. Application of acetic acid at ph 1.5 or 2.0 by blotting or dipping could inhibit pinking / browning in cut ends of whole lettuce head for 10 days, whereas the controls were pink after 2 or 3 days. It is reported that lowering the ph below 4 could inhibit browning of lettuce due to the inactivation of phenylalanine ammonia- lyase (PAL, EC ) that is responsible for phenolic metabolism in lettuce tissues (Tomas-Barberan, 1997). However, treatment with acetic acid led to dehydration and shrivelling of the treated area and outer leaves. Application of washing treatments after acid dips was evaluated to avoid the dehydration and shrivelling. Calcium hydroxide was used to improve the texture of treated lettuce tissues. It is reported that calcium treatment helps to retain texture of treated fruit and vegetable tissues (Dong et al 2000). Washing with water or calcium hydroxide lowered the effect of acid treatment and led to the browning of the cut surface. To delay browning using acid treatment, it is important to maintain the ph of treated area below ph 5 during storage. The combined effect of acid dips and heat was evaluated to enable the use of acids at a higher ph to minimise the drying effect. Dipping at 55 C for 30 sec in acetic acid at ph 2.5 helped to inhibit pink colour development on the cut end of lettuce heads. These samples retained acceptable colour for 14 days against the control of 2 days. However, application of heat and acid combined treatment at pilot scale was not very feasible on the commercial scale due to the generation of acid fume. The outer leaves were susceptible to damage during dipping treatments. The effect of gums and acid were evaluated to minimise dehydration and to overcome problems related to dehydration of treated areas and outer leaf damage caused by dipping treatments. Acid treatment at ph ( M) was able to delay the browning of the cut end of lettuce heads for 5-7 days when the control was pink at day 3, although the acid treated samples developed a yellow colour after 5 days. Calcium chloride and gums were used as moisture-retaining agents. The use of Calcium chloride was also helpful in further reducing the ph of acid solutions. Acid, calcium chloride and gum treated samples delayed brown colour development for 4-7 days where as the control was pink in <2 days. The treated samples had a slower rate of browning than the corresponding control samples. Browning rate was generally higher at 12 C than at 7 C and 4 C. Temperature control was a very important factor for the inhibition of browning in both coated and control samples. The combined use of gums at ph 2.5, as a dip was feasible at pilot scale. Considerable batch-to-batch variations were observed in the browning intensity in controls and response to the above treatments in terms of browning inhibition in treated samples. Application of the above browning inhibition treatment to lettuce heads extended the shelf life of bagged lettuce heads stored at 1 C - 4 C. The adoption of results of this study would help to extend the shelf life and reduce waste resulting from regular trimming at retail level. Extended shelf life would lead to an increase in the available transport time and allow the products to reach remote Australian and export markets. The overall outcomes of this project will be to improve profitability of the whole value Report for Horticulture Australia Limited Food Science Australia 72

79 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES chain: grower, processor and retailer. It is important to note that commercial scale trials should be conducted before commercialisation with known varieties to understand the variation of response in raw material and make recommendations. 6.2 Shredded lettuce Heat treatment at 55 C with acetic acid at ph 3.5 for 30 seconds or 20 ppm Tsunami for 30 seconds or water for 90 seconds was able to retard browning of the shredded lettuce packed in polyethylene bags and stored at 4 C for 10 days, compared to control of only 3 days. Tsunami is used as a sanitising treatment in the fresh cut industry. The microbiological counts of these samples were high and close to spoilage levels after 10-day storage but the samples did not show any sign of spoilage. The above treatments were effective in lowering the counts initially, but the counts increased with storage time. The initial selection of microorganisms due to heat treatment might have affected the final microbial counts. Dipping trials conducted with acetic acid solutions at ph 3.5 for seconds at 45 C showed stabilisation of the colour of treated shredded lettuce during a 10-day storage period at 4 C against the control of 2 days. The microbiological quality of the shredded lettuce was also maintained by treatment with ph 3.5 at 45 C for 60 sec. However, the treatment with heat imparted an undesirable translucent appearance and affected the texture of treated shredded lettuce, negatively. 6.3 Broccoli Heads The cut end of broccoli heads did not develop a brown colour due to enzymatic browning. The major limiting factor was the loss of moisture and softening or the toss of firm texture. Treatment of broccoli heads and florets with acid and gum combinations helped mainly to prevent the softening due to loss of moisture from the tissue. This treatment was also effective in reducing the whitish appearance developed during storage of broccoli florets. Report for Horticulture Australia Limited Food Science Australia 73

80 IMPROVING THE QUALITY AN SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES PART 2- IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES BY BIOPRESERVATION 3 INTRODUCTION: Minimally processed vegetable products and in particular leafy salad vegetables have high microbial loads of naturally occurring microflora that can limit the shelf-life of these products. The predominate spoilage microflora are pseudomonads and the food pathogen of greatest concern with this product category is Listeria monocytogenes due to its psychrotrophic growth properties, should it enter the final product due to a failure in hygienic processing or on-farm management practices. Minimally processed vegetables and fruits that are packaged and ready-to-eat have become a growing food category on the supermarket shelf. Due to absence of a no terminal microbial kill step in the minimal processing stage, the microbial quality and safety of the products is to a large extent dependent on good hygienic practices and effective temperature control throughout the processing and distribution chain. Washing of fresh produce with chilled chlorinated water is the only microbial reduction step currently available to improve the shelf-life, quality and safety of the product. However, a number of studies in Australia and overseas indicate that the maximum microbial reduction on the vegetable products as a result of chlorinated washing is not more than 2 log units, and the slightly lower initial microbial population after, washing will still increase rapidly over storage and reach up to 10 7 cfu/g by the time of consumption (Nguyen and Prunier 1989, Nguyen and Carlin 1994). More recent studies with peracetic acid sanitisers show comparable results with chlorine. Minimally processed vegetables products pose a particular risk with respect to psychrotrophic pathogens including Listeria monocytogenes, and psychrotrophic spoilage due to Pseudomonas spp. that can grow well at refrigeration temperature in the cold chain of distribution and storage. While no food-borne incidences have been reported with Aeromonas species and minimally processed salad products these bacteria are psychrotrophic, occur in a relatively high proportion of products and strains from water sources have been reported as pathogenic to humans. Should bacterial pathogens such as Listeria monocytogenes contaminate vegetables they are likely to survive the washing process and grow at storage abuse temperatures. Processors have expressed interest in additional food safety hurdles for minimally processed horticultural products that would also assist in increasing microbial shelflife. The effectiveness of lactic acid bacteria (LAB) as bioprotective cultures has been studied in meat products (Schillinger et al, 1991; Campanini et at, 1993; McMullen and Stiles, 1996 and Andersen 1995), fish products (Huss et al, 1995), and vegetable products (Choi and Beuchat, 1994). In particular, Vescovo et al 1995, 1996 and 1997 reported encouraging results with the application of selected strains of Lactobacillus casei, Lactobacillus plantarum, and Pediococcus spp. for biopreservation of ready-to-eat mixed salads consisting of endives, carrots and chicory. The competitive properties of the cultures rather than their fermentative characteristics are required for application to minimally processed vegetables. Also many LAB produce bacteriocins that have activity against Listeria species that may assist in their competition with these bacteria. Report for Horticulture Australia Limited Food Science Australia

81 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES The current project has investigated a number of food-grade antimicrobial compounds and competitive cultures for their effectiveness in controlling the growth of pseudomonads and Listeria spp. and the putative psychotrophic pathogen Aeromonas spp on lettuce and lettuce extract test systems. The effectiveness of using edible coatings as a delivery method for bioprotective cultures and agents was also evaluated. In addition provisional patent PR3862, Control of food-borne pathogens in horticultural produce has been lodged describing investigations not disclosed in this report. 4 METHODS: 4.1 Cultures and media. Lactobacillus alimentarius BJ-33 (FloraCarn L-2, Chr. Hansen A/S, Denmark) and Carnobacterium piscicola JG 126 (Jack et al 1996), were used as bioprotective cultures. Cultures used as challenge microflora for the competitive growth and inhibition trials included Listeria monocytogenes FSAW 2310, (Food Science Australia, Werribee culture collection) Listeria innocua ATCC 33090, E. coli ATCC 11775, Salmonella salford IMVS 171, Pseudomonas fluorescens ATCC 948 and Aeromaonas hydrophila ATCC Lactobacillus alimentarius was grown in MRS broth (Oxoid), whereas Carnobacterium piscicola was grown on MRS without sodium acetate. Listeria monocytogenes, Listeria innocua, E. coli, Salmonella salford, Pseudomonasfluorescens,and Aeromaonas hydrophila were cultivated in tryptone soya broth supplemented with yeast extract 0.6 % w/v. All cultures were grown aerobically at 30 C for 24 h. 4.2 Microbiological and ph analysis. Samples were diluted appropriately using 0.1% peptone water and viable counts enumerated (0.1 ml spread plate) for lactic acid bacteria on MRS Agar (Oxoid) 48h, 30 C anaerobically; Listeria monocytogenes and Listeria innocua on Listeria- Selective agar (Oxford formulation, Oxoid) 48h, 30 C; Aeromonas hydrophila and Pseudomonasfluorescensin GSP Aeromonas and Pseudo/nonas-Selective Agar (Merck formulation, Germany) 48h, 30 C. Pseudomonas spp. were enumerated on Pseudomonas-CFC Selective Agar (Oxoid) incubated at 30 C for 48h. S.Salford and E. coli were enumerated on Sa/mone//a-Selective Agar (XLD) and Cromocult Coliform Agar (Merck, Australia) respectively at 37 C for 24h. The measurement of ph was performed on samples removed during storage by using a Radiometer ph meter model PHM Research Model (Radiometer, Copenhagen, Denmark) 4.3 Evaluation of bioprotective L. alimentarius culture against pathogenic and spoilage bacteria in a lettuce extract test system. Fresh lettuce was obtained from a local supermarket and fresh juice was prepared from lettuce leaves processed with a domestic juicer (Breville, Australia). The extracted juice was centrifuged at 12,000 rpm for 15 min at 4 C and sterilized by Report for Horticulture Australia Limited Food Science Australia 75

82 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES vacuum filtration (SVF) using a pre-sterilized pleated capsule filter with a 0.2 urn pore size (Pall Gelman Laboratory, Australia). After filtration equal volumes of sterile distilled water was added to make 50 % v/v lettuce juice. Prior to use, sterility of the juice was checked by pour plating one ml of juice on plate count agar. Sterile lettuce juice was stored at 4 C. Freshly grown cultures of Listeria monocytogenes, Pseudomonas fluorescens, and Aeromonas hydrophila were diluted in 0.1% peptone water to achieve a suspension of approximately 5 log cfu/ml to be used as inoculum in lettuce juice test systems. A cell suspension of Lactobacillus alimentarius of approximately 7 log cfu/ml was similarly prepared. Two psychrotrophic pathogens, i.e Listeria monocytogenes FSAW 2310 and Aeromonas hydrophila ATCC 7966 and a psychrotrophic spoilage bacterium Pseudomonas fluorescens ATCC 948 were selected as micro-organisms for growth studies in the lettuce juice medium. Ten ml of lettuce juice was dispensed in 30 ml glass vials and inoculated with the Listeria or Aeromonas strains individually or in combination along with Lactobacillus alimentarius and incubated at 4 C for 7d. The level of inoculum was 4 log cfu/ml for Listeria monocytogenes, Pseudomonas fluorescens, and Aeromaonas hydrophila and 6 log cfu/ml for Lactobacillus alimentarius. Controls of these challenge micro-organisms on lettuce juice without added Lactobacillus alimentarius were also prepared and incubated at 4 C for 7d. This temperature was selected as an ideal temperature of storage in a cold chain. Lettuce juice test systems were analysed for bacterial populations on appropriate selective media at 0, 2, 5 and 7 d intervals of storage at 4 C as described above. One ml of sample was removed from the vial for each test system. Appropriate dilutions of each sample were spread plated (0.1 ml) onto selective media. 4.4 Evaluation of coating material and application method for shredded lettuce and carrot pieces. Iceberg lettuce was obtained from a local supermarket and 4-6 external leaves were removed and the whole head washed in running tap water. The lettuces were shredded into 1.5cm wide pieces with sharp stainless steel knives. Two preliminary studies were conducted to evaluate the coating properties of 5 gums and identify two suitable gum preparations for further studies. The selected coating formulations were, Xanthan gum (Keltrol, Germantown, NSW) g/l and g/ L; Alginate (Kelco, NSW) 5g/L and 10g/L, dissolved in de-ionised water at 20 C using an electric mixer. All coating solutions were stored at 4 C overnight for hydration. Shredded lettuce (200g) was immersed in 600 ml of gum solution for 1 min and drained using a mesh sieve. Drained lettuce pieces were placed in a perforated plastic bag 19.5 cm x 20.5 cm PD 961 (Cryovac, Australia) and heat-sealed. Triplicate samples per treatment were placed in a commercial spin drier (Angelo-Po, Italy) and de-watered at speed 2 (approximately 400 rpm) for 2 min or 4 min. The weight of the samples was recorded after de-watering and samples were re-packed in polyethylene bags (100g/bag) and heat-sealed. Samples were evaluated for weight gain after coating and calculated as a percentage of the original weight. Microbiological tests for total viable count were determined at day 3 and 7, after storage at 4 C. Report for Horticulture Australia Limited Food Science Australia

83 IMPROVING THE QUALITY AN SAFETY OF UNPROCESSED AND FRESH-CUT VEGETASLES 4.5 Evaluation of bioprotective Lactobacillus alimentarius against indigenous spoilage pseudomonads on shredded iceberg lettuce leaves. The growth of Lactobacillus alimentarius BJ-33 (FloraCam L-2, Chr. Hansen A/S, Denmark) was tested, with and without the addition of an edible coating. Fresh, iceberg lettuce was obtained from a local vegetable supplier and the outer leaves were removed and lettuce heads washed with ice-cold tap water before shredding. A freshly grown culture of L alimentarius was harvested by centrifuging at 3,840 x g, at 4 C for 20 minutes. The cell pellets were resuspended in sterilised distilled water to a concentration of approximately 8-log cfu/ml and suspensions were used to prepare immersion solutions. The concentrations of L alimentarius in immersion solutions were approximately 5-log cfu/ml. There was no challenge inoculation of Pseudomonas sp. but analyses were performed to determine the indigenous population of pseudomonads. Shredded lettuce leaves were inoculated by the immersion method and for each two kilograms of lettuce, six litres of immersion solution was used. Immersion solutions contained either a protective culture with or without edible coating. The coating solution contained Xanthan gum (0.125 g/l of coating solution) and lettuce leaves were immersed for five minutes with continual mixing. The lettuce was centrifuged at rpm, to remove excess coating solution (using kitchen salad spinners). Lettuce was packed in polyethylene lettuce bags PD 961 (Cryovac, Australia), weighing about 100 grams per bag, and stored at 4 C for 10 days. At time intervals of 0, 3, 7 and 10 days, samples were analysed for microbial growth. At every sampling time, analyses were performed in triplicate for each treatment. Lettuce samples were homogenised (10% w/v, 25 grams of lettuce sample and 225 ml of 0.1% sterile peptone water) in a Stomacher (Seward, UK) for 2 minutes. Appropriate dilutions of each sample were spread plated onto selective media to enumerate inoculated lactic acid bacteria in MRS Agar incubated at 30 C for 48h, under anaerobic conditions. Pseudomonas spp. were enumerated on Pseudomonas-CFC Selective Agar (Oxoid) incubated at 30 C for 48h. 4.6 Evaluation of bioprotective Lactobacillus alimentarius and Carnobacterium piscicola against Listeria innocua and indigenous Pseudomonads on lettuce leaves. The attachment and subsequent growth of Lactobacillus alimentarius and Carnobacterium piscicola was tested on lettuce leaves without added coating. Fresh, iceberg lettuce was obtained from a local vegetable supplier. Before shredding, the outer leaves were removed and lettuce heads were washed with ice-cold tap water. Immersion solutions containing L alimentarius and C. piscicola were prepared as described in above but without the addition of an edible coating. Lettuce was packed in polyethylene lettuce bags PD 961 (Cryovac, Australia), using about 100 grams per bag, and stored at 7 C for 14 days. At time intervals of day 0, 6, 7 and 14, samples were analysed for microbial growth. At every sampling time, analyses were performed in triplicate for each treatment. There was no challenge inoculation of Pseudomonas sp. but analyses were performed to determine the indigenous population of pseudomonads on lettuce leaves. Report for Horticulture Australia Limited Food Science Australia

84 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Lettuce samples were homogenised and tactic acid bacteria, Pseudomonads and Listeria spp. were enumerated as described above. 4.7 Evaluation of bioproteetive agents Polyphenon 70S, pediocin and nisin on carrot pieces. Commercially available polyphenol tea extract, Polyphenon 70S ( Mitsui Norin Co. Ltd., Japan), pediocin (ALTA 2341 a bacteriocin preparation produced by Pediococcus acidilactici, Quest International, USA) and nisin (as Nisapilin, Aplin & Barrett, UK) were tested with and without coating against an inoculated strain of L innocua (as a non-pathogenic surrogate for L monocytogenes), E. coli, Salmonella salford, Aeromonas hydrophila and against indigenous Pseudomonads on carrot pieces. Fresh carrots were obtained from a local vegetable supplier, and were washed with ice-cold tap water before and after peeling. Carrot pieces (1cm 2 and 10cm long) were prepared using a Hobart vegetable slicer and dried in air for 30 min (spreading pieces out in a laminar flow cabinet). Cells of L innocua, E. coli, Salmonella salford and Aeromonas hydrophila were harvested as described previously. The harvested cell pellets were resuspended and serial diluted in sterile distilled water to achieve a cell concentration of 10 or 10 s cfu/ml for challenge experiments. There was no challenge inoculation of Pseudomonas sp. but analyses were performed to determine the indigenous population of pseudomonads. The carrot pieces were challenged with L innocua, E. coli, Salmonella salford and Aeromonas hydrophila by immersing the carrot pieces in the celt suspension (1-kg product in 1.5 litre cell suspension) for 5 min with continuous stirring. The carrot pieces were dried under sterile conditions by holding vertically in test tube racks in a biohazard cabinet for 15 min to remove excessive culture suspension. Subsequently, the challenged carrot pieces were treated with a coating solution (10 g/l xanthan gum in distilled water) with or without Polyphenon 70S (1g per litre of coating solution), pediocin (1% w/v of coating solution) and nisin (250 ng/litre of coating solution) by immersing the carrots in the coating solution (1 kg product in 1.5 litre coating solution) for 5 min followed by draining to remove excessive coating solution in test tube racks in a bio-hazard cabinet for 30 min. The carrot pieces were then packaged in polyethylene bags (PD961, Cryovac, Australia) at 100 g per bag and stored at 4 C to 8 C for 7 to 10 days. Carrot samples were homogenised (10% w/v, 25 grams of sample and 225 ml of 0.1% sterile peptone water) in a Stomacher for 2 minutes. Appropriate dilutions of each sample were spread (0.1 ml) or pour (1mL) plated onto selective media to enumerate lactic acid bacteria, Listeria, Salmonella, E. coli, Aeromonas and Pseudomonas spp. In addition yeasts and moulds were enumerated on Oxytetracyctine Glucose Yeast Extract Agar (Oxoid) incubated at 25 C for 5 days. At time intervals of day 0,1, and 7, samples were analysed for microbial growth and surface ph measurements. At every sampling time, analyses were performed in triplicate for each treatment of inoculation. Report for Horticulture Australia Limited Food Science Australia

85 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES At time intervals of day 0,1 and 7, triplicate sample bags for each treatment were removed for determination of viable count of presumptive Listeria, Aeromonas hydmphila and Pseudomonas spp. on L/ster/a-Selective Agar, and GSP Agar (Merck, Australia) respectively. Whereas viable count of Salmonella and E. coli was determined on Sa/mone//a-selective agar (XLD) and Cromocult Coliform Agar (Merck, Australia) respectively at time intervals of day 0,1,7 and 10. Report for Horticulture Australia Limited Food Science Australia

86 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES 5 RESULTS AND DISCUSSION: 5.1 Effect of L alimentarius on L monocytogenes, A. hydrophila and P. fluorescens in lettuce extract system. In 50% lettuce juice, L alimentarius grew approximately 2 log units over 7 days in the presence of other cultures (Fig 1) Aeromonas hydrophila and Pseudomonas fluorescens increased by about 3 log cfu/ml after 5 d regardless of the absence or presence of L alimentarius (Figs 2-3). Listeria monocytogenes showed a marked reduction in the presence of both Pseudomonas and Aeromonas at 7 days (Fig 4). The sharp decrease in viable count at day 7 corresponded with a decrease in ph values ranging from 6.06 to 4.46 (Figs 5-8). While, L alimentarius inhibited all added cultures L monocytogenes was effected the least most likely due to a higher resistance to low ph than either the Pseudomonas or Aeromonas cultures. 5.2 Effect of edible coating on delivery of bacterial cultures to lettuce leaves. Application of gum coatings helps to retain more liquid in the product. When compared with the control, both gum solutions had a high level of liquid retention after de-watering (Table 1). However de-watering for 4 min reduces the amount of liquid retention on all coatings. Application of coatings appeared to cause a somewhat lower total viable count on lettuce leaves than the control samples without added coating (Table 2). The application of L alimentarius in coating resulted in a somewhat higher LAB viable count adhering to the surface of lettuce leaves compared with the addition of culture without coating (Fig. 9). No Listeria spp. were found in un-inoculated samples. The population of indigenous pseudomonads on lettuce was not substantially inhibited with the L. alimentarius. The application of coatings did not result in the reduction of pseudomonads count on lettuce leaves (Fig. 10). In general coating do not seen to have marked adverse effects on growth of added cultures or indigenous microflora and there appears to be no great advantage in adding cultures with coatings for the purpose of enhancing competitive growth properties. Report for Horticulture Australia Limited Food Science Australia

87 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES "L alimentarius + L. monocytogenes L alimentarius + A. hydrophila JP "L alimentarius + P. fluorescens Storagetime (days) 6 L alimentarius + L monocytogenes + A. hydrophila + P. fluorescens Figure 1. Growth of Lactobacillus alimentarius in presence of target microorganisms in lettuce juice at 4 C. 'P. fluorescens (control) "L. alimentarius + P. fluorescens "L alimentarius + P.fluorescens + A. hydrophila + L. monocytogenes Storage time (days) 6 Figure 2. Growth of Pseudomonas fluorescens in lettuce juice at 4 C. Report for Horticulture Australia Limited Food Science Australia 81

88 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES 10 I 9 8 "3 7 s W 6 5 ; 4i Tr \ 3 1 ( ) 1 Storagetime (days) 5 S 7 + A. hydrophita (control) " L alimentarius + A. hydrophila * L alimentarius + A. hydrophila + P. fluorescens + L monocytogenes Figure 3. Growth of Aeromonas hydrophila in lettuce }uice at 4 C. "L monocytogenes (control) "L alimentarius + L. monocytogenes ~L alimentarius + L monocytogenes + A. hydrophila + P. fluorescens Storage time (days) 6 Figure 4. Growth of Listeria monocytogenes in lettuce juice at 4 C. Report for Horticulture Australia Limited Food Science Australia 82

89 IMPROVING THE QUALITY AND SAFiTY OF UNPROCESSED AND FRESH-CUT VEGETABLES "L alimentarius + L monocytogenes l. alimentarius + A. hydrophila I a "L. alimentarius + P Storage time (days) 6 L alimentarius + L monocytogenes + A. hydrophila + P. fluorescens Figure 5. ph change during the growth of Lactobacillus alimentarius in presence of target microorganisms in lettuce at 4 C. - P. fluorescens (control) a L alimentarius + P. fluorescens - L alimentarius * P.fluorescens + A. hydrophila + L monocytogenes Storage time (days) Figure 6. ph change during the growth of Pseudomonas fluorescence in lettuce juice at4 C. Report for Horticulture Australia Limited Food Science Australia 83

90 IMPROVtNG THE QUALITY ANO SAFETY OF UNPROCESSED AND FRISH-CUT VEGETABLES 7 a. 6 ' 5 t\ i I i i i i Storage time (days) A hydrophila (control) L. alimentarius + A hydrophila A L. alimentarius + A hydrophila + P. fluorescens + L monocytogenes Figure 7. ph change during the growth of Aeromonas hydrophila in lettuce juice at 4 C. - L. monocytogenes (control) Q. - L alimentarius + L. monocytogenes - L alimentarius * L monocytogenes + A hydrophila + P. fluorescens Storage time (days) Figure 8. ph change during the growth of Listeria monocytogenes in lettuce juice at 4 C. Report for Horticulture Australia Limited Food Science Australia 84

91 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VIGETA1LES Table 1. Percentage weight gains on shredded lettuce after coating. Coating material De-watering time (min) 2min 4 min Control Xanthan gum Xanthan gum Alginate Alginate Table 2. Total plate count on coated and shredded lettuce. De-watering time (min) Coating Total plate count (cfu/g) DayO Day 5 Control 1.1 x10 B 3.5x10' Xanthan gum x x10' 2 Xanthan gum x x10 B Alginate x x10 Alginate x x 10 e Control 9.0 x x10' Xanthan gum x x10 e 4 Xanthan gum x x10 Alginate x x10 Alginate x x10 Report for Horticulture Australia Limited Food Science Australia 85

92 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES -Control Coating L.alimentarius ~L. alimentarius Time (days) 10 Figure 9. Growth of Lactobacillus alimentarius n lettuce leaves at 4 C. 9 -Control 'Coating -Coating+ L. alimentarius L. alimentarius 3 7 Time (days) 10 Figure 10. Growth of pseudomonads on lettuce leaves at 4 C. Report for Horticulture Australia Limited Food Science Australia 86

93 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES 5.3 Effect of bioprotective cultures on Listeria innocua and Pseudomonas spp. on lettuce leaves The count of lactic acid bacteria (LAB) on lettuce leaves was much higher in samples inoculated with L. alimentarius than in un-inoculated samples at time zero (Fig 11). However, the difference in the count was reduced as the lactic acid bacteria indigenous population (either LAB or Enterobacteriaceae) grew well in un-inoculated samples, reaching a count of 7-8 log cfu/g on day 7. Co-inoculation with L alimentarius on lettuce leaves resulted in a reduction of 2-log cfu/g in viable count of inoculated L. innocua compared with the sample without co-inoculation of L alimentarius (Fig. 12). No Listeria spp. were found in un-inoculated samples. The population of indigenous pseudomonads on lettuce was not substantially effected by the inoculated L alimentarius, until day 14 (Fig. 13). The L alimentarius demonstrated a notable and undesirable fermentative effect (Fig. 14) consistent with observations in lettuce juice test systems. The inoculation of lettuce leaves with the bacteriocin producing culture of C. piscicola resulted in a high count of approximately log 6 cfu/g on lettuce leaves (Fig 15). In uninoculated samples, the population of either LAB or Enterobacteriaceae reached a count of log 8 cfu/g at day 14. The inoculation with C. piscicola did not have any competitive effect on the population of indigenous pseudomonads on lettuce leaves (Fig. 16). In contrast samples co-inoculated with the protective culture showed a 3- log reduction in the count of L innocua after 14 days (Fig. 17). In contrast to the observations with L alimentarius, there was only minor ph decreases observed with C. piscicola (Fig 18). Final pn values on day 14 samples showed a slight decrease ranging from These results indicate that the inhibitory effect of C. piscicola on the growth of challenged L. innocua on lettuce leaves may have been assisted by production of the bacteriocin, piscicolin by C. piscicola. The drop in ph was implicated in the L innocua reduction. In the current study the culture Lb. alimentarius affected the ph of the product to a larger extent than that reported by Andersen (1995). This could be due to the difference in nature of the products tested, lettuce compared with meat. On lettuce leaves, protective cultures need to colonise on the product, to compete with other microorganisms for nutrients to grow. Any inhibition caused by protective cultures is applicable only to microorganisms colonised in adjacent areas. It is not certain how effective the inoculation was, in terms of density and homogeneity of distribution of the protective cultures on the leaves on each occasion. Ineffective inoculation could possibly limit the inhibitory effect of the protective cultures on lettuce leaves. Apart from the inoculated protective cultures, there are other lactic acid bacteria strains present in the product, many of them may be responsible for spoilage, and some may have protective effects. Report for Horticulture Australia Limited Food Science Australia 87

94 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 11. Growth of lactic acid bacteria on lettuce leaves at 7 C with Lactobacillus alimentarius. (Legends: Control = uninoculated, Lis = inoculated with L. innocua, Lb = inoculated with L. alimentarius BJ-33, Lis+Lb = inoculated with L. alimentarius BJ-33 plus L. innocua) Figure 12. Growth of Listeria innocua on lettuce leaves at 7 C under the influence of Lactobacillus alimentarius. (Legends: Lis = inoculated with L. innocua, Lis+Lb = inoculated with L alimentarius BJ-33 plus L innocua) Report for Horticulture Australia Limited Food Science Australia 88

95 IMPROVING THE QUALITY AND SAFfTY OF UNPROCESSED AND FRESH-CUT VEGETABLES Figure 13. Growth of indigenous pseudomonads on lettuce leaves at 7 under the influence of Lactobacillus alimentarius. (Legends: Control = un-inoculated, Lis = inoculated with L. innocua, Lb = inoculated with L. alimentarius BJ-33, Lis+Lb = inoculated with L alimentarius BJ-33 plus L. innocua) - -Control -B-Lte - -Lb -«-LisHl» Figure 14. ph changes in lettuce leaves samples with Lactobacillus alimentarius. (Legends: Control = un-inoculated, Lis s inoculated with L innocua, Lb = inoculated with L alimentarius BJ-33, Lis+Lb = inoculated with L. alimentarius BJ-33 plus L innocua) Report for Horticulture Australia Limited Food Science Australia

96 IMPROVING THE QUALITY AMD SAFETY OF UNPROCESSED AND FRESH-CUT VIGITAiLES Figure 15. Growth of lactic acid bacteria on lettuce leaves at 7 s C with Carnobacterium piscicola. (Legends: Control un-inoculated, Lis inoculated with L innocua, JG = inoculated with C. piscicola, JG+Lis = inoculated with C. piscicola plus L innocua) Figure 16. Growth of indigenous Pseudomonads. on lettuce leaves at 7 C under the influence of Carnobacterium. Piscicola. (Legends: Control = un-inoculated, Lis = inoculated with L innocua, JG = inoculated with C. piscicola, JG+Lis = inoculated with C. piscicola plus L innocua) Report for Horticulture Australia Limited Food Science Australia

97 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES 10 04,,,,,, Time (day) " tit" LjS ^H"J f'ljs Figure 17. Growth f Listeria innocua on lettuce leaves at 7 C under the influence of Carnobacterium. Piscicola. (Legends: Lis inoculated with L innocua, JG+Lis * inoculated with C. piscicola plus L. innocua) 2 4,,,,,, Tfcne(day) [-*-Control -B-Us -*-JG -B-J&HJsl Figure 18. ph changes in lettuce leaves samples with Carnobacterium piscicola. (Legends: Control = un-inoculated, Lis = inoculated with L. innocua, JG = inoculated with C. piscicola, JG+Lis = inoculated with C. piscicola plus L innocua) Report for Horticulture Australia Limited Food Science Australia 91

98 IMPROVING THE QUALITY AN SAFETY OF UNPROCESSED AND FRESH-CUT VIGETAILES 5.4 Effect of bioprotective agents Polyphenon 70S, pediocin and nisin on food pathogens and spoilage bacteria on carrot pieces with edible coatings. To investigate the effects of bioprotective agents on Listeria innocua (as a nonpathogenic surrogate for L monocytogenes) inoculated onto carrots, Polyphenon 70S (a tea polyphenol extract), pediocin and nisin (bacteriocin preparations from LAB) were added to the coating and packed in barrier film and stored at 4 C for 7 to 10 days. Polyphenon 70s had little if any inhibitory effect on any of the cultures tested on carrot pieces under the conditions used (Figs 19-23). The initial Listeria innocua load on carrot pieces was 10 3 cfu/g and a rapid decline in viable count was observed in both samples coated with pediocin and nisin. The concentration of pediocin (1% w/v in coating solution) and nisin (250 ug/litre of coating solution) was found to be sufficient to kill added Listeria cells immediately and no recovery of viable count was detected at 7 days (Fig 24). A slight increase in Listeria innocua population was observed in controls after 7 days and no Listeria spp. was found in un-inoculated samples. As would be expected, the indigenous pseudomonad viable count remained unaffected in the presence of pediocin and nisin treatments and the count rose from 3-log cfu/g to 5-log cfu/g in 7 days (Fig. 25). Yeast moulds and lactic acid bacteria are the main spoilage microflora of minimally processed carrots but viable counts for these microorganisms was maintained at < 100 cfu/g in all the sample treatments throughout the trial period. The pediocin and nisin coated samples of carrot pieces showed a minor decrease in ph compared with untreated controls (Fig. 26). In the trial on carrot pieces, pediocin and nisin in coatings both demonstrated inhibitory activity against Listeria innocua, but not on naturally occurring Gram-negative pseudomonads. Both these bioprotective agents have been earlier reported to produce an inhibitory effect on Listeria spp and some of the lactic acid bacteria (Berry et al. 1991, Schiliinger et. al. 1996, Jack et al and Coventry et al. 1995) in meat products. Torriani et. al also reported that the addition of 3% culture permeate of Lactobacillus, casei to mixed salads reduced the total mesophilic bacteria counts from 6 to 1 log unit and suppressed conforms, enterococci and Aeromonas hydrophila. Application of edible coatings with the inclusion of anti-microbial bioprotective agents could reduce the opportunity for growth of Listeria sp. The use of coatings in specific products applications to control physiological effects on products may also be used to include anti-microbial agents that provide an additional barrier to the growth of pathogens. Report for Horticulture Australia Limited Food Science Australia

99 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES -Control uncoated Carrot pieces + L. innocua -Carrot pieces + Polyphenon + L innocua o Time (days) 6 3 Figure 19. Effect of Pofyphenon 70S on Listeria innocua inoculated on carrot pieces at 4 C. 7 I 6 3 -J* Z o 4 c 3 0 o o "Control uncoated "Carrot pieces + A. hydrophila. "Carrot pieces* Polyphenon + A. hydrophila 0« Time (days) 8 Figure 20. Effect of Polyphenon 70S on Aeromonas hydrophila inoculated on carrot pieces at 4 C. Report for Horticulture Australia Limited Food Science Australia 93

100 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES -Gofiteal uncoated "Carrot pieces + A. hydrophila "Carrot pieces + Polyphenon + A. hydrophila Time (days) 6 S Figure 21. Effect of Polyphenon 70S on indigenous Pseudomonads n carrot pieces at 4 C. Report for Horticulture Australia Limited Food Science Australia 94

101 IMPROVING THI QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VlGETAiLiS 8 Lcoli 6.C0II + Potyphenon 4 6 Time (days) Figure 22. Effect of Polyphenon 70S on carrot pieces inoculated with E. coli at 8 C. 5 ^ I 0 J 3 I Y,Li^ rr^z^^+- ~** S. satford ± S. satford + Polyphenon 2 i i i i i ( ) Time (days) Figure 23. Effect of Polyphenon 70S on carrot pieces inoculated with Salmonella salford at 8 C. Report for Horticulture Australia Limited Food Science Australia 95

102 improvtng THE QUALITY AND SAFITY OF UNPROCESSED AND FRESH-CUT VEGETABLES "Control ' L. innocua + Coating L. innocua + Pediocin + Coating L. innocua + Nisin + Coating 0 Time (days) Figure 24. Effect of Pediocin and Nisin on Listeria innocua inoculated on carrot pieces at 4 C. Control ~ "" Coating * L. innocua + Coating X L. innocua + Pediocin + Coating * L. innocua + Nisin + Coating 0 Time.t«(days) 6 8 Figure 25. Effect of Pediocin and Nisin on indigenous Pseudomonads on carrot pieces at 4 C. Report for Horticulture Australia Limited Food Science Australia 96

103 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRISH-CUT VEGETABLES Control Coating X a * L. innocua + Coating X L. innocua + Pediocin + Coating "** L. innocua + Nisin + Coating Time (days) Figure 26. ph changes in carrot pieces at 4 C. Report for Horticulture Australia Limitecl Food Science Australia 97

104 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES 7 TECHNOLOGY TRANSFER 7.1 Technology Transfer - Part 1 (Improving the quality and safety of unprocessed and fresh-cut vegetables by modifying agents) Results on prevention of pink discolouration on cut end of lettuce heads were presented in the form of a poster at an industry field day (Grow for Profit) organised by Queensland Fruit and Vegetable Growers Association in As a result of the above presentation two industry parties have shown interest in conducting commercial scale trials on the use of a coating on the cut surface of whole lettuce heads. 1. A commercial exporter of whole lettuce heads to Asia from South Australia and 2. Dr. Denise Phillip in Dept of Agriculture Western Australia, who has contacts with commercial scale exporters of lettuce heads to Asia. 7.2 Technology Transfer - Part 2 (Improving the quality and safety of unprocessed and fresh-cut vegetables by biopreservation) The results of the most promising application of a food-grade antimicrobial agent for minimally processed horticultural products have been lodged as a provisional patent (PR 3 62). One overseas company and one Australian company as potential manufacturers of the antimicrobial agent and one major Australian processor of minimally processed horticultural products have been presented with a copy of the provisional patent under non-disclosure agreements. Initial interest has been expressed by some parties and further approaches will be pursued in an attempt to secure the necessary financial contribution from appropriate companies to undertake a commercial evaluation and associated industry trials. Report for Horticulture Australia Limited Food Science Australia

105 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES 8 RECOMMENDATIONS The key out comes and recommendations of the part 1 of this report: Key out comes: 1. Conduct commercial scale coating trials on whole lettuce heads under commercial scale harvesting and cooling conditions. 2. Browning prevention of shredded lettuce needs to be studied further in relation to the effects of heat treatment ontextureand appearance. 3. Dehydration of cut surfaces of broccoli heads and florets could be minimised by application of the coating developed in this study. Recommendations: It is recommended to conduct collaborative commercial scale trials between Food Science Australia and interested commercial parlies or research groups. It is recommended to conduct work with chemical companies on preprepared absorbent pads for coating the cut end of lettuce heads in commercial scale The key out comes of the part 2: Of the bioprotective agents and cultures investigated those described in Provisional patent application PR 3862 and not described in this report show the greatest potential for control of food-borne pathogens on minimally processed vegetable products. Future efforts for research into bioprotective agents should be directed at further developing and commercialising the findings reported in PR ACKNOWLEDGEMENTS We wish to acknowledge the Victorian Department of Natural Resources, Vegetable Industry Levy and Horticulture Australia for providingfinancialsupport for this project. We also wish to thank especially Dr Vic Reyes for his contribution to this project, technical assistance given by Ms Cindy Tran and administrative support given by Mr. Ian Gould. A part of this project was partially conducted as a study project by Ms Thuy Nguyen from the Royal Melbourne Institute of Technology, Melbourne. We wish to thank, Ms Daphne Piovesan of Food Science Australia, Werribee and Mr Steve Skopillanos of Keilor Valley Gardens Pty Ltd, Keitor, Vic 3036, for organising raw material supply. Report for Horticulture Australia Limited Food Science Australia

106 IMPROVING THE QUALITY AND SAFETY OF UNPROCESSED AND FRESH-CUT VEGETABLES Finally, we wish to thank members of the Food Packaging and Coating, and Food Safety and Quality sections of Food Science Australia, Werribee for their contribution during this project. Report for Horticulture Australia Limited Food Science Australia 100

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