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1 Effective sanitation for the fishing industry: Using bacteriological assessment to optimise sanitiser type for processing equipment and finfish product Prof T. V. Riley, Rachel Tonkin, Steven Munyard Project No

2 Fisheries Research and Development Corporation, PathWest Laboratory Medicine WA, and The University of Western Australia 27. ISBN Effective sanitation for the fishing industry: Using bacteriological assessment to optimise sanitiser type for processing equipment and finfish product Professor TV Riley Department of Microbiology Queen Elizabeth Medical Centre Nedlands WA 69 Telephone: Fax: triley@cyllene.uwa.edu.au Project No This work is copyright. Except as permitted under the Copyright Act 1968 (Cth), no part of this publication may be reproduced by any process, electronic or otherwise, without the specific written permission of the copyright owners. Neither may information be stored electronically in any form whatsoever without such permission. The Fisheries Research and Development Corporation plans, invests in and manages fisheries research and development throughout Australia. It is a statutory authority within the portfolio of the federal Minister for Agriculture, Fisheries and Forestry, jointly funded by the Australian Government and the fishing industry.

3 TABLE OF CONTENTS OBJECTIVES:...2 NON TECHNICAL SUMMARY:...2 OUTCOMES ACHIEVED TO DATE...2 KEYWORDS...3 ACKNOWLEDGMENTS...4 BACKGROUND...4 Cleaning and sanitation in the seafood industry...4 Compounds used in this study...6 NEEDS...7 OBJECTIVES...7 METHODS...7 1) Sanitisers used...7 2) Microbiological analysis...7 3) Quality Index Determination...8 4) Experimental Design...9 RESULTS Efficacy of sanitisers on processing equipment Protocol Efficacy of sanitisers on processing equipment Protocol Use of sanitisers on fresh product to increase shelf life Effect of sanitisers on Herring The effect of adding sanitisers to refrigerated sea water on the shelf life of Herring The effect of adding sanitisers to ice on the shelf life of Herring Effect of sanitisers on the spoilage of Tailor Effect of sanitisers on the spoilage of Spangled Emperor DISCUSSION Use of Sanitisers on Seafood Processing Equipment Use of Sanitisers on Raw Product to Improve Quality - Herring Addition of Sanitisers to Refrigerated Sea Water and Ice Impact on Herring Quality Use of Sanitisers on Raw Product to Improve Quality Tailor and Spangled Emperor BENEFITS FURTHER DEVELOPMENT PLANNED OUTCOMES CONCLUSIONS REFERENCES: APPENDIX 1: Intellectual Property Issues APPENDIX 2: Staff APPENDIX 3: Herring Quality Index... 5 APPENDIX 4: Tailor Quality Index Scheme APPENDIX 5: Spangled Emperor Quality Index Scheme

4 SIDF Effective sanitation for the fishing industry: Using bacteriological assessment to optimise sanitiser type for processing equipment and finfish product PRINCIPAL INVESTIGATOR: Professor Thomas V Riley ADDRESS: Microbiology & Immunology The University of Western Australia and Division of Microbiology & Infectious Diseases PathWest Laboratory Medicine Queen Elizabeth II Medical Centre Nedlands 69 Western Australia Tel: Fax: OBJECTIVES: 1. To assess the effectiveness of sanitising agents on fishing industry equipment and product. 2. To determine if the shelf-life (as measured by the quality index) of selected finfish species can be extended with the use of sanitisers. 3. To extend the results of 1 and 2 to the fishing industry. NON TECHNICAL SUMMARY: OUTCOMES ACHIEVED TO DATE 1. The effectiveness of three sanitisers used in the seafood industry was assessed. All reduced bacterial levels on fish processing equipment. 2. The impact of the preparation of the three sanitisers in artificial seawater was assessed and found to be negligible. 3. Evidence that the soaking of whole finfish in sanitisers prior to refrigeration can increase shelf life. 4. Evidence that the storage of whole finfish in ice containing sanitiser increases shelf life and reduces overall bacterial numbers. Under the FSANZ Australian Primary Production and Processing Standard seafood businesses are required to comply with Standards and of the Food Standards code (FSANZ, 26). These standards relate to cleaning, sanitising and maintenance of processing areas. The application of sanitisers as part of the cleaning process helps eliminate bacteria from processing surfaces, reducing the risk of product cross contamination with pathogenic or spoilage microorganisms. Little information exists regarding the application of sanitisers in the seafood industry. In a previous small trial, six different sanitisers were tested for their anti-microbial effect on bacteria of seafood origin. A significant difference in the effectiveness of the different sanitisers was noted. This study aimed to confirm and extend these observations by - 2 -

5 testing a number of these sanitisers for their effectiveness on seafood processing equipment, and to investigate the use of sanitisers as an aid to increase the shelf life of whole finfish. Three sanitisers were investigated to quantify their ability to kill bacteria present on contaminated seafood processing equipment. The active ingredients of the three sanitisers were bitter orange extract, chlorine dioxide, and a quaternary ammonia compound. Knives and chopping boards similar to those used for seafood processing were contaminated by cutting and smearing the gut and gills of Australian Herring (Arripis georgianus) on the equipment. The efficacy of the sanitisers was assessed by performing microbiological counts on the equipment prior to sanitiser, and then at intervals following. The quaternary ammonia compound product was the most effective sanitiser, reducing the microbial loads by over 8% within 5 minutes of application. The second most effective was the bitter orange extract sanitiser, and the chlorine dioxide based sanitiser was the least effective, although bacterial levels were still significantly reduced when compared with the original microbial load. These sanitisers were applied in a worst possible condition scenario, as the equipment was not washed prior to application of the sanitisers, possibly causing partial inhibition of the sanitisers due to a high organic load. The dilution of sanitisers in artificial seawater did not have a significant impact on the efficacy of the sanitisers. The use of bitter orange extract and chlorine dioxide sanitisers as a means of increasing the shelf life of refrigerated whole finfish was evaluated. The shelf life of fish was assessed using quality index (QI) scores. The fish studied were Herring, Tailor, and Spangled Emperor. Sanitisers were applied to the fish as a dip prior to storage in refrigeration. The application of sanitisers to all of the tested species consistently resulted in the reduction of the QI score when compare the QI score of untreated fish. The chlorine dioxide based sanitiser consistently gave marginally better results than the bitter orange extract based sanitiser. The effect of sanitisers on bacterial counts was also examined. The results were highly variable and inconclusive for all fish species and sanitiser s. This variability indicates that either the QI is not affected by microbiological spoilage, or that the microorganisms that cause spoilage were not being sensitively detected by the count methods employed in this study. The addition of sanitisers to ice prepared from artificial seawater and subsequently placed over stored Herring was also examined. Storage of Herring in treated ice resulted in an increase in the shelf life of the Herring as determined by QI scores. The response to sanitisers was similar to that seen in previous soaking trials, with chlorine dioxide containing ice producing better QI scores than ice containing bitter orange extract. The storage of Herring in treated ice also had a significant impact on the growth of bacteria during storage. For all bacterial counts performed, the total number of bacteria present on fish stored in ice containing either sanitiser was significantly lower than the counts of fish stored in untreated ice. KEYWORDS: Sanitisers, spoilage, finfish, processing equipment

6 ACKNOWLEDGMENTS We would like to thank Eric Mustoe of Geraldton Fish Market for his assistance with the supply of fish used in this study. We are also grateful to Brian Bignell of Citrofresh International Ltd, Jean Pendlebury of Chemform, and Mitrol Technology (Aus) Pty Ltd for supplying the sanitisers used in this study. Thanks also to Dr Alan Bremner for his comments and assistance during the development of this study. BACKGROUND Cleaning and sanitation in the seafood industry With the introduction of the Australian Primary Production and Processing Standard for Seafood by FSANZ on 26 May 26 all seafood businesses from primary production through to retail must now have a documented food safety management plan. These businesses are obligated to produce food that is not unsafe or unsuitable for its intended use. Under this standard, seafood businesses are required to comply with Division 5 of Standard and of the Food Standards Code relating to cleaning, sanitising, and maintenance (FSANZ 26). Benefits such as improved quality and reduced bacterial loads have increased the use of sanitisers within the fishing industry. There are many sanitisers used within the industry and this project looked at several of these to determine their efficacy. Sanitation generally involves the use of detergents and sanitisers to clean both the fishing boat and also the processing plant to reduce microbial loads. The two different compounds used for cleaning and sanitising are applied to different situations. A detergent is a compound or mixture of compounds intended to assist cleaning; they can consist of different components which have varying properties, including surfactants, abrasives, oxidants, substances to modify ph and enzymes. Detergents, often in association with manual cleaning, are responsible for removing grease and dirt by forming an emulsion between them and the water. Sanitisers are applied to surfaces following cleaning to reduce microbial numbers ( Detergents and sanitisers are beneficial in eliminating bacteria provided they are handled in accordance with manufacturer s instructions and comply with government regulations. There are some sanitisers that are not able to be used on fish due to the effect they have on fish, for example phenolic compounds alter the fish s odour which may affect the taste. When choosing a sanitiser it is best to find one that works well in the conditions it will most frequently be used. Since fishing boats have ready access to seawater, this will obviously be the water source of choice. As a consequence, the compound must remain effective when mixed with seawater. Seawater has a ph of around 8.5, so compounds should be chosen that are effective at this ph. For example, chlorine products are not very effective as the optimum ph for chlorine powders such as those used in swimming pools is only - 4 -

7 approximately ( HandlingFreshFish/HandlingFreshFishPage5.php?5). A previous pilot study into sanitisers used in the seafood industry made the following findings Sampling at a commercial seafood processing establishment found significant levels of bacterial contamination on product and equipment despite the sanitising protocols being followed. Six sanitising agents (active ingredients were hydrogen peroxide, quaternary ammonium compounds, sodium hypochlorite, bitter orange extract, chlorine dioxide and mixed oxidiser species) were tested for effectiveness (when compared to untreated control) in decreasing bacterial numbers on contaminated fish cutting knives and boards. There were significant differences in effectiveness of the sanitiser types. The same sanitising agents were tested for effectiveness in killing bacteria isolated and cultured from commercial samples of mulies (Sardinops sagax). Again there were significant differences in effectiveness. There were differences in effectiveness between the equipment trials and against the bacteria isolated from fish. Fresh samples of Tailor (three s - control, treated with bitter orange extract and treated with chlorine dioxide) were stored at 4 C. Bacterial numbers were enumerated daily and the quality index (QI) (as based on a scheme developed for mullet) was assessed at the same time as the bacterial samples were collected. The results showed that: total bacterial numbers did not correlate with the QI, and there was a significant effect on the QI in the sanitiser treated samples. Bacteria found on fish The bacteria present on fish, and their metabolites, have a marked impact on the quality and safety of seafood products. Bacteria are typically found on the skin and gills, and in the gastrointestinal tract of all fish. Bacteria commonly found on fish include Acinetobacter, Flavobacterium, Moraxella, Shewanella, Pseudomonas, Vibrio, Photobacterium, Bacillus, Micrococcus, Clostridium, and Lactobacillus (Aguas, Capell and Davies, 1998). Bacteria such as Shewanella purtrefacians, Pseudomonas, Photobacterium phosphoreum, and Lactobacillus are commonly identified as causes of spoilage in raw and processed fish (Aguas, Capell and Davies, 1998). Pathogenic bacteria commonly found on fresh caught fish can include Clostridium botulinum, pathogenic Vibrio species, Plesiomonas shigelloides and Aeromonas species (Ababouch, Gram and Huss, 23). Although these organisms are dangerous human pathogens, they generally occur naturally in low levels on fish. Other pathogenic bacteria, found in the general environment, and often passed onto fish, include Listeria monocytogenes, Clostridium perfringens and Bacillus species. These bacteria, along with their non pathogenic counterparts, are also found in low numbers in the environment (Ababouche, Gram and Huss 23). Bacterial species such as - 5 -

8 Salmonella, Shigella, Escherichia coli, Campylobacter and Staphylococcus aureus are not normally present on fish. Consequently cross contamination is the usual source of these organisms, generally occurring in the processing plants. Fish deteriorate rapidly with contamination of such species and so rejection of sale due to off odours and flavours prevent contaminated fish to be sold to the public. Compounds used in this study Previous sanitation experiments identified three compounds that were effective in eliminating bacteria and testing was continued on successful products (Tonkin et al. 25). These three compounds included a sanitiser with bitter orange extract as the active ingredient, a sanitiser with chlorine dioxide as the active ingredient and a sanitiser with quaternary ammonium compounds as the active ingredient. The bitter orange extract is a natural sanitiser, now being introduced into the industry. This sanitiser is responsible for breaking down and destroying the cellular membrane of a micro-organism. It also has a strong residual effect, working effectively for up to 48 hours. This property is an advantage for maintaining quality in fresh produce and foods. The bitter orange extract provides the sanitiser with a broad spectrum anti-microbial activity, the ability to break down biofilms and an ability to maintain effect when organic matter is present. It is also non-toxic, non-carcinogenic, non-mutagenic, non-corrosive and non-tainting and does not change the flavour of the food or produce ( Products which contain chlorine dioxide as their main ingredient are advantageous because they work within a wide range of ph values (2-12). They are also less affected by organic matter than other chlorine-based sanitisers (Hugo & Russell 1999). Such products are becoming increasingly popular due to the effectiveness these products have in maintaining high quality and extending shelf life. Chlorine dioxide does not dissociate in water, it has a very high efficacy against vegetative cells such as bacteria, fungi, yeasts and moulds, viruses, algae and protozoa, and has no effect on human, animal or fish cells. Chlorine dioxide is also considered to be less harmful to the environment (Hugo & Russell 1999) ( Mainly used on surfaces, quaternary ammonium compounds are very effective at reducing bacterial loads. Benefits of these sanitisers include their broad spectrum activity, fungicidal and bactericidal (Hugo and Russell 1999). It appears to have no adverse effect on painted or polished floors, and does not have a strong odour. Special corrosion inhibitors are added to minimize corrosion. It is instantly soluble in water and may be stored for long periods of time without losing strength

9 NEEDS An increase in the shelf life of low value fish and the consumer s perceptions of the freshness and quality of these species could result in increased consumption of these under utilised species, resulting in higher demand for the species and a higher return to the producer. This project addresses aspects of the strategic challenges identified in the FRDC industry development plan, specifically, making better use of under-utilised fish, and producing safe, high quality seafood. OBJECTIVES 1. To assess the effectiveness of sanitising agents on fishing industry equipment and product. 2. To determine if the shelf-life (as measured by the QI) of selected finfish species can be extended with the use of sanitisers. 3. To extend the results of 1 and 2 to the fishing industry. METHODS 1) Sanitisers used The following sanitisers were used in this study: Chlorine dioxide AquaPlus 5 Bitter orange extract Citrofresh Quaternary ammonium compounds Superquat Products were diluted and used as per manufacturers instructions. The concentrations of sanitisers used are detailed in each experiment below. 2) Microbiological analysis Surface Total Viable Count Contact (RODAC) Plate Method Plate Count Agar (PCA) RODAC plates were purchased from Excel Laboratory Products (Perth). Plates were allowed to reach room temperature prior to use. Tests were performed as described by Sveum et al (1992) and plates incubated at 25 C for 48h. Surface Total Viable Count Swab Method The surface swab method was performed as described by Roberts, Hooper and Greenwood (1995) using MRD (sterile.1% peptone /.85% NaCl solution). The area sampled for each experiment is detailed in each experiment below. A sterile cotton swab was dipped into 1mL of MRD in a screw caped tube containing 5 glass balls. Excess fluid was squeezed out of - 7 -

10 the swab against the inside of the tube. The moistened swab was run over the surface turning the swab whilst rubbing. The swab was broken off into the MRD tube. A second dry swab was rubbed over the same surface area as the moistened swab and placed broken off into the same MRD as the first swab. The MRD tube was shaken vigorously for 3 45 sec to remove the bacteria from the swab. The sample was further diluted by serial 1/1 dilutions (1mL in 9mL MRD) if required and plated onto appropriate media by either the spread plate or spiral plate method. Spread Plate Method Spread plates were prepared as described in Australian Standard AS (1991). 1µL of MRD was pipetted onto duplicate agar plates and spread using sterile hockey sticks. Plates were incubated at appropriate temperatures as detailed below. Spiral Plate Method Spiral plating was performed on a Whitley Automated Spiral Plater (WASP) (Don Whitley Scientific, Shipley) in accordance with manufacturer s instructions. A total deposition volume of 5µL was plated onto each agar plate. Plates were incubated at appropriate temperatures as detailed below. Total Plate Count (TPC) The total plate count was determined by inoculating PCA plates by either the spread plate or spiral plate method and incubating plates at 3 C for 2 days. Psychrotrophic Plate Count (PPC) The total plate count was determined as described by Cousin, Jay & Vasavada (1992). PCA plates were inoculated by either the spread plate or spiral plate method. Plates were incubated at 7±1 C for 1 days. Total Viable Count on Modified Long and Hammer (mlh) agar (mlhtvc) mlh agar was prepared as described by Koutsoumanis and Nychas (1999) with 2g proteose peptone (Oxoid), 4g gelatine (Davis), 1g K 2 HPO 4, 1g NaCl,.25g ammonium ferric citrate, 15g agar (Oxoid) in 1L distilled water. The ph was adjusted to 7. as required and the media were autoclaved at 121 C for 15 minutes. mlh plates were inoculated by either spread or spiral plating and incubated at 7 C for 1 days. 3) Quality Index Determination The quality index schemes were developed from the following sources. Australian Herring, adapted from the Eurofish QMI scheme for herring; Tailor, adapted from the Mullet Quality Index scheme (Tonkin et al., 26); Spangled Emperor, scheme in development by Dr H. Williams and Mr P. Anthonisen (School of Public Health, Curtin University of Technology). All QI assessments were performed by the same person for each experiment. All QI schemes had not been validated at the time of performing them. Research members performing the QI determinations had not undergone specific training

11 Interpretation of the QI parameters were discussed and agreed to prior to commencement of experiments. 4) Experimental Design a) Efficacy of sanitisers on processing equipment Protocol 1 All three sanitisers were tested for the surfaces experiment. Each product was appropriately diluted to obtain optimum efficiency and reduce toxicity based on manufacturer s instructions. Chlorine dioxide was diluted 4ml/L; bitter orange extract was diluted 2ml/L and the quaternary ammonia compound (QAC) 12ml/L. Stainless steel knives and plastic chopping boards showing minimal signs of wear were washed and sprayed with 7% alcohol to avoid contamination. The equipment was allowed to air dry for 15 3 minutes. A fresh Australian Herring (Arripis georgianus) (Herring) was de-headed and the knives and chopping boards were smeared with the Herrings gills and gut contents, to imitate contamination likely to occur in a processing plant. The knives and boards were then left for approximately 1 minutes on the bench at room temperature. To test for initial levels of bacteria, a RODAC plate was applied to each chopping board and knife. The knives were then sprayed with a sanitiser, and the chopping boards were soaked in tubs full of 2 litres of sanitiser. The knives and chopping boards were left for 5 minutes before using RODAC plates on an area untouched by previous contact plates, to test for the numbers of bacteria present. A control was also prepared for this experiment, with the knife being sprayed and with the chopping board dipped in tap water. Following the second sampling, the knives were then left on the bench overnight at room temperature and the chopping boards were left to soak overnight in the tubs. Samples were taken using contact plates the next morning to determine the bacterial load present on each knife and chopping board. b) Efficacy of sanitisers on processing equipment Protocol 2 The method for processing equipment protocol 2 was similar to protocol 1 with minor changes. Chlorine dioxide was diluted 4ml/L, bitter orange extract, 2ml/L and the quaternary ammonia compound was diluted 12ml/L in both tap water and artificial sea water (ASW). The fish were cut on plastic chopping boards using the stainless steel knives, imitating practices used in the processing plant. However, instead of leaving the chopping boards and knives for 1 minutes before applying the sanitiser, the equipment was left for 5 h before the sanitisers were applied. There were no other differences between the two methods. The detergent SAN-E-FOAM was used on the chopping boards in conjunction with bitter orange extract. The detergent was diluted in both seawater and tap water according to the manufacturer s instructions. The chopping boards were coated in the foam of the detergent, as directed, and left for 3 minutes on the bench. The detergent was then washed off the chopping boards and sanitiser was sprayed and left for 5 minutes before a - 9 -

12 sample was taken. Instead of soaking the boards overnight in sanitiser, once the boards were sprayed they were left overnight to dry on the bench. The knives tested with bitter orange extract were dipped in a solution of sanitiser, diluted in either seawater or tap water. The knives were left for 5 minutes before a sample was taken and the knives were then left to soak overnight in the solution. c) Use of sanitisers on fresh product to increase shelf life Protocol 1 Chlorine dioxide and bitter orange extract were tested to see if they could be incorporated into a product wash system to increase shelf life. The sanitisers were diluted into 1.5 litres of artificial seawater. Chlorine dioxide was diluted 2ml/L and bitter orange extract was diluted 4ml/L. Three freshly caught Herring purchased from a fish market were used for this experiment. Prior to commencement of testing the Herring were assessed for their QI. The QI was assessed according to the index detailed in Appendix 3. All three fish were swabbed over a 2cm x 2cm area with a cotton swab. The swab was placed in a bottle containing 1ml of MRD containing glass beads and then vortexed for at 15-2 seconds. Each sample, from the sanitisers or the control, was plated using the spiral plater onto PCA to perform both the total plate and psychrotrophic plate counts as detailed above. The area swabbed was marked on the fish using a sterile scalpel to ensure re-sampling of the site did not occur. Following the initial swabbing, the fish were dipped in 1.5 litres of either ASW for the control, or ASW containing either chlorine dioxide or bitter orange extract. The fish were soaked in these solutions for approximately 1 minute, after which they were placed in plastic tubs without lids and refrigerated at 4ºC. The fish were QI checked and swabbed at 24h intervals for 12 days and tested as described above. d) Use of sanitisers on fresh product to increase shelf life Protocol 2 Protocol 2 was a replicate of Protocol 1 with minor changes. The sanitisers were diluted in 3L of artificial seawater, compared to the 1.5L used in protocol 1. Chlorine dioxide was diluted 2ml/L and bitter orange extract diluted 2ml/L. The fish were swabbed (2cm x 2cm) and the swab was placed in 1ml of sterile MRD and glass beads and vortexed for 15-2 s. The samples were spiral plated onto two PCA plates, one Iron Agar plate and one mlh agar plate. Each plate was incubated as described above for the TPC, PPC, and mlhtvc. The area swabbed was marked on the fish using a sterile scalpel to ensure re-sampling of the site did not occur. Following the initial swabbing, the fish were dipped in 3 litres of either ASW for the control, or ASW containing either chlorine dioxide or bitter orange extract. The fish were soaked in these solutions for approximately 1 minute. The fish were placed in a 4 C cold room, layered with ice on top of them to prevent them from drying out. Swabs were taken at 24h intervals for up to 12 days. Fresh ice was placed on the fish daily. The QI of the fish was also recorded at each sampling point. e) Use of sanitisers on fresh product to increase shelf life Protocol 3 The method for protocol 3 was a replicate of protocol 2. However, one whole side of the fish was swabbed when a sample was taken instead of 2cm 2 areas. Fish of equal side surface area were used for each experiment

13 Enough fish were included in the experiment so that one side was swabbed once. The swabs were placed in 1ml of sterile MRD and plated as per protocol 2. Tailor (Pomatomus saltarix) and Spangled Emperor (Lethrinus nebulosus) were also tested using this procedure. As the Spangled Emperor was significantly larger than the other two species tested, each side of the fish was halved horizontally and each half was swabbed as one sample. f) Use of sanitisers on fresh product to increase shelf life Protocol 4 The effect of adding sanitisers to refrigerated artificial sea water (RSW) on the shelf life of raw Herring was studied. Sanitiser was added to 3L of artificial sea water held at 4 C at concentrations detailed in fresh product protocol 2 above. Swabs and QI assessments were taken as described in Protocol 3 prior to. The Herring was placed into RSW (with or without sanitiser) and held at 4 C for 1 h. The RSW was then removed and a swab taken. The fish were then covered in ice and held at 4 C. Sampling and QI assessments were performed at day 1 and then every second day. Fresh ice was placed on the fish daily. The effect of adding sanitiser to ice on the shelf life of raw Herring was also assessed. Sanitisers were added to ASW at the concentrations described in protocol 2 above. Ice was prepared from each ASW solution and crushed to a size similar to that produced by ice making machines. Swabs and QI assessment were performed as described in Protocol 3 prior to the fish being overlayed with the prepared ice. Swabs and QI assessments were performed at day 1 and then every second day

14 RESULTS Efficacy of sanitisers on processing equipment Protocol 1 All three sanitisers were tested on both stainless steel knives and plastic chopping boards to determine their effectiveness on common production surfaces. The results for knives are summarised in Figure 1a. The initial time point on all chopping boards, equivalent to t= h, had high levels of bacteria present. However, the 18h time point indicated that bitter orange extract and QAC sanitisers eliminated bacteria extremely well over night. The decrease in bacterial load using chlorine dioxide was limited. The control for this experiment also showed a significant decrease in bacteria after the overnight soaking calling into doubt the validity of the results. Figure 1b demonstrates the effectiveness of the three sanitisers on stainless steel knives. Samples taken 5 minutes following exposure to the sanitisers showed that chlorine dioxide decreased the total bacterial load by more than half. The bitter orange extract sanitiser showed a significant decrease of more than 1/3 of the original bacterial load, and the QAC sanitiser had a reduction of 94%. A further sample was taken after allowing the knives to dry in air overnight (18h). The results showed that chlorine dioxide and QAC eliminated most bacteria. The bitter orange extract sanitiser showed a significant decrease in the levels of bacteria present on the knife compared to the 5 minutes sample however, the decrease in numbers was less than the decrease seen in the control Before Treatment After Treatment QAC Figure 1a: Effect of sanitisers on bacterial loads on contaminated plastic chopping boards Protocol

15 Before Treatment 5 min After Treatment 18h After Treatment 4 2 QAC Figure 1b: Effect of sanitisers on bacterial loads on contaminated knives Protocol 1 Efficacy of sanitisers on processing equipment Protocol 2 The results obtained from this second surface experiment were similar to those of the first. There appeared to be no major differences between results for sanitisers diluted in tap water and those diluted in artificial seawater. Results from the chopping boards demonstrated that the QAC sanitiser was the most effective at eliminating bacteria from the chopping boards. All bacteria were eliminated within 5 minutes, compared to bitter orange extract where bacteria were present at 5 minutes, but were eliminated by 18h, and chlorine dioxide which still showed low levels of bacteria present after the 18h soaking (Figures 2a & 2b). In comparison to the first experiment, the control did not decline significantly overnight (18h) for both chopping boards dipped in the seawater and tap water. The results obtained from the knives experiment also showed little variation between sanitisers diluted in the tap water or artificial seawater. In general, the products showed little variation between this experiment and the first. Chlorine dioxide efficiently eliminated the majority of bacteria within 18h, however bacteria were still present 5 minutes following, compared to the QAC sanitiser, where bacteria were eliminated by 5min in sea water (with only 1% surviving in tap water). Low levels of bacteria were still present on knives treated with bitter orange extract prepared in tap and sea water after 18h for both experiments. The bacterial count for the control for the knives also appeared to drop after holding the knives for 18h indicating

16 desiccation of the bacteria as a possible cause of microbial reduction for all s at 18h (Figure 2c-2d) Before Treatment 5 min After Treatment 18h After Treatment 4 2 QAC Figure 2a: Effect of sanitisers prepared in tap water on the bacterial loads of contaminated chopping boards Protocol 2. Error bars represent standard deviation of Original Bacterial % Load 1 8 Before Treatment 5 min After Treatment 18h After Treatment QAC Figure 2b: Effect of sanitisers prepared in sea water on the bacterial loads of contaminated chopping boards Protocol 2. Error bars represent standard deviation

17 Before Treatment 5 min After Treatment 18h After Treatment 4 2 QAC Figure 2c: Effect of sanitisers prepared in sea water on the bacterial loads of contaminated knives Protocol 2. Error bars represent standard deviation Before Treatment 5 min After Treatment 18h After Treatment 4 2 QAC Figure 2d: Effect of sanitisers prepared in tap water on the bacterial loads of contaminated knives Protocol 2. Error bars represent standard deviation

18 Use of sanitisers on fresh product to increase shelf life Effect of sanitisers on Herring Protocol 1 To test the effectiveness of each sanitiser in their ability to maintain quality and freshness, bacterial counts and a QI were recorded. Protocol 1 protocols were used to assess the effect of sanitisers. Quality Index From the QI results the control gradually increased at a steady rate, in comparison to both sanitisers which were consistently low up to around day 4 at which time they increased at a more dramatic rate. However, both still had a better QI recorded by day 4 than the control which recorded 8 as its QI (Table 3a, Figure 3a). From day 6 all products and the control appeared to have in increase of bacterial load at a steady rate and by days 1, 11 and 12 there was little difference in QI between the three. By day 12, chlorine dioxide and bitter orange extract gave the same QI and the control was only one QI point higher. Day Chlorine dioxide Bitter orange extract Table 1a: Effect of sanitisers on Herring QI Protocol

19 Quality Index 1 8 Chlorine dioxide Bitter orange extract Figure 3a: The effect of sanitisers on Herring QI Protocol 1 Experiment 1 Bacterial Counts Swabs taken from the Herring at 24 h time points and incubated at 7 C (PPC) and 3 C (TPC) indicated the levels of bacteria present had no correlation with the QI. Initial bacterial levels were 15, CFU/cm 2 for the TPC and 2, CFU/cm 2 for the PPC. There was a decrease seen in the psychrotrophic bacterial load on the fish treated with both sanitisers from plates incubated at 7 C (Figure 3b). The bacteria appeared to be significantly decreased by day 2 and bacterial levels remained low until day 6 when a spike occurred and the bacterial load increased. This spike was not as obvious in swabs taken from fish treated with chlorine dioxide as it was with fish treated with bitter orange extract and the control. Chlorine dioxide appeared to maintain lower levels of bacteria in the longer term. In comparison, bitter orange extract showed spikes in bacterial levels at around day 7 and day 12. The control for this experiment also showed a decrease in bacteria by day 2, then a plateau before another increase at day 7 and again at day 1, as shown in Figure 3b. A similar trend occurred with PCA plates incubated at 3 C (TPC) (Figure 3c), with fish treated by both sanitisers showing a marked decrease in bacteria by day 2. Chlorine dioxide treated Herring remain consistently low over the 12 day period, however bitter orange extract treated Herring had TPCs that fluctuated at days 7 and 11, similar to the PPCs. The control also showed a decrease in bacteria by day 2. However, the control appeared to have an extra spike at around day 4, followed by another on days 6 and

20 16 14 % of Original Bacterial Numbers Before After Figure 3b: Herring PPCs Protocol 1 Experiment % of Original Microbial Load Before Treatment After Treatment Figure 3c: Herring TPCs Protocol 1 Experiment 1 The experiment was repeated a second time. The QI of Herring from the second shelf life experiment is listed in Table 3b. The QI for this experiment

21 was, in general, lower than the first, however, the trends were similar. The control increased at a steady rate with deterioration in appearance seen from day 2. Chlorine dioxide maintained a low QI for the first few days until approximately day 5 where the QI began to increase and deterioration was becoming visible on the fish. Bitter orange extract performed slightly better than chlorine dioxide, with the QI remaining low until approximately day 6. From day 6, the QI for fish treated with either sanitiser increased at a steady rate until both finished with a QI of 13 compared to the final value of 14 for the control. Day Chlorine dioxide Bitter orange extract Table 3b Effects of sanitisers on the QI of Herring Protocol 1 Experiment Quality Index 8 6 Chlorine dioxide Bitter orange extract Figure 3d: The effect of sanitisers on Herring QI Protocol 1 Experiment

22 Before Treatment After Treatment Figure 3e: Herring TPCs Protocol 1 Experiment Before Dunking After Dunking Figure 3f: Herring PPC Protocol1 Experiment 2-2 -

23 % Original Bacterial Load Before Treatment After Treatment Figure 3g: Herring ml&htvc Protocol 1 Experiment 2 Figures 3e-3g illustrate the levels of bacteria found on each fish at each 24 h time point over a 12 day period. Figure 3e shows the Total Plate Count grown at 3 C on PCA, Figure 3f the Psychrotrophic plate count grown at 7.5 C on PCA and finally Figure 3g shows bacteria grown on the Long Hammer agar incubated at 7.5 C. The values represented on these graphs are expressed as a percentage of the total number of bacteria present on the fish prior to. Bacterial loads for Herring on the TPC plates (3 C incubation) saw all samples start with an initial bacterial load of around 18, CFU/cm 2. Following bacterial levels dropped by more than 6% for sanitiser treated Herring and by a little over 3% for the control. The TPC for all s appear to drop at around day 3. By day 6 TPC levels appear to have increased and are present at levels seen prior to day 3. Bacterial levels for all s appear to be relatively low until approximately day 9 where a rapid increase in growth can be seen. By day 11 Bitter orange extract appears to plateau compared to Chlorine dioxide and the control which both appear to have continuously rising bacterial levels after day 9. Psychrotrophic plate counts for the Herring prior to were around 2. CFU/cm 2. Following Herring treated by either sanitiser had reduction in PPC of more than 5%. Following the control PPC increased slightly. The bacterial levels are fairly low throughout the experiment until day 5 (day 6 for the bitter orange treated Herring). All s saw a significant increase in the psycrotrophic plate count from

24 day 6 to day 12 with final counts of more than 15, CFU/cm 2 recorded for all treated fish. Figure 3g, illustrates the amount of bacteria grown on Long Hammer agar at 7.5 C. Figure 3g shows a slight decline at around day 3 for all products and the control, with a steady increase in bacterial after day 5. On day 11, the sample taken from Chlorine dioxide appeared to have considerably less bacteria than previous days, however levels rose again by day 12 and may be due to sampling error. Use of sanitisers on fresh product to increase shelf life Protocol 2 Small changes to protocol 1 were introduced primarily to reduce the drying out of the fish during refrigerated storage. Ice was layered over the fish during storage. Figure 4a shows the QI of the fish during storage. As shown previously the QI for the sanitiser treated fish was lower than the control fish. Bitter orange extract had a consistently lower QI value than the control throughout the experiment, up until day 12 where the QI equalled that of the control (QI value = 14) Chlorine dioxide produced the lowest final QI (14 at day 12). It also produced consistently lower QI values than the fish treated with bitter orange extract with the exception of the first day following. The chlorine dioxide treated fish had a peak difference in QI values of 8 at days 5 and 6 when compared with the control fish Quality Index Figure 4a: The effect of sanitisers on Herring QI Protocol 2 Figures 4b, 4c, and 4d show the effect of the sanitisers on bacterial numbers present on the treated fish. The values are expressed as a percentage of the

25 total number of bacteria present on the fish prior to. The untreated fish had a TPC (Figure 4b) of approximately 2, CFU/cm 2 prior to. Immediately following the chlorine dioxide treated fish had a reduction in bacterial numbers of approximately 4%, the bitter orange treated fish had an approximately 25% reduction, and the control did not significantly reduce (<.1% reduction). Bacterial numbers on all treated fish (including the control) dropped significantly 1 day following to approximately 7% of initial levels, and then peaked at day 11 with counts 6 to 1 times higher than prior to. There appeared to be no direct correlation between TPC and QI as the control fish had the smallest total increase in TPC over time. PPCs (Figure 4c) for the fish prior to were around 18, CFU/cm 2. Following, the chlorine dioxide and bitter orange extract treated fish had a 6% reduction in PPC. The PPC for control fish doubled post. Following a reduction in counts for all s after 1 day, the counts steadily increased for all s with a final increase in the PPC to 11 times the starting count. Similar results were observed for the Long and Hammer agar counts (Figure 4d) although the chlorine dioxide treated fish increased its count immediately following in line with the results of the control (36% increase). Initial counts were approximately 3, CFU/cm 2 reaching a maximum of 2, CFU/cm 2 for all s after 11 days Before Treatment After Treatment Figure 4b: Herring TPCs Protocol

26 Before Treatment After Figure 4c: Herring PPCs Protocol Before Treatment After Treatment Figure 4d: Herring ml&htvc Protocol

27 Use of sanitisers on fresh product to increase shelf life Protocol 3 To see if the sharp reduction in all observed counts 1 to 3 days after was due to the position the swab was being taken on the fish, one whole side of a Herring was swabbed at each sampling point. Fish were treated and held under identical conditions to protocol 2. The QI results were similar to those observed previously in protocol 2. The QI for the control fish increased steadily from day 1 to a maximum of 17 at day 12. The QI for bitter orange extract treated fish was as good as or better than all s up to and including day 3 after which it was consistently better than the control but did not perform as well as the chlorine dioxide. Chlorine dioxide had the lowest rate of QI increase and had a maximum difference in QI of 7 at days 6 and 7 compared to the QI of the control fish at these days Quality Index Figure 5a: The effect of sanitisers on Herring QI Protocol 3 Bacterial count results are summarised in Figure 5b, 5c, and 5d. No sharp falls were observed post sanitiser following this protocol. The TPC for fish prior to was 6, CFU/side. Following, both sanitiser treated fish had reduced counts by approximately 75% while the control counts reduced by 15%. All counts, including the control, reduced to less than 25% of the original count at day1. The bitter orange treated fish had a TPC that was only 13% of the original count. After this point the TPC steadily increased for all s, with the increase greatest on the chlorine dioxide treated fish. PPCs were approximately 16, CFU prior to. Following, the counts fell by 55% for the sanitiser treated

28 fish, and 34% for the control. The PPCs rose on all s to result in a final count approximately 2 times the original Before treatement After Figure 5b: Herring TPCs Protocol Before dunking After dunking Figure 5c: Herring PPCs Protocol

29 Before After Figure 5d: Herring ml&htvc Protocol 3 The effect of adding sanitisers to refrigerated sea water on the shelf life of Herring The addition of sanitisers to refrigerated artificial sea water (RSW) had a positive impact on the QI of the treated Herring. Figures 6a and 6b show the QI values for Herring from batches of Herring received on different days. Both show a difference in the QI for sanitiser treated fish compared with the fish that were placed into RSW without sanitiser (control). Chlorine dioxide produced the largest reduction in the rate of QI score change. There was a marked difference in the QI values between both batches with the first batch having a much higher final QI score and a more rapid onset of deterioration. The final QI values for the first batch of fish were 14 for chlorine dioxide treated fish, 15 for bitter orange extract treated fish, and 18 for the control fish. The second batch of fish had QI values of 8, 11, and 12, respectively. This trend was also evident in the onset of deterioration with the first batch of fish showing onset of observable deterioration on day 4 for chlorine dioxide treated fish, day 3 for bitter orange treated fish, and day 2 for the control fish. The second batch of fish had deterioration onset beginning at days 4, 5, and 5, respectively, for each type

30 Quality Index Figure 6a: QI of RSW treated Herring Batch Quality Index Figure 6b: QI of RSW treated Herring Batch 2 The bacterial counts for the two batches of Herring are summarised in Figures 6c - 6g. Figures 6c and 6d summarise the TPC results, Figures 6e and 6f summarise the PPC results, and Figures 6g and 6h summarise the Long and Hammer agar count results. As was observed earlier, there appeared to be no direct correlation between the bacterial counts and QI values. The first batch of Herring tested by this protocol had significantly lower starting bacteria

31 counts compared with the second batch, with starting counts of 2 CFU, 3 CFU, and 1 CFU for the TPC, PPC, and ml&htvc counts, respectively. In contrast, the batch 2 fish had initial counts of 5, CFU, 6, CFU, and 2, CFU, respectively Before After Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Figure 6c: Effect of sanitiser treated RSW on Herring TPCs Batch Before After Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Figure 6d: Effect of sanitiser treated RSW on Herring TPCs Batch

32 Before After Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Figure 6e: Effect of sanitiser treated RSW on Herring PPCs Batch Before After Day 1 Day 3 Day 5 Day 7 Dy 9 Day 11 Figure 6f: Effect of sanitiser treated RSW on Herring PPCs Batch 2-3 -

33 Before After Day 1 Day 3 Day 5 Day 7 Day 9 Day11 Figure 6g: Effect of sanitiser treated RSW on Herring ml&htvc Batch Before After Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Figure 6h: Effect of sanitiser treated RSW on Herring ml&htvc Batch

34 The effect of adding sanitisers to ice on the shelf life of Herring The effect of adding sanitisers to storage ice on QI is summarised in Figure 7a below showing the average QI value for repeated ice experiments. The addition of sanitisers to the storage ice had a positive impact on the overall QI value. Chlorine dioxide showed the most marked improvement in QI value with a peak difference of 5 points at day 6 and a final average QI value of 12, 3 points below the peak for the control and 1 point lower than that of the bitter orange ice. Overall this reduction is no greater than that seen in all other Herring experiments (see Figures 3a, 4a, 5a, 6a, and 6b). Compared to the previous Herring sanitiser projects the addition of sanitisers to storage ice had a marked impact on the growth of bacteria on stored fish. Figures 7b, 7c, and 7d represent the bacterial growth of the TPC, PPC and Long and Hammer plate count for one of the batches tested. Whist these data represent only one of three batches tested, the results were consistent across all batches. The growth of bacteria in ice containing either sanitiser was greatly reduced across all bacterial counts performed when compared to the growth observed on the control fish. Neither of the sanitisers was found to give constantly better results Quality Index Figure 7a: QI of Herring held in ice containing sanitiser