Reducing spoilage and contamination risks of fresh vegetables in China and Australia

Activities to date have concentrated on meeting project objectives:

1) Analysis of production and handling systems to determine risk factors leading to spoilage and contamination, and 3) Improved monitoring of human pathogen contamination risks during fresh vegetable handling and marketing. The project formally began with a start-up workshop held in Beijing during September and October 2004. Participants included staff from the partner organisations and from the Ministry of Agriculture (MoA). Discussions and site visits gave participants an understanding of past and current project interests and the capabilities of each organisation. Structured sessions reviewed the current science on each project topic and developed the experimental details for each subproject. After the workshop, Chinese and Australian partners collaboratively conducted process audits of 4 fresh vegetable supply chains in the Beijing area. These audits mapped the process flow from production to retail, identified the human and plant health hazards, the control points and the verification steps practised. Steps requiring investigation and / or improvement were identified.

Following the process audit, researchers from the China Agricultural University began a microbiological contamination audit on three types of fresh vegetables (pak choi, carrots and tomato) from these supply chains. The audit consisted of sampling vegetables at harvest, during washing and at retail and also sampling from 5 environmental sources (soil, organic fertiliser, irrigation water, wash water before use and used-wash water). Tests are determining the total plate count, E. coli/coliform counts and enterococci populations in the samples. In addition, the presence/absence of pathogens (Salmonella and Listeria monocytogenes) will be determined on the products at retail.

CAAS IVF scientists have completed experiments to investigate the influence of cultivar, inoculum concentration (Erwinia carotovora), packaging, temperature and time on the incidence and severity of bacterial soft rot of pak choi and carrot. An experiment is planned for July 2005 to determine the influence of these factors on Botrytis rot of tomato. Tomatoes were planted at the Nankou station of IVF in April to prepare for this experiment.

DPI scientists completed a trial demonstrating a strong influence of plant nutrition on the susceptibility of pak choi to bacterial soft rot. Statistical analysis is pending, however, high levels of calcium in plant tissue have been shown to increase resistance to pathogens, most probably due to its role in strengthening and stabilising the cell wall and membranes, while high rates of nitrogen fertilisation have been associated with decreased resistance. The interaction of these two nutrients can play a part in determining the storage life. Generally high levels of nitrogen lead to very vigorous growth and depressed root development, both of which may lead to lower levels of calcium being distributed to expanding leaves. This is likely to lead to low nitrogen and high calcium treatments being more disease resistant.

FSA scientists have conducted a comprehensive literature review on Enterococcus spp and their significance for fresh vegetables. Enterococcus faecalis has a limited host range, being more specific to human, dog and chicken faeces, suggesting E. faecalis is an ideal contamination indicator for microbial source tracking. A collection of presumptive enterococcal cultures has been isolated from fresh pak choi obtained from supermarkets in Melbourne. A genetic database search has identified up to three regions within the 16S rRNA gene unique to E. faecalis and appropriate PCR primers have been developed. A PCR system targeting a region of the 16S rRNA gene has been established for the specific detection of E. faecalis. The PCR system has been demonstrated to project partners from CAU during their visit to Australia in March 2005. In addition, during the visit to CAU in June 2005, the FSA sub-project leader assisted and established capabilities at CAU for isolation of Enterococci from vegetable and environmental samples, and for the E. faecalis PCR system.

China partners from IVF and CAU visited Melbourne from March 10th to 22nd 2005. In addition to participating in experiments described above, the project team observed supply chains from farm to retail. An excursion to the Western Irrigation District Recycled Water Scheme examined the production of class A reclaimed water for the vegetable industry. Discussions and site visits to DPI and FSA gave the IVF and CAU partners a detailed understanding of the interests and capabilities of DPI and FSA.

1 Analysis of production and postharvest systems to determine risk factors leading to spoilage and contamination and development of recommendations for risk management.
In the second year, work on this objective investigated:

The influence of plant nutrition during production on the susceptibility to postharvest disease and quality.
The concentration of human pathogens and faecal indicator organisms on vegetables in the supply chain and levels of these organisms in the farm environment.

Calcium and nitrogen have critical roles in plant structure and function which influence the plants resistance to spoilage. DPI scientists conducted hydroponic experiments to observe the influence of these minerals and their interaction on the development of postharvest rots in pak choi (a leafy vegetable) and tomatoes (a fruiting vegetable). Pak choi plants given low nitrogen / high calcium treatments, better resisted attack by the spoilage bacterium Erwinia carotovora and maintained their quality longest during a shelf life test. However tomatoes from plants fed with moderate and high doses of nitrogen were less susceptible to rotting by Botrytis cinerea.

An experiment was also conducted to observe the effect of the irrigation water source on postharvest quality. Iceberg lettuce irrigated with river water, reclaimed water and a mixture of the two was assessed for quality after storage and simulated marketing. The lettuce irrigated with river water had more severe rot development. Work is continuing to investigate the reason for this.

A comprehensive microbiological contamination audit on pak choi, carrots and tomato from three representative farms is being completed in China by CAU. The audit collected samples of produce at 3 points of the supply chain (harvest, after washing and retail) and sampled 5 environmental sources (soil, organic fertiliser, irrigation water, clean wash water and post-wash water). Samples were analysed for total plate counts, E. coli, coliform, and enterococci populations. Vegetables from retail were also tested for the presence of pathogens (Salmonella and Listeria monocytogenes). With the exception of clean wash water, all samples were positive for Enterococcus spp and E. coli, suggesting potential faecal contamination of the vegetables.

2 Development and testing of strategies for decontaminating wash-water and introduction of hygienic postharvest systems for vegetables.
IVF scientists are completing experiments to investigate the influence of washing and sanitising on rot development of carrot, tomato and pak choi. Experiments investigated the impact of washing method, sanitiser type and contact time on the incidence and severity of rot. Results to date demonstrated that:

100 ppm sodium hypochlorite (NaOCl) is the best sanitiser for carrots cleaned by brushing, whilst clean water is sufficient for hand washing carrots (small farmer practice)
Sanitised water was better than tap water for washing pak choi -the optimal treatment was soaking for 1 minute then agitating for 5 seconds in 5 ppm CLO2.

An experiment on tomato is underway.

3 Improved monitoring of human pathogen contamination risks during fresh vegetable handling and marketing.
DPI has been testing the hypothesis that Enterococci may persist longer on vegetables than E. coli, and would therefore make a better sanitary indicator. Inoculated Cos lettuce was stored at 4, 21 and 37C, over a period of 3 weeks. The persistences of both species were similar whilst the lettuce was in a marketable condition. An experiment on tomatoes is nearing completion. CAU have conducted complementary work which has shown after selective growth enrichment Enterococci and E. faecalis were detected more frequently on fresh vegetables than E. coli.

FSA have developed individual Enterococcus (genus specific) and E. faecalis (species specific) PCR methods. These have been further developed into a multiplex PCR which enables the simultaneous detection of both Enterococcus and E. faecalis in one single test. The multiplex PCR has been tested on a selection of reference Enterococcus spp. and closely related non-Enterococcus cultures (Streptococcus thermophilus, Aerococcus viridans and Listeria monocytogenes). The project has collected a further 15 reference Enterococci from (BCCM), 22 Enterococcus wild-type cultures obtained from sampling in 2004/05 and a further 97 putative wild-type Enterococcus cultures isolated from vegetable, soil and irrigation water samples. These cultures will be used for the further validation of the multiplex PCR. The E. faecalis specific PCR has been used by CAU scientists for specific detection of E. faecalis in vegetable and environmental samples in China.

4 Development of technical capacities for hygienic handling of fresh vegetables and human pathogen contaminant monitoring
There were three exchange visits made by project staff in the reporting period and these have enabled learning through collaborative research, in-depth discussion of experimental methods and statistical design and comparative analysis of the vegetable supply chains of both Australia and China.

The most significant training event was a workshop conducted in Beijing on the 8th and 9th of December 2005. This was hosted by IVF CAAS and conducted in collaboration with project team members from DPIV. The main focus was on quality and contamination risks and ultimately the development of a framework for good agricultural practice (GAP). The participants were the potential users and trainers from leading organisations.

Analysis of production and postharvest systems to determine risk factors leading to spoilage and contamination and development of recommendations for risk management.
DPIV investigated the influence of the irrigation water source on postharvest quality of iceberg lettuce, cos lettuce and broccoli. Vegetables irrigated with river water, reclaimed water and a mixture of the two (shandy) were assessed for harvested yield and quality retention after storage and simulated marketing. There was a trend of reduced yield in both recycled and shandy treatments, although yield reduction was only significant in iceberg lettuce irrigated with recycled water. Following storage, quality and storage life were similar in all treatments, however, lettuce irrigated with river water developed more severe rot. It is theorised that this lettuce which was higher yielding, was more susceptible to disease.
Vegetables were examined for pathogen indicators (thermotolerant coliforms, E. coli, Enterococcus spp., Clostridium perfringens, and Salmonella spp.). Out of 85 vegetable samples, 5 samples (1 river, 3 shandy, 1 recycled) had detectable levels of one or more indicators.

DPIV has been investigating the persistence of E. coli and enterococci on vegetables after harvest. In year 3, experiments were conducted on lettuce, tomato and carrot stored at 4, 21 and 37C, for 3 weeks. On carrot, populations of both pathogens declined steadily over time at all temperatures. On tomato, however, populations of both pathogens changed little over 21 days. On both tomatoes and lettuce enterococci were more persistent than E. coli at high temperature, suggesting enterococci would be a better indicator on these vegetables. DPIV also investigated the influence of mechanical damage in the field on the establishment and persistence of E. coli on lettuce heads after harvest. Lettuce plants were injured to simulate chewing insect damage at several intervals prior to inoculation with E. coli. Plants were watered with a mixture of E. coli strains. Watering with contaminated water within 2 days following injury or within 5 days of harvest was shown to be high risk.

CAU completed a comprehensive microbiological contamination audit of fresh vegetables (54 samples of 6 vegetables from 2 farms, preharvest, after preparation for market and at retail). The 6 vegetables were cucumber, green pepper, radish, celery, lettuce and purple cabbage, all of which could be consumed fresh. Counts were made of total aerobic, yeasts and moulds, total coliforms, Enterococcus and Enterococcus faecalis. Total aerobic counts were higher on vegetables after trimming and preparation and from retail shelves than before harvest. The increase was attributed to workers' hygiene and the transfer of soil onto the leaves during trimming. The washing process was also not thorough. At retail, vegetables were at room temperature, which may have encouraged microbial growth. Enterococcus was found on cucumber, lettuce and purple cabbage sampled preharvest, after handling and from retail, and green pepper sampled after handling and from retail. Enterococcus faecalis was found on cucumber pre-harvest.

2. Development and testing of strategies for decontaminating wash-water and introduction of hygienic postharvest systems for vegetables.
IVF scientists completed experiments to investigate the influence of washing and sanitising on rot development of tomato. Experiments investigated the impact of washing method, sanitiser type and contact time on the incidence and severity of rot. Sodium hypochlorite (NaOCl) was shown to be the best sanitiser for tomato. The optimal treatment was soaking for 1 minute in 100ppm NaOCl at 35C. Tomatoes treated this way developed less than 10% rot following 7 days storage at ambient temperature.

3. Improved monitoring of human pathogen contamination risks during fresh vegetable handling and marketing.
In order to validate PCR for the identification of E. faecalis, FSA scientists isolated 110 wild-type putative Enterococci cultures from fresh vegetable, soil and irrigation water samples and identified the species using the API biochemical tests (bioMerieux). A total of 58 of these wild-type Enterococci cultures have been tested using the multiplex E. faecalis/Enterococci species PCR system developed in this project.
Experiments are also in progress using the most probable number (MPN) technique in combination with the multiplex PCR for determination of detection sensitivity on naturally existing Enterococci, and challenged 5-strain Enterococci mix in fresh vegetable samples. The method successfully detected Enterococcus spp and E. faecalis inoculated at 10 to 100 cfu/g in fresh salad vegetables.

4. Development of technical capacities for hygienic handling of fresh vegetables and human pathogen contaminant monitoring
Mr Zhang Xuejie (IVF CAAS) visited Australia in February 2007 to participate in experiments and attend a project planning and review meeting. Mr Zhang learned hygiene assessment techniques in studies conducted on harvest containers and packing lines. At FSA he trained in PCR techniques with project team. Project staff attended 3 technical conferences to improve their knowledge.

Following a review in May 2007 the project has been extended to enable knowledge transfer.
The objectives of the extension are to:
maximise the uptake of improved postharvest practices by the vegetable industry in the Beijing district, reducing food safety and spoilage risks.
to continue building the technical capacities within the partner organisations for research and development in postharvest technology, postharvest pathology and food safety
to attain recognition for the project and inform the research community by publishing findings in scientific journals.
Activities in 2007/08 concentrated on evaluating the processes in the vegetable supply chain from a food safety and product quality perspective and identifying the needs and priorities for process improvement. While there are over 40,000 vegetable growers in the Beijing region, 35% of produce is consolidated and distributed by fewer than 12 large companies. Eight of these companies participated in the process evaluation workshops and will benefit from an ongoing involvement in the project. The evaluations are also being used to develop guidelines for the production of safe, quality vegetables, which will be published later this year.

Experiments are underway to investigate the potential for ultrasonic washing to reduce spoilage and contamination risks. Tertiary students are assisting with these experiments on pak choi, carrot, spinach and tomato.

Mr Zhang Xuejie from the Institute for Vegetables and Flowers led a study tour by executives from the Chinese Academy of Agricultural Sciences to Australia in April 2008. The group examined vegetable supply chains in operation and met with the food safety and postharvest horticulture researchers at DPI and Food Science Australia to overview current research. The purpose was to assist the Chinese partner organisations to develop and to gain organisational support for postharvest horticulture projects.

Two scientific papers and one conference abstract have been submitted for publication. Further research outcomes will be presented in two international conferences to be held in Beijing in the second half of 2008; The China International Food Safety and Quality Conference and Expo (September 24-25, 2008) and Vege2008Beijing (October 14-17, 2008).