updateMicrobialfruit & salad

International Food Hygiene — Volume 25 Number 3 9

Fruit and fresh produce consumptionincreases year on year. There are manyreasons for this, but key is the ‘healthy

eating’ advice currently being given, wherethe ‘five portions per day’ message is widelyknown, indeed even seven portions a dayhave recently been mentioned. The expansion of the fresh produce mar-

ket place over recent years has resulted in awide variety of fruit and fresh produce beingavailable throughout the year (not just in thelocal growing season) and in more exoticfruits and vegetables being stores. Thedemand for ‘easy to use’ fruit fresh producehas also created the market for ‘prepared’ready to eat products which can be washed,cut and otherwise processed to enable theconsumer to simply open the pack and eatthe contents.There is no doubt that the consumption of

increasing amounts of fresh fruits and veg-etables is beneficial to the health of the con-sumer. Microbiologically, however, therecan be some real challenges in the produc-tion of fresh produce.

Growth

These foods are generally grown in soil andoutdoors. Both scenarios will allow freeaccess for a whole range of micro-organismsto the crop. Soils will contain a very highmicrobiological microflora and will be indirect contact with parts of the plant for itswhole life cycle, whilst the outside environ-ment is almost uncontrollable from a micro-biological viewpoint. The impact of soil flora

Dr Roy Betts, Head of Microbiology,Campden BRI, Chipping Campden,Gloucestershire, GL55 6LD, UK.Produced as a service to the food industry by Thermo Fisher Scientific.

and the external environment on the micro-biology of a plant will vary depending on avariety of conditions; the major onesinclude:l The closeness of the plant to the soil:lower areas of a plant may be expected tohave a greater chance of contamination withsoil flora than higher parts.l Rainfall: whilst rain is often required toprovide water for plant growth, it will alsocause soil splash onto lower parts of theplant.l Irrigation: in drier areas or at particularparts of the growing cycle, it may be neces-sary to irrigate the plants. This provides anumber of potential microbiological chal-lenges. Firstly, the water used will not usu-ally be from a mains drinking supply. It maycome from farm reservoirs, or be extractedfrom streams or rivers, so the microbiologi-cal quality of the water may impact on themicrobiology of the plant. Secondly, the waythe water is provided to the plant may affectits microbiology. If it is sprayed on from thetop, it has great contact with all of the upperexternal surfaces, and will deposit anyorganisms contained in the water on theseareas; additionally the ‘splash issue’ notedwith rain will also occur. If the water is dripirrigated onto the soil directly under theplants, it will not contact upper plant sur-faces, and splash is very unlikely.l Plant/soil nutrition: it is common to placefertilisers on or in soils to give a better plantgrowth. There has been a move away fromthe use of artificial fertilisers to the use ofnatural manures. In their ‘fresh state’manures contain all of the faecal organismsfrom the animal from which they came. Thetransformation of faeces into manure shouldinclude a period under suitable conditionsthat allow sufficient temperatures todevelop, to eliminate potential pathogens.Additionally, after adding manures to soils, itis unusual to grow produce immediately; aperiod of time is left to allow a natural soilbacterial flora to develop.

l The need to obtain common cropson a year round basis and the con-

sumer demand for ‘exotic’ fruitsand vegetables has resulted in aworldwide sourcing of produce

items. This means that some itemseasily available from stores willhave been grown in areas wherethe natural microbiology is very

different to that at the point of con-sumption, and where standards ofhygiene may be different.

l Animals and people: field growth gives

every opportunity for animals (birds,rodents, wild mammals, even domestic petssuch as dogs) to come into contact with theproduce. This can result in animal faecalmaterial being deposited onto growingplants. Additionally much fresh produce ishand picked, so that the hygiene of the pick-ers is an important issue to consider. Manygrowers now install field toilets completewith good hand wash facilities to improvehygiene in this area.l Some crops are now grown in hydro-ponic conditions. This considerably reducescontamination issues relating to the environ-ment, but such material will still requirepicking, therefore picker hygiene is stillimportant.l Once picked, produce will have to betransported to pack houses/processingareas where the material will be portionedand packed ready for the consumer.Vehicles and containers used for this needto be kept clean and should not be used totransport other items (for example dirtyitems, agricultural equipment, animals etc.)All of these potential points of microbio-

logical contamination may be of reducedimportance if the plant or crop is to becooked before consumption. The cookingprocess will usually be effective at eliminat-ing any potentially pathogenic organisms,making the food safe to eat. However,much fresh produce (salad crops, fruits etc)is eaten in a raw state, and here the pres-ence of contaminating micro-organisms,particularly pathogens, could cause prob-lems for the consumer.

Microbiology

As fresh produce is a natural material, it willalways have its own natural microflora. Thismeans that it would not be unusual to findtotal viable counts on ready-to-eat freshproduce of over 106/g. This is perfectlyacceptable. The types of organisms will generally

include pseudomonads, Enterobacter spp.,Klebsiella spp. Serratia spp., Flavobacteriumspp. Xanthomonas spp., Chromobacteriumspp. Alcaligenes spp. and Leuconostoc spp.,and yeast and mould species includingCryptococcus, Candida, Rhodotorula,Sporobolomyces, Cladosporium,Aureobasidium, Aspergillus, Penicillium,Phoma, Botrytis, Fusarium, Epicoccum andGeotrichum. It should be noted that it iscommon to find Enterobacteriaceae on

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fresh produce; they form part of the normalflora of plants, and their presence shouldnot be looked upon in the same negativeway as a high Enterobacteriaceae count on,for example, a ready to eat meat product.The natural flora of fresh produce are an

essential part of the plant, indeed someresearchers have hypothesised that thepresence of high levels of generally harmlessorganisms could help to inhibit the develop-ment of possible spoilage organisms or evenhuman pathogens on such material.

Significance of pathogens

Of course, no discussion on fresh producemicrobiology would be complete withoutsome discussion of the significance ofpathogens in these materials. As detailedpreviously, the way produce has to begrown makes much of it open to potentialcontamination from the soil or organismsoriginating directly from animals; this caninclude a range of potential humanpathogens. There has been considerableresearch concerning the potential for plantsto take up pathogens directly from the soilinto the roots. There is concern that if thiscould occur, the ‘internalised’ pathogenswould be very difficult to treat or remove inany way. There is little conclusive data toconfirm that internalisation from the rootuptake occurs; however, it is certainly possi-ble that micro-organisms could becometrapped in damaged plant tissue or plantstructures like stomata, and thus be very dif-ficult to remove.Good reviews of the range and types of

pathogens that can be found on fresh pro-duce are available, but can include bacterialpathogens: Listeria monocytogenes,Clostridium botulinum, Shigella, E. coliO157:H7, Salmonella, Staphylococcusaureus, Bacillus cereus, Vibrio cholerae,Campylobacter; viral pathogens: Hepatitis Aand Norovirus; and Protozoan pathogens:Cryptosporidium, Giardia, Cyclospora. Inaddition to contamination of the growingplant, seeds are known, on occasion, to becontaminated with human pathogens. This can be an issue if the seed is to be

eaten directly, or made into a ready to eatproduct (for example. there have been sal-monella contamination issues and outbreaksof salmonellosis, linked to sesame seeds andsesame products such as tahini and hou-mous, and to germinated alfalfa seeds con-sumed as sprouted seeds).There have been several well-documented

outbreaks of illness throughout the world,linked to consumption of raw fruits and veg-etables. These include salmonella from let-tuce, tomatoes, bean sprouts and melons; E.coli O157:H7 from spinach, radish sproutsand fresh unpasteurised apple juice (applecider in USA); listeria from lettuce, shigellafrom lettuce, and Clostridium botulinumfrom vegetable juice. The range of pathogens causing illness is

large. In many occasions the conditions in oron the food would appear to be antagonisticto the growth of the organism; however,survival and consumption of the pathogen issufficient to cause illness in many cases.Some types of fresh produce will offer con-ditions that allow growth, and this can be aparticular problem. The conditions used to sprout seeds

require moisture and some warmth, idealfor microbial growth. In fruit juices the pH isusually low enough to stop growth of mostpathogens (the organisms may survive);however in many vegetable juices the pHwill be higher and be in a range in whichmicrobial growth can occur. Such productsrequire care in setting shelf life and process-ing/storage conditions. In the last few years there have been an

increasing number of issues related to softfruits contaminated with either norovirus orHepatitis A. Often the implicated fruits havebeen frozen in the country of growth andshipped to other countries for use, causingin some cases, an international outbreak.The major issue with virus contamination

is that compared to bacteria, microbiolo-gists have less understanding of methods foreliminating or rendering these agents non-infective. Thorough cooking will destroythem; however, when ‘fresh’ fruits arerequired to prepare a food, this may notalways be a suitable option.In the production of ready to eat, raw

fresh produce there is no step that will elim-inate pathogens. Any form of process thatcould do this will usually change theorganoleptic properties of the product tosuch an extent that it would become unac-ceptable to the consumer. The only stepopen to producers of raw fresh produce toreduce microbial loading is washing.Washing will remove dirt and debris from

the surfaces of produce, and has some effectat removing surface microflora. Instead ofrelying on washing solely with water, mostproducers of ready to eat fresh produce addforms of sanitiser to the water used forwashing. This helps to keep the microbialloading low, and helps to decrease the riskof pathogen presence in these products.Over recent years there has been much dis-cussion on the use of chlorine in vegetablewashing; this has led to considerableresearch both into chlorine washing itselfand its effects and into alternative ways tohelp remove microflora from fresh fruits andvegetables.

Produce washing

Until recently, chlorinated water was themost common form of wash agent used toreduce the microbial loading of fresh pro-duce. The most common form of chlorineused is sodium hypochlorite, which reacts inwater to form different types of chemicalcomponent such as sodium hydroxide(NaOH) and hypochlorous acid (HOCl). It is the hypochlorous acid (free chlorine)

that is the active biocide. This free chlorinecan bind with other compounds such asorganic matter; this product is then termedtotal chlorine. The amount of hypochlorousacid formed is pH-dependent and morehypochlorous acid remains in solution at pH5 than at pH 7. If too much acid is present, then this can

corrode the washing tanks and it is there-fore best practise to control chlorine solu-tions at pH 7.0, where 78% of thehypochlorous acid remains in solution andthis achieves a good decontamination effect. The natural tendency of chlorine solution

is to be alkaline and it is best practice toensure that washing systems are maintainedat pH 7 by the addition of citric acid. It must be noted that monitoring total

chlorine gives no information on the disin-fecting power of a solution. In order tomonitor this, free chlorine must be moni-tored. It is thought that most of the biocidalactivity occurs within the first few minutes ofwashing and increasing concentration abovea few hundred ppm does not increase thebiocidal effect. Washing fresh produce in chlorinated

water can reduce microbial levels by 10 to100-fold, as long as the washing is carefullycontrolled. Washing studies at CampdenBRI have shown that a 10-fold (1-log) reduc-tion in salmonella, E. coli and listeria levelscan be achieved by washing inoculated let-tuce in water containing 100ppm free chlo-rine for five minutes.Over the last decade there has been an

increasing interest in the use of alternativenon-chlorine based sanitisers that could beused in fresh produce washing

Alternatives to chlorine

Current popular alternatives to chlorinewashing include chlorine dioxide and organicacids. Chlorine dioxide is considered a use-ful alternative to hypochlorite as it is notaffected by pH and does not react withorganic matter to the same degree ashypochlorite. Chlorine dioxide can be unstable and

requires on-site generation, but stabilised(liquid forms) are now commercially avail-able. There is also a trend to move towardsthe use of organic acids for the washing offresh produce; some commercially availablewash agents containing mixes of organicacids are available and some of these can beused to wash organic produce.There are many other techniques that are

being investigated as potential alternativesto chlorine; these include: UV, ultrasound,ozone, irradiation, electrolysed water, per-acetic acid, hot water, bio control agentsand natural compounds like essential oils.With many of these it is too early to say

whether widespread commercial use willever occur. Sequential washing, wherebyproduce is washed a number of times witheither the same wash agent or different

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agents, is also being studied, and may havesome added benefits to microbial reductionin some cases. With any washing system, itis of the greatest importance to ensure thata proper validation of the washing system isdone before it is used commercially, andthat the systems used commercially aremonitored and controlled. In most cases, with ready to eat fresh pro-

duce, the washing step is the only point inthe production process where the level ofmicro-organisms can be controlled; as suchit is of the greatest importance.

Sprouted seeds

No current article on fresh produce wouldbe complete without some mention of themicrobiological risks associated withsprouted seeds. Seeds themselves will usu-ally be considered a microbiologically stablematerial. They are dry and whilst they mayhave a microbiological flora, it will be unableto grow due to the low water activity.Undoubtedly however, seeds will contain arich microbiological flora which will originatefrom the growing environment and mayinclude organisms from soil, fertilisers, irriga-tion water, wild animals, pickers and thetransportation/distribution system. There is a risk that some of these organ-

isms could be human pathogens. If we move

from the seed itself to the ‘sprouting’process, this will usually require seed irriga-tion and an elevated temperature, ideal con-ditions to allow the resident flora on theseed to grow. Research has indicated thatany pathogens present on seeds couldincrease in numbers by 100 to 1000 foldduring the first 24 hours of germination.Outbreaks of food poisoning have been

associated with sprouted seeds for decades.In 1973 soy, mustard and cress was associ-ated with a small outbreak of Bacillus cereuspoisoning, since then outbreaks of yersinio-sis, salmonellosis and verocytotoxin produc-ing E.c oli (VTEC) poisoning have beenassociated with sprouted seeds. Perhaps the largest occurred in Japan in

1996 where estimates suggest between6000 and 9000 people were affected with E.coli O157 food poisoning. A large Europeanoutbreak outbreak began in Germany inMay 2011, and was linked to an unusualserogroup of VTEC, O104.H4, contaminat-ing fenugreek seeds. It affected around 4000people with a severe food poisoning withapproximately 25% of those infected havinga kidney related symptom known ashaemolytic uraemic syndrome (HUS). These issues have led to various agencies

such as the UK Food Standards Agency rec-ommending that consumers should not eatraw sprouted seeds but to always cookthem before eating, and the EU enactingnew legislation aimed at increasing the sur-

veillance of imported seeds and their micro-biology.

Conclusions

There is no doubt that increasing the con-sumption of fresh fruits and vegetables isgood for a healthy diet. We should allunderstand that as a raw product that isgenerally grown in an outside environment,fruits and vegetables will have a large andvaried microflora on their outer surfaces.Some of these organisms may, on occasion,be human pathogens. Understanding good agricultural practices

should help reduce the instances whenpathogens can contaminate fresh produce,and this combined with good washing prac-tices and chilled storage during production,retail sale and domestic storage, will resultin the production of a healthy, nutritious andsafe product. It is clear from the amount ofready to eat fresh produce available to con-sumers, and the relatively low number ofoutbreaks, that many manufacturers under-stand the importance of good microbiologi-cal controls, and employ them effectively.However we must always remain aware of

the potential current and new microbiologi-cal hazards and maintain vigilance to ensurethat we employ suitable and effective con-trols at all times. nb food.testing.admin@thermofisher.com

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