Project Number: s-295 Title: Enhancing Food Safety Through Control of Food-borne Disease Agents Duration: October 1, 2000 to September 30, 2005

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Collaborative efforts among scientists have been formed from the original S-263 project. These collaborations have resulted in external funding and publications for the investigators. Additionally the collaborative efforts have made significant progress towards understanding food-borne pathogens and how to reduce their occurrence in the environment.

Objective 1. Pre-harvest reduction of food-borne pathogens in animals and the environment.

E. coli O157:H7 seldom causes disease in cattle (Cray and Moon, 1995). Prevalence in individual beef and dairy cattle in the U.S. is low, from 0.3 to 2.2% (Faith et al., 1996; Griffin and Tauxe, 1991; Hancock et al., 1994). E. coli O157:H7 exists on about 7% of farms (Faith et al., 1996). Typically, shedding of serotype O157:H7 strains from cattle is sporadic and of limited duration, lasting for approximately one month (Besser, 1997; Shere et al., 1998).

Two recent studies have investigated the effect of diet on acid resistance of E. coli shed from cattle. Diez-Gonzalez and co-workers (1998) examined the effect of high-grain diets on shedding of acid resistant E. coli from cattle. They found that the proportion of acid resistant E. coli increased in cattle fed a high grain diet, but that a switch to a diet of hay decreased the number of acid resistant bacteria. It is unclear if the results observed for E. coli in these experiments are relevant to serotype O157 strains of E. coli. Hovde et al. (1999) examined the acid resistance and the duration of shedding of E. coli O157:H7 from experimentally inoculated cattle fed hay or grain. They found no difference in the proportion of acid resistant E. coli O157:H7 between these two groups, but observed that animals fed hay shed E. coli O157:H7 longer than animals fed grain. Further studies are needed to clarify the role played by acid resistance on E. coli O157:H7 shedding from cattle.

A collaborative effort between John Foster at the University of South Alabama and Auburn investigators Stuart Price, Jim Wright, and Fred DeGraves resulted in a USDA-NRI Competitive Grant award in 1997. In that project, the Auburn investigators utilized an experimental O157 infection model in calves. We and others showed that the organism is detectable in the feces for 2- 3 weeks following inoculation with 1010 CFU ( Cray and Moon, 1995; Brown et al., 1997; Price et al., 2000), although calf-to-calf variation in shedding occurs. Just-weaned calves appeared to shed the highest number of organisms, and shedding quantity and duration decreases with age of the animal (Cray and Moon, 1995). Inoculation of calves with the high numbers of E. coli O157:H7 used in these infection models allows for extended periods of shedding at appropriate quantities for enumeration and comparison of shedding between concurrently administered strains.

Using this experimental model of O157 shedding from calves, the Auburn investigators examined the calf fecal shedding of acid resistant mutants of E. coli O157:H7 constructed by John Foster’s lab. These strains had mutations in genes involved in conferring acid resistance on O157. Results from the calf studies indicate that (1) RpoS, the stress sigma factor of E. coli, is required for survival of the pathogen in the animal, as is GadC, the antiporter protein involved in the glutamate-dependent acid resistance system; (2) inactivation of one of the two glutamate decarboxylase genes, gadA, in E. coli does not alter shedding, probably because the glutamate decarboxylase expressed by gadB is sufficient for survival in a calf, and (3) the arginine-dependent acid resistance system is not required for E. coli survival in a calf. In addition, the Auburn investigators have preliminary evidence showing that acid resistant wild-type cells of E. coli O157:H7 survive the calf gastrointestinal tract 100-fold better than do acid sensitive wild-type cells. The results of this collaborative effort have helped to better define the role played by acid resistance in survival of E. coli O157:H7 under environmental stress.

Additionally, the Auburn researchers have isolated five O157-specific strains of bacteriophage from bovine feces and begun characterizing their properties. These phage form clear plaques on 6 different E. coli O157:H7 strains, but do not form plaques on 6 non-O157 E. coli strains. Amplification of these phage strains results in titers ranging from 5 x 1010 to 1 x 1011 pfu/ml. Initial in vitro killing studies with two of these phage strains has shown that one of them is capable of killing 99% of O157 cells growing exponentially at 37oC. These preliminary results demonstrate the feasibility of isolating O157-specific phages, and, in conjunction with our expertise with the experimental calf model of O157 shedding, place us in a strong position to examine the practical application of phage-mediated O157 reduction in cattle. In fact, successful therapeutic use of phage in animals and humans was accomplished before the advent of antibiotic therapy in the 1940's, with few side effects reported (Alisky et al., 1998). Phage therapy is once again being considered as an alternative to antibiotic therapy due to the increase in antibiotic resistance.

E. coli O157:H7-specific bacteriophage have been isolated by several research groups (Kudva et al., 1999; Yu et al., 1998). Kudva et al. (1999) described a set of phage isolated from cattle that lysed O157 in vitro. However, complete lysis of O157 cultures was not achieved unless a mixture of three lytic phages was used, and the bacteria/phage mixture was incubated at 4oC for 5 days. The authors mentioned that this phage cocktail might function as an anti-O157 agent active at refrigerator temperatures on fresh vegetables contaminated with infected feces. And they suggested that other O157-specific phages that are active anaerobically at 37o C might be useful in removing O157 from carrier animals.

During studies with E. coli O157:H7-inoculated calves, the Auburn investigators noticed occasional “shedding spikes”, where the quantity of O157 being shed from calves increased by ten-fold or more over a 2-3 day period, then returned to the original shedding level. Although we could not link these intermittent increased shedding episodes to a specific stimulus, we speculated that changes in the intestinal flora precipitated increased levels of shedding. In a preliminary experiment designed to investigate this hypothesis, O157-infected calves were treated with the popular bovine antibiotic tilmicosin, suspecting that it would induce transient commensal flora changes. Half of the calves treated with tilmicosin showed O157 shedding spikes approximately 3 days post-antibiotic injection. This timing coincides with the appearance of tilmicosin in the intestinal tract following parenteral administration. The finding that a commonly used bovine antibiotic may induce O157 shedding from infected individuals is an important discovery, requiring further investigation.

As mentioned previously, Salmonella and Campylobacter cause a significant number of food-borne illnesses. Reduction of these organisms can also begin in a pre-harvest environment. The theory of probiotics, competitive exclusion (CE) and oligosaccharides have been well-documented (Goren et al., 1984; Miles, 1993; Newman, 1994; Spring, 1995). Single, two or three strains of lactic acid bacteria (LAB) have been successful in controlling pathogens in certain instances, but researchers have advanced other methods to aid in the battle against enteric pathogens. Bacteria other than LAB have also been used as feed supplements. Bacillus subtilis seemed to be an attractive prospect as a feed additive due to the fact that it is a spore-former with greater heat stability than most lactic acid cultures. However, some trials with Bacillus species have demonstrated no consistent benefit on growth performance, other studies have demonstrated increased survival rate in baby pigs which may involve the same sort of non-specific immune stimulation as seen with other peptidoglycans or oligosaccharides (Pollman D.S. et.,al. 1980; Pollman, D.S., 1986; Newman, 1995).

None of the above methods of controlling pathogens are 100% effective at eliminating the problem, but that does not mean that advances are not being made. In a survey of broiler hatcheries done in 1990 and 1995, salmonella positive samples in 1990 ranged from 91 to 67%, but the same hatcheries sampled in 1995 ranged from 52 to 13% (Cox et al., 1996). The authors attribute these reductions to the aforementioned management practices, the use of more effective sanitation chemicals, improved ventilation and should also include methods that control pathogens in the feed and prevent the spread of infection in a flock or herd. The use of organic acids, oligosaccharides and CE cultures are all tools that have been demonstrated to decrease or eliminate certain pathogens. The challenge of the future is to make further advances on pathogen control with the ultimate benefits for the animal and the consumer.

Competitive exclusion cultures are mixed microbial populations taken from adult mature birds that are found to be Specific Pathogen Free (SPF) have also demonstrated excellent results in protecting poultry species from Salmonella and Campylobacter infection. The theory behind the use of these cultures is to provide a more rapid and consistent intestinal microflora since it has been observed that healthy mature birds are less likely to become infected from pathogen exposure than a young bird with a dynamically changing microflora. Many trials have been completed which show marked reductions in pathogen colonization in birds treated with CE cultures (Hume et al., 1996; Newman et al., 1996; Stern et al., 1996). From these trials it seems clear that the effective dose rate is between 107 and 108 CFU/bird. Corrier and co-workers (1996) noted that in dose titration studies with a characterized CE culture, the most consistent results were observed when birds received these rates of application.

In addition to competitive exclusion, recently a number of progressive studies involving oligosaccharides have been reported. Spring (1995) reported a decrease in Salmonella typhimurium concentrations in challenged chicks receiving mannanoligosaccharide (MOS) in the diet compared with unsupplemented chicks. Further studies have demonstrated that chicks challenged with S. dulblin and Escherichia coli also benefited from MOS supplementation. The use of mannanoligosaccharide for pathogen control stems from several studies examining the use of simple sugars such as lactose or mannose, to reduce pathogen infection (Oyofo et al., 1989; Corrier et al., 1990). The mechanisms involved in this phenomenon involve either lowering of intestinal pH and alterations in volatile fatty acid (VFA) profiles of the intestine, as is seen with lactose (Atkinson et al., 1957; Corrier et al., 1990) or by occupying mannose specific receptors on certain pathogens mediating their adherence to the intestinal epithelium (Freter and Jones, 1976; Newman, 1994). Complex carbohydrates such as fructooligosaccharides (FOS) have also been investigated for nutritional manipulation of the gastrointestinal tract to inhibit pathogens. Oyarzabal and co-workers (1995) found that Salmonella spp. could not use a purified fructooligosaccharide preparation for growth but were able to utilize a commercial preparation of FOS.

Mechanisms involved in Campylobacter infection and resistance have also been studied by the scientists in this group at the pre-harvest food safety level. CipR . High Level CipR (HLCR) isolates (defined as MIC 16 ug/ml) were generated in the laboratory on Brucella Agar with Sheep’s Blood (BASB) containing 2 ug/ml Cip via spontaneous mutation at a frequency of approximately 1 to 5 per 108 viable cells. This rate of mutation suggests that HLCR can develop by a single step mutation. HLCR isolates have been generated from 4 other C. jejuni strains at similar frequency. Eleven independent HLCR isolates contained the same C to T transition in codon 86 of the QRDR of gyrA. 30 CipS C. jejuni strains and did not contain the codon 86 mutation (or any other QRDR mutation). HLCR could be acquired by CipS isolates of the parent strain via natural transformation using chromosomal DNA at high frequency (1 per 107 recipient cells) but not by CipS isolates of a different strain.

A rapid assay for identification of C. jejuni and CipR has been developed. The QRDR region in the gyrA gene is highly conserved among isolates of C. jejuni (Wilson et al., 1999) but differs significantly from C. coli and other bacteria at 3 specific locations. Based on this unique region, we designed a rapid, real-time polymerase chain reaction (basic TaqMan assay), that can identify and enumerate Campylobacter jejuni using DNA purified from bacterial isolates. The assay detects the genomic DNA from the equivalent of 10 cells consistently and as few as 1 cell in certain assays. A modification of the basic assay, called “Allelic Discrimination”, simultaneously identified C. jejuni and discriminated between wild-type and mutant codon 86. The assay specifically detects HLCR C. jejuni isolates (Patent Application Submitted). Recently we demonstrated that both assays (basic TaqMan and Allelic Discrimination) work effectively on cells from isolated bacterial colonies without the need to purify genomic DNA. The assay can be performed in a single afternoon reducing the time of analysis for CipR by several fold when compared to our microbroth dilution assay.

c. ErmR. A novel gene encoding a putative rRNA di-methylase (tentatively called ermF) was recently identified by a Blast search (a computer aided search for sequence identity) of the C. jejuni genome using ermF2 from Bacteroides fragilis (Rasmussen et al., 1986) as the query sequence. An open reading frame was identified with similar size and significant identity (30% identity, 42% similarity) to ermF2 and to erm genes which confer MLS type of acquired ErmR . Because ErmR Campylobacter isolates are cross-resistant to lincosamides and streptogramin B (hallmarks of MLS type acquired resistance) and because not all ErmR isolates studied to date contain mutations in Domain V of the 23S rRNA gene (Trieber and Taylor, 1999) we propose that ErmF, a putative RNA di-methylase, contributes to acquired ErmR in C. jejuni via methylation of 23 S rRNA.

At Michigan State University, they determined the minimum inhibitory concentration (MIC) values for 5 antibiotics (Cip, Tet, Kan, Erm, Chloramphenicol) on 24 isolates in our Campylobacter strain collection using a microbroth dilution assay based on National Committee for Clinical Laboratory Standards (NCCLS). The assay normally can be completed in 48 hours on bacterial isolates.

Additionally, they have successfully transformed C. coli and C. jejuni isolates using DNA isolated from antibiotic resistant isolates (KanR and CipR) derived from the same strain (intra-strain transfer) but have not been successful at inter-strain or inter-species transfer suggesting that natural “barriers” to transfer exist. In future work we will characterize the mechanisms that underlie the apparent “barrier” to inter-strain transfer and to determine if environmental stress can increase the rate of DNA uptake or integration into the chromosome. Collection of Campylobacter isolates is underway through a collaboration with researchers at MSU, the Michigan Department of Agriculture, and with Dr. Paula Cray at the USDA in Athens GA, and Dr. Fred Angulo at CDC. Further collaborations will be developed with other scientist in the S-263 group who are collecting Campylobacter isolates from the pre-harvest and food processing environment.

Objective 2. Chemical and Physical Decontamination in Food Processing Plant Environments

As stated earlier, a wide range of chemical treatments to reduce the bacterial load on raw foods have been investigated. Relatedly, a wide range of methods have been used to determine efficacy of the various treatments. Although many of these treatments have been efficacious in laboratory experiments, few of these carcass disinfectant treatments have been adopted by the food industry. Below is a brief discussion of some of the approaches to food decontamination that have been researched. The purpose of this discussion is to illustrate the wide variety of treatments that are potentially available to food processors if there were better testing means available, and to also illustrate that conflicting data are often reported. This latter problem is also methods-related.

Decontamination of Meat and Poultry. To address control and elimination of bacterial contamination of poultry and beef, a number of antimicrobial treatments for carcasses have been studied, including chlorine (Mead et al., 1975) organic acids/salts (Adams et al., 1990; Lillard et al., 1987; Robach, 1979; Zeitoun and Debevere, 1992), ozone (Sheldon and Brown, 1986), hydrogen peroxide with sodium bicarbonate ( Bell et al., 1997, Russell et al., 1993), air scrubbing (Dickens and Cox, 1992), trisodium phosphate (Bender, 1992), etc. Today, chlorine continues to be the predominant, product-contact disinfectant used for commercial poultry processing in the U.S. Water, steam, and to a lesser extent, organic acids are used sparingly during the processing of beef.

For poultry processing, chlorination of chill water has been widely studied. It is known that bacteria washed from carcasses into the chill water can be controlled by chlorination of the water, thereby preventing cross contamination (Lillard, 1982). Mead and Thomas (1973) concluded that 40 - 50 ppm of total residual chlorine and 5 liters of fresh water per carcass effectively destroyed bacteria. Comparable results were found using 25 - 30 ppm and an overflow of 8 liters. These overflow rates are higher than the 0.5 gallon per bird required by USDA. Lillard (1980) reported that significant bacterial reductions in chill water were obtained using 20 ppm chlorine and an overflow rate of 0.5 gallon. May (1974) reported that 18 - 25 ppm chlorine added to immersion chilling systems significantly reduced bacterial counts on poultry carcasses. Similar results were reported by Kotula et al. (1962). Water containing chlorine in excess of 50 ppm has been shown to be more effective in reducing bacterial populations than in water containing lower amounts. Ranken et al. (1965) evaluated levels of chlorine ranging from 50 to 800 ppm in a slush ice immersion for 4 hours. Reductions in bacteria were not significant until the level of chlorine reached or exceeded 200 ppm. At 200 ppm chlorine, no off-flavor was reported. Conversely, Izat et al. (1989), reported that100 ppm chlorine in chill water effectively reduced salmonellae, but resulted in carcasses that exhibited a strong chlorine odor. Chlorine at 100 ppm, 200 ppm, and 400 ppm reduced bacterial counts significantly on beef forequarters within 24 hours of the chlorinated wash (Emswiler et al., 1976). Stevenson et al. (1978) , however, reported no significant reductions in bacterial counts when using a 200 ppm chlorine spray on beef carcasses.

The effectiveness of chlorine as a bactericidal agent is dependent upon the conditions in which it is used in processing, including concentrations of chlorine, temperature, and chemical composition of the water. Low levels of chlorine are effective in reducing bacterial counts on carcasses, but only if the amount of water per carcass is high and the relative amount of organic matter is low. Higher levels of chlorine are effective, but produce off-flavor and carcass discoloration. Furthermore, a high level of chlorine must be handled properly by plant personnel because of skin irritation, and equipment corrosion.

Organic acids have been investigated because of their bactericidal activity and because they are generally recognized as safe (GRAS). Acetic and propionic reportedly have the most inhibitory effect against salmonellae (Chung and Goepfert, 1970). Mountney and O'Malley (1965) reported acetic, adipic, and succinic as the most effective, however, when applied in poultry chill water skin discolorations were observed. Significant reductions in bacteria were reported by Dickson and Anderson (1992) with pre- and post- sanitizing of beef with 55oC acetic acid. Dickson (1991) reported reductions in S. typhimurium, up to 3 logs, when acetic acid was included as a sanitizer in a beef spray chilling system. Lillard et al. (1987) were unable to detect salmonellae in scald water containing 0.2% and 0.5% acetic acid, however, the treatment did not reduce salmonellae numbers on the carcasses. The bactericidal activity of acids has been shown to increase with an increase in concentration (Dickson and Anderson, 1992). However, the higher concentrations of acids which are usually more effective produce undesirable product (sensory) characteristics (Bilgili et al., 1996).

Decontamination of Produce. A variety of different microorganisms comprise the natural microflora of fresh fruits and vegetables. However, it is contamination with pathogen microorganisms, specifically bacteria, that is at issue in regards to food safety. Produce can be contaminated during production, harvest, packing, post-harvest processing, and distribution. The primary bacteria of concern are those of fecal origin (human or animal). At preharvest, primary sources of pathogens include feces from wild or domestic animals, human feces, soil, and irrigation water. At postharvest, pathogens can arise from feces, workers, equipment, animals, processing water, ice, and other food products (Beuchat, 1996; Tauxe et al., 1997)

Although fresh produce can harbor as many as 106 microorganisms/g at time of packing (Brackett, 1994), little attention has focused (until recently) on antimicrobial treatments for fresh produce. Because fresh fruits and vegetables are often soiled at packing, washing treatments are applied. Washing treatments result in overall reductions in microbial counts, but washing alone typical does not produce adequate sanitation of the product (Beuchat, 1992). For disinfection, a number of treatments have been researched, including chlorine, hydrogen peroxide, trisodium phosphate, ozone, acidified sodium chlorite and gamma irradiation.

At the University of Nebraska, hydrogen peroxide was found to be an effective decontaminant of produce. Hydrogen peroxide was used at the 3% level as a wash for a variety of produce, alone or in combination with 2 or 5% acetic acid. The washes were applied by dipping or spraying. Green peppers and lettuce were inoculated with Shigella spp., broccoli and cherry tomatoes were inoculated with Salmonella spp. The most effective washes were the combination washes with the 3% hydrogen peroxide and 5% or 2% acetic acid. A 4-5 log reduction was common and generally eliminated the inocula except on the melon skins. A 4 log reduction was found on the smooth skin and only a 3 log reduction on the rough skin. The 3% hydrogen peroxide alone was very effective on the green peppers and tomatoes.

Chlorine-based compounds are the most widely used product disinfectants. However, efficacy tends to be highly variable. Chlorine can reduce populations on lettuce and tomato surfaces (Beuchat and Brackett, 1990, 1991). However, Zhuang et al. (1995) found that Salmonella survived on tomatoes treated with < 200 ppm chlorine, and Brackett (1987) found L. monocytogenes to be resistant to 50 ppm chlorine on brussel sprouts. Beuchat (1996) recommended 200-300 ppm chlorine as a wash water sanitizer. However, Hong and Gross (1998) found that hypochlorite treatment of tomatoes negatively affects physiological and biochemical properties, which in turn can influence ripening.

In addition to potential interference with ripening processes, aqueous chlorine’s bactericidal activity decreases in alkaline conditions and/or at high levels of organic matter. Furthermore, potentially toxic/mutagenic reactions products, including trihalomethanes (THMs), are formed during chlorine treatment of food components. Health concerns for such reaction products indicates a need to explore alternative disinfectants. Due to its advantages over aqueous chlorine, chlorine dioxide (ClO2) has received much attention from the food industry. ClO2 was seven times more potent than aqueous chlorine in killing bacteria in poultry processing chill water (Lillard, 1979). Bactericidal activity of ClO2 was not affected by alkaline conditions, such as the pH 6-10 of most food processing operations, and/or the presence of high levels of organic matter (Costilow et al., 1984). Unlike chlorine, ClO2 does not generate THMs from interaction with organic compounds.

Currently, the Food and Drug Administration (FDA) allows the use of 5 ppm ClO2 as a disinfectant in rinse water that comes in contact with whole, unpeeled fruits and vegetables, provided the final product is rinsed with potable water. Potatoes (cut and peeled) can be rinsed with 1 ppm ClO2. The National Food Processors Association (NFPA) in its food additive petition to FDA requested the agency to approve the use of ClO2-treated water for cut or peeled produce, and to eliminate the potable water rinse requirement when ClO2 is used at 5 ppm (Food Chemical News, 1994). FDA announced on March 3, 1995 the clearance of a food additive petition allowing the use of 3 ppm residual ClO2 to control the microbial population in process waters contacting whole poultry carcasses (FDA, 1995). The Bio-Cide International, Inc. also filed petition with FDA for approval of using ClO2 for seafood processing and washing. ClO2 is currently used as a component of a sanitizer solution, a bleaching agent for flour, and is approved by the EPA for use in potable water treatment plants.

Trisodium phosphate (TSP) has recently been approved and marketed as a food grade antimicrobial. Presently it is being adopted by the poultry industry as a means of reducing pathogens numbers on chickens during processing and seems to be effective against attached pathogens (Tamblyn et al., 1997). The use of TSP as a produce sanitizer has also been investigated. Dipping tomatoes into a 15% TSP solution for 15 seconds provided effective kills of S. montevideo on surfaces of the fruits (Zhuang and Beuchat, 1997).

Ozone through its oxidative properties has excellent ability to kill many types of microbes, however, its oxidative nature also limits its use in many foods. Fresh produce, which rely on many oxidation-sensitive mechanisms to ripen are therefore negatively affected by direct application of ozone (Hovrath et al., 1985). Ozone does appear to have potential for treating water for reuse.

Acidified sodium chlorite (ASC) has recently been developed by Alcide, Inc., and is being marketed as a antimicrobial for poultry processing under the trade name Sanova. ASC is effective against bacteria attached to chicken skin (Alcide, unpublished data) and more recently Alcide has been granted FDA and EPA approval of ASC as an antimicrobial treatment for fresh produce. Data collected in field trials indicate that ASC has potential as an effective sanitizer for a variety of fresh fruits and vegetable (Alcide, unpublished data).

In contrast to the amount of previous work reported about the use of antimicrobial treatments, there is little information available on the validation of implemented HACCP plans. The National Advisory Committee for the Microbiological Criteria for Foods (NACFMS) standardized the seven HACCP principles (NACMCF, 1998). These principles can be utilized by the food processing industry to identify hazards that might occur in their particular food and/or process and to identify critical control points (CCPS) that can prevent that hazard from occurring. The National Academy of Sciences has recommended that government agencies adopt HACCP so that all food processors operate under these principles (NRC, 1995). These recommendations were based on years of development of HACCP principles by experts in the areas of food microbiology and processing.

On December 18, 1995, the FDA published a final rule requiring all seafood processors to operate under HACCP while the USDA published a similar final rule in July of 1996 that required HACCP implementation in meat and poultry processing plants. Many meat and poultry processors have been faced with challenges in HACCP implementation because of the lack of validated CCPS and the lack of scientific evidence to support critical limits or to dictate when a corrective action was needed.

Specifically, there is little information available related to room temperature and product temperatures and how that impacts microbial growth. Currently many processors set a critical limit of 50oF as the critical limit for processing room temperature. If the processing room temperature reaches 51oC, then all product processed in that room since the last recorded observation is subject to a corrective action. This is a costly consequence. Product may have to be reworked, destroyed, or held for further analyses. Many times the room temperature can be reduced within a few minutes and the product temperature was probably not significantly impacted. However, as the regulations are currently written, a corrective action still must be taken because there is not scientific evidence that the product temperature was safe when the room temperature increased. Many relationships between time and substrate temperature and between substrate temperature, and time and room temperature (unsteady-state heat transfer) have been observed and established. Many thermal properties of meat as a substrate are known. However, the integration of unsteady-state heat transfer relationships for meat products in preparatory environments with microbial activity relationships has been very limited or nonexistent.

Developing time/temperature formulas that would assist processors in correlating the room temperatures to product temperature would be a valuable tool that could save time and money and would provide a scientific basis for taking corrective actions and for making scientifically sound decisions about whether or not a product is safe.

There is also little information related to the overall reduction of microbial risks due to HACCP implementation. Dormedy et. al. (2000) established microbial baselines in a beef slaughter facility, a beef fabrication facility, and in a ground beef facility in Nebraska prior to HACCP implementation. University extension specialists assisted the processors in writing HACCP plans and then collected samples to compare to the baseline 1, 3 and 6 weeks after HACCP implementation. While all of the processors noted no change in the processing environment after HACCP implementation, the total plate counts on the products were significantly reduced after HACCP implementation. Generic E. coli and coliform counts did not significantly increase or decrease after implementation, but they were well below USDA baselines prior to HACCP implementation. This type of data validates the use of HACCP to create a cleaner environment using HACCP in a food processing facility. The data was used by the processing facilities to support their HACCP plans and to help them meet USDA requirements.

Dormedy et al. (2000) also determined the effectiveness of acid rinses on beef carcasses as critical control points in HACCP systems. While there was a wealth of information published about the impact of acid rinsing on small pieces of meat inoculated with pathogens, there was no information indicating that it would effectively reduce microbial loads naturally present in the plant environment and that the product would remain safe during further processing. They reported that a 2% lactic acid rinse of beef carcasses reduced total counts, coliform counts, generic E. coli counts, pseudomonads, psychrotrophic organisms, lactic acid bacteria, and acid tolerant organisms compared to carcasses that were not acid washed. Additionally, the same microbial populations remained low after chilling and during further processing of the carcasses into sub-primal cuts and ground beef. While elimination of microbial hazards is not possible in a fresh meat system, their results validated the use of acid rinsing as an effective CCP in a fresh meat system to reduce microbial hazards.

Processing facilities also need data to support changes in their HACCP plans. A poultry processor was utilizing a 40 psi carcass wash as a CCP to meet USDA requirements for 0 fecal contamination. The wash resulted in quality problems in the final product so the processor wanted to reduce the pressure to 30 psi. The USDA would not allow a change in the HACCP plan without statistically sound, scientific data to support the change. The food microbiology laboratory at the University of Nebraska compared microbial and visual data on 1250 carcasses from the plant that had been subject to the two different wash pressures. Data indicated that the 30 psi resulted in a carcasses that was both visually and microbially equivalent to a 40 psi wash. After review by the USDA regional inspector, the plant was allowed to adjust the CCP and to increase the quality of their product.

In plant data needs to be gathered nationwide to validate CCPs, change CCPs and provide information that define critical limits that will reduce hazards in food processing plants through the use of HACCP plans. Studies provide a model of how to collect in-plant data and how to work with regulatory agencies to use the data to change HACCP plans. Information can be used in not only the meat and poultry industry, but in all food processing operations.


Objective 1: Pre-harvest reduction of food-borne pathogens in animals and the environment

Aim 1: Development and optimization of therapeutic methods to eliminate or reduce E. coli O157:H7 from cattle.

  1. Effect of acid resistance-inhibiting analog drugs on E. coli O157:H7 shedding from experimentally infected calves.

  2. Screening and use of O157-lytic bacteriophage to clear O157 from infected calves.

  3. Management of tilmicosin treatment of cattle to prevent antibiotic-induced O157 shedding from infected calves.

Aim 2: Preventive natural barriers to the colonization of food borne pathogens

Aim 3: Defining food borne pathogen survival in manure and manure-amended soil use for fruit and vegetable production

Aim 4: Development of Methods to detect pathogens in pre-harvest environments and monitor rates of development and transfer of resistance to antibiotics.

Objective 2. Chemical and Physical Decontamination in Food Processing Plant Environments

Aim 1. Develop Method for Determining the Efficacy of Pathogen Reduction (Decontamination)Treatments for Raw Food Commodities

Aim 2. Validation of the effectiveness of HACCP Systems in Food Processing Plant Environments

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