EP0439597A1 - Method of enhancing microbial stability of partially prepared refrigerated foods - Google Patents

Abstract

Method for inhibiting the growth of pathogenic bacteria in refrigerated food using a hydrolysis mixture of an aldonic acid and its lactones or a precursor thereof and the food thus produced.

Description

"METHOD OF ENHANCING MICROBIAL STABILITY OF PARTIALLY PREPARED REFRIGERATED FOODS"

BACKGROUND OF THE INVENTION

Recently, consumer demands have been directed toward foods that are nutritious, retain their original color and taste, are ready to eat with minimum preparation, but are not frozen, dried, or canned. The food industry's major thrust in response to this demand is the generation of new types of foods preserved primarily through refrigeration. These new foods, which may be called partially prepared foods often, but not always, receive a blanch or heat process or some other preservation treatment which reduces their microbiological load, but does not produce commercial sterility. They are non-sterile and require refrigerated storage to avoid rapid bacterial deterioration. Such foods have included salads, ready-to-eat vegetables, sauces, gravies, seafood items, and other products. Even under refrigeration, the shelf life of some of these foods is only a few days or, at most, a few weeks.

The long established methods of food preservation such as freezing, drying and canning have minimized the problems of microbial spoilage of foods. In the case of canning, exhaustive studies have been made of the species of organisms that can grow under the anaerobic conditions inside a can and of the heat conditions in a retort necessary to kill these organisms. Thus, although in the past there have been occasional and disturbing exceptions, microbial contamination of canned foods by pathogenic organisms in commercially canned foods is rarely a problem. If the prescribed thermal process is correctly applied, the canned food product is rendered commercially sterile and the food contents are contained under anaerobic conditions until the container is opened. Upon opening, however, the canned food is subject to airborne contamination by aerobic organisms and must be refrigerated the same as the partially prepared, chilled foods that are the main subject of this application.

Refrigerated partially prepared foods are not sterile and may contain many types of organisms not found in the sterile canned food. From the advent of mechanical refrigeration after World War I until recently, it was assumed that refrigeration in the range of 35°F to 45°F was adequate to prevent the growth of pathogenic bacteria and would provide shelf life for partially prepared foods (as well as leftovers) of a few days up to several weeks, depending on the type of product and the amount of bacterial contamination present.

Typically, serious cases of food poisoning have been associated mainly with bacteria like Ataphylococcus aureus and Salmonella tvphimurium. These organisms do not propagate at refrigeration temperatures, but are commonly associated with handling abuse.

In recent years, however, a number of illnesses and deaths in the consuming public have been traced to psychrotrophic pathogens. Psychrotrophs are defined as organisms which may propagate at temperatures normally regarded as refrigeration temperatures, i.e. 34 to 45 degrees F. These organisms, because of their unique nature, are capable of not only surviving but actually thriving under refrigeration. Such organisms have been encountered in foods on a large scale only recently with the introduction of refrigerated foods on a large scale.

Temperature abuse, that is, exposure to temperatures above

45°F, tends to accelerate the growth of most of these organisms. Furthermore, slight temperature changes can have a profound effect on the growth of psychrotrophic pathogens and spoilage organisms. In one dramatic example, a strain of Pseudomonas fluorescens, a psychrotrophic spoilage organism stored at 32 F and 32.9°F exhibited generation times of 30.2 and 6.7 hours

respectively. Thus, a temperature increase of only 0.9°F resulted in nearly a five-fold increase in the bacterial growth rate.

The hazards associated with foodborne psychrotrophic pathogens have only recently been recognized and publicized by federal regulatory agencies. Yersinia enterocolitica, Listeria monocytoqenes, enteropathogenic Escherichia coli, Clostridium botulinu type E, and Aeromonas hydrophila are such psychrotrophs.

There follows a brief description of some of the microbes which cause difficulties to the processor of partially prepared foods.

Yersinia enterocolitica and other Yersinia enterocolitica- like bacteria are known to cause gastroenteritis accompanied by violent abdominal pains that mimic appendicitis. In a recent outbreak, chocolate milk served at school lunches was found to be contaminated. Reconstituted powdered milk and turkey chow mein were the vehicles in the second outbreak which occurred in a children's day camp. In both outbreaks, several children underwent appendectomies before the bacterial nature of the illness was ascertained. Since 1985, the status of L ster a monocy oqenes as changed from being regarded as an organism known to only a handful of microbiologists to being a fully recognized foodborne pathogen of concern. The ability to grow at refrigerated temperatures and tolerance to the preserving agents sodium chloride and sodium nitrite, make Listeria of particular concern as a contamination agent for refrigerated food. Its pathogenicity includes meningitis, fetal abortions, and other diseases with a total 35% mortality rate.

Bacteria of the species Escherichia coli were long thought to be relatively harmless, but now it has been discovered that enterotoxigenic strains of Escherichia coli have the ability to grow and produce toxin under refrigeration conditions. Enterotoxigenic strains of E. coli produce heat labile and/or heat stable enterotoxins. The organism is believed to be the cause of most travelers' diarrhea and other dysentery-like symptoms.

In the last 5 years, much has been written about the hazards of psychrotrophic organisms in refrigerated foods as the analysis of foodborne illnesses becomes more sophisticated. Very little has been published regarding solutions to these problems, beyond

the rather obvious factors of good sanitation, lowering refrigeration temperatures, avoiding temperature abuse, and the addition of microbiological "hurdles" such as acidification, reduction of water activity, addition of preservatives, and modified atmosphere packaging.

While acidification has long been recognized as a means to slow the growth of many genera of bacteria, its broad use has been avoided because of the flavor problems created when most common food grade acids are added to partially prepared foods. Furthermore, in the past, it has been shown that different acids have different inhibiting effects when used to achieve the same pH in a given food, and that different genera of bacteria react differently to an acid environment. However, very little research has been done to explore the effect of specific food acidulants on the psychrotrophic pathogens that have been so recently discovered as the cause of many foodborne illnesses and deaths. This invention deals with the finding that hydrolysis mixtures of aldonic acids and their lactones are exceptionally effective in preventing the growth of many psychrotrophic pathogens in food, even at abuse temperatures, and that these mixtures can be used in a wide variety of chilled foods without destroying the flavor appeal of the food.

The need for providing such additional barriers or hurdles to slow microbiological growth of psychrotropic pathogens and other organisms is highlighted by the well documented difficulties in controlling the processing and distribution of partially prepared chilled foods.

Food products are subject to bacterial contamination at any of several points in their route from the garden or other source to the table. These danger points include processing, packaging, transportation, handling for display and vending and final consumer preparation for consumption.

Processing, packaging, and at least the initial phases of transportation, are normally under the control of trained professionals so that the food is relatively safe during these steps of the route. Display in the retail marketplace, be it a large supermarket, a small convenience store, a delicatessen, or other food shop, is often under conditions which expose the food to temperature abuse. The food which may initially have been frozen or refrigerated is often displayed in refrigerated cabinets or salad bars where it is subjected to intermittent or continuous temperatures well above temperatures which are

regarded as refrigeration conditions. The food may reach temperatures as high as 50°F or even higher during^ the display phase of the cycle. Additionally, during this display period, the food is subject to handling by the vendors and the consuming public, for instance in self serve salad bars. Such handling may lead to direct or cross contamination.

In the hands of the household or institutional consumer, the food may undergo even more serious temperature abuse before it is consumed, often without any cooking or further preparation.

Each of these temperature abuse intervals presents pathogenic bacteria the opportunity to grow in the food.

Several procedures have been devised to assure the safety of food by providing barriers to bacterial growth.

One of the earliest methods of preserving foods is the fermentation procedure such as is employed in the production of sauerkraut or pickles. In this procedure, the food is permitted to ferment and produce lactic acid through the action of lactobacilli thereby to decrease the pH and inhibit growth of other bacteria. Neither this procedure nor the analogous

procedure of directly adding acids εuch as lactic, acetic, citric, and the like are satisfactory with most partially prepared foods since conventional acids impart a strong acid taste which is unacceptable to many consumers and difficult to mask.

Another procedure is to package the food in an oxygen-free or limited oxygen atmosphere. This so-called modified atmosphere technique is not always satisfactory because it adds another step to the processing and packaging procedure, is expensive, and requires the use of gas barrier films and bags to provide protection against oxygen. Moreover, it does not protect against anaerobic bacteria such as Clostridium botulinum type E, which do not require oxygen for growth. Such organisms flourish in an oxygen-free modified atmosphere environment because they may grow without competing for nutrients with aerobic bacteria which would otherwise be naturally present in the food in the absence of modified atmosphere packaging.

The art has also employed fixed date of sale procedures which suggest that the food not be sold after a date imprinted on the package or on a label attached to a package. This procedure is subject to the exigencies of the trade. In some retail

outlets, it is followed strictly and provides an additional safety barrier to the consumer. In other outlets, it may be virtually ignored.

Perhaps the least successful of the techniques has been attempts to train food handlers in delicatessens, school cafeterias, restaurants, and similar food outlets, or to educate the consumer. The efficacy of the training procedure is reduced by the rapid turnover of personnel normally employed by such establishments. Consumer education has not been completely successful due to the inattention of consumers or their inability to apply the preferred procedures. Most household refrigerators cannot steadily maintain temperatures as low as 40°F or lower during frequent use periods.

The likelihood of ingesting large numbers of pathogenic bacteria from refrigerated foods designed to be heated prior to consumption has been increased by the recent broad use of microwave ovens by consumers. Microwaves can warm refrigerated foods rapidly to serving temperature without ever subjecting pathogenic organisms which may be present in the food to the long exposures at high temperature necessary to destroy the pathogens.

Moreover, many bacteria can contaminate food and cause illness without causing any change in the outward appearance or flavor of the food. No amount of training or education can completely overcome these obstacles to food safety. It is, of course, impractical and prohibitively expensive to keep a trained professional equipped with the necessary instrumentation and other bacterial testing tools in every retail food outlet.

One of the more difficult aspects of the problem of bacterial contamination of refrigerated foods is the problem of liability in the event of consumer illness or death attributable to contaminated food. This problem has discouraged some qualified food processing companies from entering the field and has caused others to withdraw their products. Companies may produce their products under the most carefully controlled conditions, adhering conscientiously to all good manufacturing practice techniques and government regulations. However, once the product has entered the transportation, selling and consumer preparation stages, even though it may no longer be subject to company control, the processing company still bears the legal risk for damages because their name is on the label of the product.

The art, therefore, has long sought a procedure for producing non-sterile, refrigerated, partially prepared foods which are convenient and attractive in taste and appearance while, at the same time, avoiding the problems aforesaid.

THE INVENTION

It has now been discovered that the growth of bacteria, particularly pathogenic psychrotrophic bacteria, can be inhibited in non-sterile, refrigerated, partially prepared foods, especially salads containing fruits, pasta, vegetables, meat, poultry or fish products, which will be subject to refrigeration with or without prior freezing, by combining the food during processing, i.e., any operation which takes place prior to refrigeration, with a sufficient amount of a hydrolysis mixture of an aldonic acid and its lactones or a precursor thereof to reduce the pH of the foodstuff prior to refrigeration to a value which is less conducive to propagation of pathogenic organisms.

The process of the invention is applicable to partially prepared non-sterile foods, including salads such as pasta and seafood salads; sauces; meats; and meat mixtures such as meat balls, meat loaves and stuffed peppers. The process of the

invention is particularly useful with salads, particularly pasta, meat, potato, poultry, fruit, vegetable, and seafood or other salads containing these foodstuffs. It is particularly useful for foods of the type typically displayed in freezer cabinets, lunch counters, and salad bars in supermarkets, delicatessens and school cafeterias. It is these foods which are often subject to handling temperature abuse thereby providing the opportunity for pathogenic microorganisms to multiply.

It has been discovered that utilization of hydrolysis mixtures of aldonic acids and their lactones, or precursors thereof can be employed to create an environment in the foodstuff which is not conducive to bacterial growth especially growth of the newly recognized psychrotrophs such as those mentioned above. The preferred agent for acidification is a hydrolysis mixture of gluconic acid and its lactones or precursors thereof. This mixture is generally referred to as GDL and will be εo identified in this specification and claims. For convenience, the invention will be principally described as it applies to GDL. It will be understood, however, that what is said about GDL is also applicable to the other pH reducers used in the invention.

The particular advantage of the process of this invention is that the pH reduction sufficient to inhibit the growth of pathogenic microorganisms which multiply at refrigeration temperatures or under temperature abuse conditions can be effected without imparting an objectionable acid taste to the treated food such as results from utilizing even small amounts of those acids normally employed for the acidification of food such as acetic, citric, lactic, malic, and tartaric acid.

As will be apparent from the examples, the extent of the reduction in pH necessary to inhibit bacterial growth will vary from food to food and from one species of bacteria to another. This is because each food has its own natural pH and bacteria vary in their nutritional and pH requirements under refrigeration conditions. For example, foods with a natural pH above 4.6 are generally regarded as low acid foods, and those with a natural pH below 4.6 are regarded as acid foods. For purposes of this disclosure, the natural pH is the pH of the food or food mixture e.g. the salad. If there is no mixture, the natural pH is the pH of the applicable food. Generally speaking, it has been observed that reducing the natural pH of the food from about .5 to 2.5 pH units in accordance with the invention is sufficient to retard and often to inhibit pathogenic bacterial growth, particularly pεychrotropic bacterial growth. It is surprising to find that with the acidification agents of this invention, it is possible to reduce the pH to an effective inhibitory or lethal value without significantly adversely affecting the flavor or other organoleptic values of the foods since acids normally impart an acid flavor to foods which limits their acceptability for human consumption. The natural pH of a foodstuff may be determined by measuring the pH of the food prepared without added GDL.

Clearly there are advantages to reducing the pH as much as possible since the lower the pH, the more inhibitory^* the environment for most bacteria. It is, therefore, a major advance in the art to discover an antibacterial acidification agent which can be employed to achieve these desirable pH values without objectionable interference with organoleptic values.

Another factor in the practice of the invention is the period of time the treated food is reasonably expected to be exposed to the danger of contamination. In fast turnover situations, it may not be necessary to lower the pH as much as it would be lowered when the period of possible contamination is longer.

Still another is "leftovers," i.e., food which is removed from the home refrigerator or commercial display case for serving, a portion of which is then returned to refrigerated storage. The food returned to storage will invariably have been subjected to temperature abuse and the possibility of further bacterial contamination.

The aldonic acids which can be employed in this invention are prepared for example by oxidation of sugars or aldoses, preferably from those having six carbon atoms, although they could be prepared from those having five carbon atoms. Those acids prepared from sugars having six carbon atoms are talonic, galactonic, idonic, gulonic, mannonic, gluconic, altronic and allonic (although currently these acids, with the exception of gluconic, may be unavailable commercially) . These acids are respectively derived from their aldoses — talose, galactose, idose, gulose, mannose, glucose, altrose and allose. Sugars having five carbon atoms are lyxose, xylose, arabinose and ribose. Those skilled in the art will understand from this disclosure regarding six and five carbon atom aldonic acids, that other acids which form their own lactone(s) and mixtures of other acids and their lactones, which perform the same functions and objectives of this invention, particularly regarding lowering the

pH and regarding lack of an objectionable acid taste in the treated foodstuff, would be within the scope of this invention. For example, aldariσ acids, i.e., dibasic acids such as glucaric which forms saccharo lactone, might be employed.

The preferred method for providing the aldonic acid and its lactones to the foodstuff is to combine the foodstuff with a precursor of the aldonic acid. A precursor of the acid herein means a liquid, material or compound which adds the acid to, or forms or provides it in the foodstuff with which it is combined. Again, when the acid contacts moisture or water in or of the foodstuff, it will convert partially to and will co-exist with its lactones.

Precursors of these acids which can be employed include their lactones themselves (which can be said to be latent acids since they hydrolyze in water to form a mixture of the acid and its lactones) , mixtures of these lactones, and salts of the acids in combination with certain strong acids. For example, precursors of the preferred gluconic acid which can be employed include glucono-delta-lactone, glucono-gamma-lactone, mixtures of these lactones, and gluconate salts in combination with the strong acid, hydrochloric. By far, the most preferred precursor

for this invention is glucono-delta-lactone (GDL) . It is commercially available in food grade as a free-flowing, odorless white powder. It has an initial sweet taste. Food grade solutions of GDL are also commercially available and can be employed. GDL is an inner ester of gluconic acid which when hydrolyzed forms gluconic acid. Hydrolysis occurs when GDL is combined with water, for example that of an aqueous brine or in the foodstuff. Hydrolysis of the glucono-delta lactone results in an equilibrium mixture of from about 55% to about 60% (by weight) gluconic acid and from about 45% to about 40% (by weight) of a mixture of glucono-delta lactone and glucono-gamma lactone. The rate of acid formation during hydrolysis is affected by the temperature, the pH value and concentration of the solution. Hydrolysis of delta lactones tends to be more rapid than hydrolysis of gamma lactone.

Examples of those salts which can be used in combination with certain strong acids (each suitable for food use) include sodium, potassium and calcium salts, for example, sodium, potassium and calcium gluconates. An example of an acid considered herein to be "strong" is one which will react with the acid salt and provide enough available hydrogen ions to form the desired aldonic type, manner and/or amount of strong acid(s)

employed should be such that in accordance with the objectives of this invention, a sharp, strong or objectionable acid taste is not imparted to the foodstuff. If hydrochloric acid is used as the strong acid, all of it should be converted so that no such acid would remain, only some derived salt.

Any suitable method of material can be employed to bring the aldonic acid and its lactones into combination with the foodstuff, including spraying, dipping or the use of the dry acid or its precursor. While the acid might be added by itself (since the acid, when in contact with moisture or water in the foodstuff, will be converted to a mixture of the acid and its lactones) , doing so currently does not appear practical since aldonic acids are not known to applicants to be commercially available in crystalline form or in food grade. This is the case with the preferred gluconic acid. These acids may be commercially available in technical grade in aqueous solutions. For example, gluconic acid is so available in aqueous solutions stated to be about 50% (by weight) gluconic acid. These aqueous solutions of the acid are equilibrium mixtures of gluconic acid and its lactones, glucono-delta lactone and glucono-gamma lactone.

The most important aspect of this invention is that sufficient GDL be mixed with the foodstuff at some stage in its preparation so that while the foodstuff is under refrigerated storage, its pH is sufficiently low to inhibit the growth of pathogenic, psychrotrophic bacteria. It is apparent, therefore, that the optimum method of adjusting the pH may vary appreciably with the particular foodstuff.

The term "partially prepared foodstuff" as used herein is intended to encompass a wide variety of foodstuffs, some of which require very little additional "preparation" by the consumer before ingestion. In the case of sauces or gravies, the consumer preparation will be little more than heating. With salads, the consumer may need only to stir to improve the mixture before serving. With other foodstuffs, the consumer may add milk, water, or other liquid before heating.

As stated above, the pH of the foodstuff refers to the negative logarithm of the hydrogen ion concentration during refrigeration. The pH can be determined using any of the procedures conveniently employed by those skilled in the art. See 21 CFR 114.90, revised April 1, 1987. A pH meter is, perhaps, the most suitable method. With liquid foods, the pH is

readily determined by placing the electrodes in the liquid. With solid and semi-solid foods, a representative sample is blended and the pH of the blend determined. If the blend does not provide sufficient liquid medium, a small amount of neutral distilled water may be added to the sample. If the solid or semi-solid foodstuff is a salad or other mixture with a plurality of components, it is essential that the sample selected for blending be large enough so that all components are represented in the sample.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will now be demonstrated by the following examples which illustrate the invention without limiting it. The examples illustrate the applicability of the invention to a wide variety of foodstuff substrates. Those skilled in the art will readily understand and be able to apply the benefits of the invention to other foodstuffs by utilization of the principles illustrated by these examples.

Several of the examples were conducted with a ground meat mixture such as is commonly employed for the preparation of stuffed peppers. The meat mixture, after cooking, is used to stuff cored peppers which have been blanched for 3 minutes. The resulting product is then usually frozen during storage and shipment. At the retail outlet, it is placed in a refrigerated display case and allowed to thaw and to come to the temperature of the display case. The product may remain in the case for varying periods of time, e.g., 1 to 10 days before purchase. During that period, the stuffed peppers are often subject to temperature abuse or to contamination by handling.

The meat mixture was prepared from the following components:

100.0% 77.8 oz.

The celery and onion were pre-acidified by soaking overnight in a solution of GDL at the same concentration as used in the meat mixture recipe. The ground beef, celery, and onion mixture was stir fried in a large skillet until the meat was brown and then the excess fat was drained off. The other ingredients were

stirred in and the mixture cooked for about 7 minutes at about

190°F to 212°F until the liquid components were absorbed by the solid components, principally the rice and the entire mixture became workable, i.e., for able into balls by hand. The pH of the meat mixture was taken after the cooked mixture had cooled to

25°C. It was found that use of a 2% solution of GDL in the recipe yielded a pH of 4.6. Use of a 1.1% solution yielded a pH of 4.9. Distilled water was used in the recipe for normal pH meat mixture.

Subsequent handling was designed to mimic the sequence of events that would take place in the normal consumer chain. The mixture was frozen and then thawed in a refrigerator for the selected period of time. Since the experiments were designed to allow testing for bacterial growth, the mixture was inoculated with the test organism after freezing and thawing but before refrigerated storage.

EXAMPLE 1

GROWTH OF ESCHERICHIA COLI IN A PREPARED STUFFED PEPPER MEAT MIXTURE AT NORMAL pH AND ACIDIFIED TO pH 4.6 WITH GDL.

Procedure

A. Preparation of stuffed pepper meat mixture

1. The meat mixture was prepared as described above. Ingredients were purchased at local foodstores.

2. For the acidified product, a 2% GDL agueous solution was substituted for distilled water. The chopped celery and onion were pre-acidified in a 2% GDL solution for 24 hours at 42°F.

3. Meat mixture pH's: Normal pH = 5.5

Acidified pH = 4.6

. The normal pH and acidified prepared meat mixtures were each distributed in 22 gram quantities in sterile Whirl-Pak bags and stored frozen.

5. Inoculum: The inoculum was a suspension of E. coli diluted to approximately 1.0 x 10 cells/ml.

6. Inoculation: Each thawed sample in a Whirl-Pak bag was inoculated with 0.1 ml of the suspension to provide approximately 2.2 x 10 5 cells per bag or 1.0 x 104 cells per gram. The suspension was thoroughly mixed into the meat mixture.

7. Incubation temperatures: Samples were incubated at normal refrigeration temperature of 42°F or an abuse temperature of 58°F.

8. Growth determination: Duplicate samples were tested at each sample time. 88 ml. of sterile distilled water were added directly to the samples in bags at the appropriate sampling times to form the primary dilution. The samples were blended in the bags in a Stomacher Lab

Blender 400 for one minute. Appropriate decimal dilutions from the blend were surface plated in duplicate on Eosin Methylene-Blue Agar (EMB) for selective recovery of the test strain at 90°F. Colonies were counted after 24 hours. Decimal dilutions of the blend were also plated in duplicate in Plate Count Agar (PCA) for an aerobic plate count determination. PCA plates were incubated at 90 F and counted after 48 hours.

9. Controls: Uninoculated control samples from each pH variable (pH 5.5 and 4.6) were incubated concurrently at the same incubation temperatures and sampled using the same procedures as the inoculated samples. The controls were used to determine the numbers of naturally occurring organisms in this product under the test preparation methods and conditions.

Results

As illustrated in Figure 1, there was no detectable growth of the E. coli test strain in the meat mixture at normal pH or acidified to pH 4.6 during incubation at a normal refrigeration temperature of 42 F. for 20 days. The numbers of organisms remained relatively constant throughout the test period suggesting there was very little cell death or growth. The aerobic plate counts alεo remained constant.

During incubation at 58°F, representative of a storage or handling abuse temperature, the numbers of the test organism increased from 6.50 x 10 3 per gram to 1.04 x 109 per gram in seven days in the unacidified product variable (Table 1 and Figure 2) . Growth was inhibited, however, in the product acidified to pH 4.6 and the numbers of organisms recovered remained fairly constant during the sampling period. There was no significant difference between the selective isolation counts on EMB agar and the aerobic plate counts, suggesting there was no growth of the competing background flora at either pH.

TABLE 1

GROWTH OF ESCHERICHIA COLI AT 58°F. IN PREPARED STUFFED PEPPER MEAT MIXTURE AT NORMAL pH (5.5) AND ACIDIFIED TO H 4.6 WITH GDL

* EMB = Eosin Methylene-Blue Agar at 90 F

** APC = Aerobic Plate Count in Plate Count Agar at 90°F.

EXAMPLE 2

GROWTH OF ESCHERICHIA COLI IN A PREPARED STUFFED PEPPER MEAT MIXTURE AT NORMAL pH AND ACIDIFIED TO pH 4.9 WITH GDL.

Procedure

A. Preparation of stuffed pepper meat mixture.

1. The frozen meat mixture was prepared as in Example 1. The water solution of GDL had a concentration of 1.1% to yield a pH of 4.9.

2. Meat mixture pH's: Normal pH = 5.4 Acidified pH = 4.9

3. Incubation temperatures: Normal refrigeration = 43°F

Abuse temperature = 58°F

4. Aim inoculum: A suspension of E. coli diluted to

5 provide approximately 2.2 x 10 cells per bag or

₄ 1.0 x 10 cells per gram.

5. All other procedures were as described in Example 1.

Results:

As illustrated in Figure 3, there was no measurable growth of the E. coli test strain in the meat product at the acidified pH of 4.9 during incubation at 43 F for 20 days. The fluctuations in counts noted at sampling times probably represent normal sample to sample and procedural variations. There appears to be a slight increase in numbers of the test strain followed by a decrease, in product at the normal pH. This contrast may be caused in part by sample to sample variation. In addition, the samples were incubated in the "40 F" cold room (which was running at 43°F) in an effort to use a slightly higher "normal" refrigeration temperature to facilitate bacterial growth. This storage room is subject to temperature fluctuations as a result of everyday working conditions and personnel (i.e. opening and closing of the door, etc.).

The numbers of the E. coli test strain increased from 2.8 x

10 3 to 9.0 x 108 per gram during incubation at the abuse temperature in the product at its normal pH (Table 2 and Figure

4) . The increase in numbers in this product variable is similar to that noted in Example 1 in the unacidified product. Growth was inhibited at 58°F in the product acidified to pH 4.9 for the first three days of the incubation period. Between three and ten days the numbers of the test strain increased from 3.1 x 10 3 to approximately 2.6 x 10 7 per gram of product. The aci.di.ficati.on to an intermediate pH of 4.9 appears to be somewhat effective initially in limiting growth during temperature abused storage, but is not as inhibitory as acidification to pH 4.6, where, as reported in Example 1, no growth was detected during the 10-day test period. There was no significant difference between the selective isolation counts on EMB agar and the aerobic plate counts, suggesting there was very little growth of the competing background flora at either pH.

In summary, there was no apparent growth of the E. coli test strain in the meat mixture at the normal pH of 5.4 or at the acidified pH of 4.9, when incubated at a normal refrigeration temperature (43°F) for 20 days. At an abuse temperature (58°F) , the numbers of E. coli increased from 2.8 x 10 3 to 9.0 x 108 per

gram in 10 days in the meat mixture at pH 5.4. Growth was inhibited for approximately three days in product at pH 4.9, then numbers increased from 3.1 x 10 3 to 7.6 x 108 in the remaining seven days of the test period. No significant increases in the numbers of naturally occurring microorganisms in the product were noted at pH 5.4 at 43°F or pH 4.9 at either incubation temperature. The natural flora increased from less than 10 to 1.6 x 10 7 per gram in meat mixture at pH 5.4 at the abuse temperature in 10 days.

TABLE 2

GROWTH OF ESCHERICHIA COLI AT 58°F IN A PREPARED

STUFFED PEPPER MEAT MIXTURE AT NORMAL pH (5.4)

AND ACIDIFIED TO pH 4.9 WITH GDL

*EMB = Eosin Methylene - Blue Agar at 90°F.

**APC = Aerobic Plate Count in Plate Count Agar at 90°F. = Estimated number from the highest sample dilution plated.

EXAMPLE 3

GROWTH OF YERSINIA ENTEROCOLITICA IN A PREPARED STUFFED PEPPER MEAT MIXTURE ADJUSTED TO pH'S 4.3, 4.4, 4.6 and 4.7.

Procedure

5 A. Preparation of Stuffed Pepper Meat Mixture

1. The frozen stuffed pepper meat mixture was prepared as described in Example 1, with the following exceptions:

a. Varying concentrations of GDL solutions (shown below) were used in the recipe for the acidified o meat mixtures and used to pre-acidify the chopped onions, celery and rice.

b. The spices and sodium chloride were omitted from the formulation to eliminate any inhibitory effects they may impair.

2. Incubation temperature: 46°F.

3. The inoculum was a suspension of Y. enterocolitica ATCC . 27739 diluted to provide approximately 2.2 x 10 5

4 cells per bag or 1.0 x 10 cells per gram of meat mixture.

4. Growth determination: Duplicate sample bags were tested at each sample time as described in Example 1. Appropriate serial dilutions of the samples were surface plated in duplicate on Cefsulodin-Iragasan-Novobiocin (CIN) Agar for selective recovery of the test strains at

90°F. Colonies were counted after 24 hours. Decimal dilutions of the samples were also plated in duplicate

in Plate Count Agar (PCA) for an aerobic plate count determination. PCA plates were incubated at 90°F and counted after 48 hours.

All other procedures were as described in Example 1.

Results

Growth of the Yersinia entercolitica test strain was inhibited in the stuffed pepper meat mixtures acidified to pH's 4.3, 4,4 and 4.6 with GDL at 46°F (Table 3 and Figure 5). A reduction in the numbers of organisms recovered with time was observed for each of these pH variables. The rate of cell death increased as pH decreased. In the meat mixture acidified to pH 4.7, a decrease in the numbers of the test strain recovered was seen for the first 4 days of the test period (Figure 5) . After 4

₃ days, growth was observed with numbers increasing from 6.9 x 10

5 to 2.0 x 10 per gram in plate count agar (PCA) at 7 days (Table

3). At the 10 day sampling time the count had decreased to 2.7 x

10 3 per gram followed by an increase to 1.8 x 106 per gram (PCA) at 14 days.

It was also observed that acidification of the meat mixture to pH 4.7 was more effective in extending the time for growth to occur at 46 F than acidification to pH 5.0. Further acidification to levels less than pH 4.7 was effective in inhibiting growth of the test strain and resulted in cell death.

There was no evidence of growth of naturally occurring organisms in the meat mixtures at pH's 4.3, 4.4 and 4.7 during the test period. The numbers of the test strain recovered from the inoculated samples at each sampling time were lower on the Cefsulodin-Iragasan-Novobiocin (CIN), Yersinia-selective medium than in PCA. The selective constituents of the CIN agar are probably inhibitory to the acid-streεεed cells and reduce their development under these conditions. Since there was no interference from the background flora during the study, the counts in the PCA probably more accurately reflect the actual number of survivors. As a result, the data from the PCA was used to draw Figure 5.

TABLE 3

GROWTH OF YERSINIA ENTERCOLITICA AT 46°F IN A PREPARED STUFFED

PEPPER MEAT MIXTURE ACIDIFIED TO PH'S 4.3, 4.4, 4.6 AND 4.7

Sample Average Number Yersinia entercoLitica per gram Meat Mixture

Time

Days pH 4.3 pH 4.4 pH 4.6 pH 4.7

CIN APC CIN* APC CIN* APC CIN APC

0 1.4x10 1.6x10 1.1x10 1.5x10 1.5x10 1.7x10 1.3x10^** 1.8x10 c 3 4 4 4

1 --- --- — — 8.7x10 1.7x10 1.1x10 1.8x10

2 est.^d330 4.3x10 est .200 8.5x10 2.1x10³ 1.5x10* 6.5x10³ 1.5x10*

3 — 5.1x10² 1.3x10* 2.7x10³ 1.3x10*

2 3 3 3

4 — --- --- — 3.8x10 9.7x10 est.1.9x10 6.9x10

5 <100 est. 1.80 <100 est.750 ---

2 3 5 5

7 <50 est..38 <50 est.190 est.1.4x10 est.1.6x10 1.5x10 2.0x10

9 <50 <10 <50 est. 20 ---

2 3 3 0 --- <50 est.2.4x10 1.8x10 2.7x10

2 <50 <10 <50 <10

4 --- <50 est. 38 1.4x10⁶ 1.8x10

5 <50 est. 10 <50 <10

SUBSTITUTE SHEET ^aCIN: Cefsulodin-Iragasan-Novobioσin Agar at 90°F. APC: Aerobic plate count in plate count agar at 90 F.

^■: Not tested, est. : Estimated count from the dilutions plated. <: Less than.

EXAMPLE 4

EFFECT OF pH ON CLOSTRIDIUM BOTULINUM TYPE E GROWTH AND TOXIN FORMATION IN A PREPARED STUFFED PEPPER MEAT MIXTURE AT 54°F.

Procedure

A. Preparation of Stuffed Pepper Meat Mixture

1. The frozen stuffed pepper meat mixture was prepared as described in Example 1 with the following exceptions:

a. The spices and sodium chloride were omitted from the formulation to eliminate any inhibitory effects they may impart.

. adjusted to pH 5.7 with ION NaOH to increase the pH to a more favorable range for growth. The unacidified meat mixture pH normally ranged from 5.4 to 5.5.

c. A 1.2% and 0.6% GDL εolution were substituted for water in the recipe and used for the pre-acidification of the vegetables, for the intermediate pH mixtures of 5.0 and 5.3, respectively.

d. The meat mixtures were supplemented with 1% glucose, 1% yeast extract, and 0.1% sodium thioglycollate to enhance growth of the test strain.

e. Prepared meat mixtures were each distributed in 66 gram quantities in Nalgene wide mouth dilution bottles and sterilized 50 minutes at 250°F to eliminate interference from naturally occurring aerobic sporeformers during recover of the test organism.

2. Inoculum: The inoculum was a heat-shocked (13 min. at 140°F) suspension of Clostridium botulinum type E Beluga strain 070 spores diluted to provide approximately 1.0 x 10 spores per gram of meat mixture.

3. Incubation Temperature: 54 F (representε an abuεe temperature)

4. Growth Determination: Duplicate samples were tested at each sample time. The contents of each bottle were aseptically transferred to a sterile Whirl-Pak bag containing 1.2 ml of a sterile 10% Triton X-100 solution

(to facilitate fat break-up) with 132 ml of diluent containing 0.9% Brain-Heart infusion, 0.05% L-cysteine hydrochloride and 0.06% agar. The samples were blended in the bags in a Stomacher Lab Blender 400 for one minute. After blending, an additional 132 ml of diluent was added to each bag to form the primary dilution.

Appropriate decimal dilutions from the blend were cultured in 16 x 125 mm tubes with Beef-Heart Proteose Peptone (BHPP) agar supplemented with 0.1% sodium thioglycollate and 0.14% sodium bicarbonate for recovery

of the test strain at 90°F. Decimal dilutions of the blend were also plated in duplicate in Plate Count Agar (PCA) at 90°F for detection of post-sterilization contamination.

5. Controls: The procedures usd to analyze uninoculated control samples from each pH variable were the same as described for the inoculated εamples.

6. Toxin Detection: Product samples (1:5 dilutions) were centrifuged to obtain supernatants for toxin assay. 3.6 ml of each supernatant was mixed with 0.4 ml of Difco

1:250 Trypsin (10% solution) and adjusted to pH 6.2-6.5 with sterile sodium hydroxide. The digest was incubated at 90 F for one hour. Each sample was tested for botulinal toxin by injecting each of two 20-25 gram mice intraperitoneally with 0.5 ml of the trypsinized samples. Mice were observed for symptomatic death for 72 hours.

Results

As shown in Table 4 and Figure 6, growth was evident at three days and toxin was detected at five days in meat at pH 5.7 held at 54°F. When the meat was acidified with GDL to pH 5.3, growth and toxin formation occurred at some point in time between 7 and 10 days. Further acidification of the meat mixture to pH 5.0 prevented growth and toxigenesis at 54°F during the 21-day storage period. It was asεumed that 21 days would be the maximum shelf life for non-sterile, refrigerated foods of this type. The reεultε demonεtrate that acidification of this product with GDL to pH 5.0-5.3 can effectively delay or inhibit growth and toxin formation from spores of C^ botulinum Type E Beluga during abuse storage at 54°F.

GROWTH AND FORMATION OF TOXIN BY CLOSTRIDIUM BOTULINUM TYPE E

AT 54 F IN A PREPARED STUFFED PEPPER MEAT MIXTURE WITHOUT

GDL (pH 5.7) OR ACIDIFIED TO pH 5.3 OR 5.0 WITH GDL

Sample

Time H 5.7 H 5.3 H 5.0

SUBSTITUTE SHEET - 48 -

NT 1.4x10

NT 1.4x10* NT 5.8x10* NT 2.8x10*

NT 1.1x10⁵

NT 1.3x10⁵ NT NT NT 1.4x10

NT = Not Tested

SUBSTITUTE SHEET EXAMPLE 5

EFFECT OF pH ON THE GROWTH OF YERSINIA ENTEROCOLITICA AT 45° and 54°F IN POTATO SALAD ACIDIFIED WITH GDL.

Procedure

A. Preparation of Potato Salad

1. The potatoes used to prepare the salad were diced white Idaho potatoes packed in 303 x 406 cans with 1% sodium chloride and a varying brine concentration of GDL for pH control or no GDL for the normal pH samples. All variables were processed 24 minutes at 220°F.

2. Target Potato Salad pH's:

a. Normal pH (no added GDL): 5.5-5.6

b. Intermediate pH (0.3% GDL) in brine of canned potatoes: 5.0-5.3

c. Acid pH (1.2% GDL) in brine of canned potatoes:

4.2-4.4

3. Mayonnaise: pH 3.9

4. The 303 x 406 cans were opened aseptically and drained. 22 g. potatoes were added to sterile Whirl-Pak bags.

5. Inoculum: The inoculum was a suspension of Y. enterocolitica ATCC 27739 diluted to provide approximately 2.2 x 10 5 cells per bag or 1.0 x 104 cells per gram of potato salad.

6. Inoculation: 0.1 ml of working suspension added to each bag containing 22 g. of potatoes. Potatoes were mixed with inoculum in bags before adding mayonnaise.

7. 1.1 g. of mayonnaise were aseptically added to each bag containing potatoes and inoculum. The contents of each bag were mixed by kneading to coat the potatoes with mayonnaise thoroughly.

SUBSTITUTESHEET 8. Incubation Temperatures:

Normal refrigeration, 45°F. Abuse temperature, 54 F.

9. Growth Determination: Duplicate samples were tested at appropriate time intervals. 88 ml. of sterile 0.1% peptone were added directly to each bag and blended 1 min. in a Stomacher Lab Blender 400 to form the primary dilution. Appropriate decimal dilutions were surface plated on Celsulodin-Irgasan-Novobiocin (CIN) Agar for selective recovery at 90 F. Decimal dilutions were also plated in duplicate in Trypticase Soy Agar (TSA) at 90°F for non-selective recovery.

10. Controls: Uninoculated control samples from each pH variable were prepared and sampled as described above.

Results

As shown in Table 5 and Figure 7, the numbers of the Y. enterocolitica test strain increased approximately 3 logs (8.4 x 10 /g to 4.6 x 10 /g) in six days in the unacidified (no added GDL) potato salad (pH 5.5) at 45°F. At 54^CF, a comparable increase in numbers was attained in three days (Figure 8) .

When the potato salad was acidified to an intermediate pH (5.1) using GDL acidified potatoes, growth was observed after 48 hours at 45 F and numbers increased to 1.5 x 10 in six days (Table 7 and Figure 7). At 54°F, growth was detected after 24 hours and numbers increased from 6.3 x 10 3/g to 2.8 x 108/g (or

4.6 logs) in five days (Figure 8).

Further acidification of the potato salad to pH 4.3, with GDL (1.2% in brine) acidified potatoes, inhibited growth of the test strain at a normal refrigeration temperature (45°F) for 28 days (Table 6 and Figure 7) . The numbers of Y. entercolitica recovered at each time interval remained fairly constant throughout the test period. Under the condition of temperature abuse (54 F) , a 2 log increase in numbers was noted at 10 days, a 0.4 log increase at 21 days, and a 3.3 log increase at 23 days.

The numbers recovered during the other sampling intervals for this variable were comparable to the initial number, suggesting growth had not occurred in those samples. These conditions of pH 4.3 and 54°F may be marginal for the growth of this organism.

There was no evidence of growth in the uninoculated control samples in the acidified potato salads, pH's 5.1 and 4.3, at either temperature, indicating an absence of competitive flora. In the unacidified (pH 5.5) control salads, there was no growth at 45°, however, background flora were recovered at eight days and nine days from one sample at each time stored at 54°F. The organiε s recovered were an aerobic sporeformer and a non-heat resistant coccus.

The results show that the acidification of the potato salad to pH 4.3 with GDL-processed potatoes effectively inhibits growth of this organism under normal refrigeration for 28 days. Growth may occur at this acid pH if the potato salad is temperature abused for a prolonged period. Acidification to an intermediate pH of 5.1 will not prevent growth at 45°F or 54°F. The organism grew readily at 45°F and 54°F in unacidified potato salad.

* CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90°F ** TSA = Trypticase Soy Agar at 90°F NT = Not Tested

TABLE 6

GROWTH OF YERSINIA ENTEROCOLITICA IN POTATO SALAD ACIDIFIED TO pH 5 . 1 WITH GDL AT 45°F AND 54 °F

*CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90 F **TSA = Trypticase Soy Agar at 90°F NT = Not Tested est . = Estimated from the dilutions plated.

TABLE 7

GROWTH OF YERSINIA ENTEROCOLITICA IN POTATO SALAD ACIDIFIED TO pH 4 .3 WITH GDL AT 45°F AND 54°F

*CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90 F **TSA = Trypticase Soy Agar at 90°F

EXAMPLE 6

EFFECT OF pH ON GROWTH OF YERSINIA ENTEROCOLITICA AT 45°F AND

54°F IN SURIMI PASTA SALAD ACIDIFIED WITH GDL.

Procedure:

A. Preparation of Surimi Pasta Salad:

1. Pasta: Elbow macaroni packed in 300 x 407 cans with a brine containing 1% sodium chloride in water and the following amounts of GDL:

Control 0% Intermediate pH 0.40%

Acid pH 0.65%

was thermally processed at 222°F for 15 minutes to product macaroni in which the pH of the whole can blends were as follows:

Control 6.33

Intermediate 4.80

Acid 4.29

2. Surimi: 8-ounce packages of frozen Kibun Brand Sea Tails - Salad Style purchased at local food store.

a. Pasteurization: Surimi was thawed in the package at 42°F. overnight, repackaged in 6 1/2 x 8 inch Kapak (3M) plastic pouches and heat sealed. The pouches were pasteurized 15 minutes at 180°F. in a water bath to reduce any microbial load that might be present on the product.

b. Acidification: To prepare the acidified salads, pasteurized surimi was aseptically removed from the pouches and pre-acidified by soaking in a pre-deter ined concentration of GDL solution for

24 hours at 42°F.

3. Surimi Pasta Salad - target pH values:

a. Normal pH (no added GDL): 6.0-6.3 b. Intermediate pH: 5.0-5.2 c. Intermediate pH: 4.7-4.9 d. Acid pH: 4.1-4.3

B« Test P ckage: Macaroni, surimi, and mayonnaise were asepti *lly added to sterile Whirl-Pak bags, 15 g. of macaroni 5 g. of surimi, and 2.4 g. of mayonnaise per bag. Each bag was mixed by kneading to coat the pasta and surimi with mayonnaise.

C. Inoculum: The inoculum was a suspension of Y. enterocolitica

ATCC 27739 diluted to provide approximately 2.2 x 10 5 cells

4 per bag or 1.0 x 10 cells per gram of salad.

D. Inoculation: 0.1 ml of working suspension was added to each bag and mixed with the macaroni and surimi before adding the mayonnaise.

E. Product storage temperature:

1. Normal refrigeration: 45°F.

2. Abuse temperature: 54°F.

F. All other procedures were as described in Example 5 except that for growth determination, triplicate rather than duplicate samples were tested at each sampling period.

Results

Growth of the Y. enterocolitica test strain in the unacidified salad (pH 6.3) was noted in two days at 45°F. and at one day at 5 °F. (Table 8 and Figures 9 and 10) . Growth increased by approximately 5 logs in 7 days at both temperatures.

When the salad was acidified to pH 4.9, growth increased slightly

(0.06 log) somewhere between two and six days, and then markedly

5 after six days reaching a maximum of 3.4 x 10 /g. (1.4 logs) by nine days at 45°F. (Table 8 and Figure 9) . At 54°F. (Table 9

and Figure 10) , growth occurred after 24 hours , the number of cells increasing from 1.4 x 10 4 to 3.0 x 107/g. (3.3 logs) in six days.

In salad acidified to pH 4.7 and held at 45°F., there was no evidence of growth during the first three days of storage (Table 10 and Figure 9). There was a slight increase at six days (0.5 log) , but no further evidence of growth during the remaining 17 - day storage period, suggesting retardation of growth rather than complete suppreεεion. At 54 F. growth at pH 4.7 was retarded

5 until day eight after which it increased sharply to 6.8 x 10 /g.

(1.7 logs) and then declined (Table 10 and Figure 10).

As shown in Table 11 and Figure 9, there was no growth of the test organism in the salad acidified to pH 4.1 during the 28-day storage period at 45 F. At the abuse temperature (54°F.), slight growth (0.2 log) was noted at nine days, but, in general, growth was held in check by the acid (Table 11 and Figure 10) .

The results show that acidification of refrigerated (45°F.) surimi paεta salad with GDL provides an additional barrier to growth of this psychrotroph, the degree of inhibition or retardation being a function of GDL level as measured by pH. At

abuse temperatures as high as 54 F. , acidification to pH 4.9 would have little or no effect on growth. Adjusting the salad pH to 4.7 retards growth for about a week, and acidification to a pH of 4.1 provides a greater degree of protection against proliferation of this organism.

TABLE 8

GROWTH OF YERSINIA ENTEROCOLITICA IN UNACIDIFIED

SURIMI PASTA SALAD (pH 6 . 3 ) AT 45°F AND 54°F

* CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90 F ** TSA = Trypticase Soy Agar

NT¹ = Not Tested

TABLE 9

GROWTH OF YERSINIA ENTEROCOLITICA IN SURIMI PASTA SALAD ACIDIFIED TO pH 4 . 9 WITH GDL AT 45°F AND 54°F

* CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90°F ** TSA = Trypticase Soy Agar

-

* CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90 F ** TSA = Trypticaεe Soy Agar

NT = Not Tested

TABLE 11 GROWTH OF YERSINIA ENTEROCOLITICA IN SURIMI PASTA

SALAD ACIDIFIED

To pH 4 . 1 WITH GOL-1 AT 45°F AND 54 °F

* CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90 F ** TSA = Trypticase Soy Agar

Claims

What is Claimed is:

1. A method of inhibiting the growth of pathogenic bacteria in non-sterile, partially prepared, refrigerated fruits, pasta, vegetables, meat, poultry and fish during refrigerated storage, display and consumer use which comprises combining the foodstuffs during preparation with a hydrolysis mixture of an aldonic acid and its lactones or a precursor thereof in an amount sufficient to lower the pH of the foodstuff to a value at which such growth is inhibited.

2. A method as in claim 1 wherein the pH is lowered from about .5 to 2.5 pH units.

3. A method as in claim 1 wherein the hydrolysis mixture comprises gluconic acid, glucono-delta lactone and glucono-gamma lactone.

4. A method as in claim 2 wherein the hydrolysis mixture comprises gluconic acid, glucono-delta lactone and glucono-gamma lactone.

5. A method as in claim 1 wherein the precursor is glucono-delta lactone.

6. A method as in claim 2 wherein the precursor is glucono-delta lactone.

7. A method as in claim 1, 2 , 3, 4, 5 or 6 wherein the pathogenic bacteria is a psychrotropic bacteria.

8. A method as in claim 1, 2, 3, 4, 5 or 6 wherein the foodstuff is poultry.

9. A method as in claim 1, 2, 3, 4, 5 or 6 wherein the foodstuff is potato salad.

10. A method as in Claim 1, 2, 3, 4, 5 or 6 wherein the foodstuff is a salad.

11. A method as in Claim 1, 2, 3, 4, 5 or 6 wherein the foodstuff is a seafood salad.

12. A method as in Claim 1, 2, 3, 4, 5 or 6 wherein the foodstuff contains meat.

13. A non-sterile, partially prepared fruit, pasta, vegetable, meat, poultry or fish foodstuff for refrigerated storage, display and consumer use which has been treated during preparation with a sufficient amount of a hydrolyεis mixture of an aldonic acid and its lactones or a precursor thereof to lower the pH of the foodstuff to a value at which the growth of pathogenic bacteria is inhibited.

14. A foodstuff of claim 13 wherein the pH is lowered from about .5 to 2.5 pH units.

15. A foodstuff of claim 13 wherein the hydrolysis mixture comprises gluconic acid, glucono-delta lactone and glucono-gamma lactone.

16. A foodstuff of claim 14 wherein the hydrolysis mixture comprises gluconic acid, glucono-delta lactone and glucono-gamma lactone.

17. A foodstuff of claim 13 wherein the precursor is glucono-delta lactone.

18. A foodstuff of claim 14 wherein the precursor is glucono-delta lactone.

19. A foodstuff as in claάm 13, 14, 15, 16, 17 or 18 wherein the pathogenic. acteria is- a psyehrotropic bacteria.

20. A foodstuff as in claim 13, 14, 15, 16, 17 or 18 wherein the foodstuff is poultry.

21. A foodstuff as in claim 13, 14, 15, 16, 17 or 18 wherein the foodstuff is potato salad.

22. A foodstuff as in claim 13, 14, 15, 16, 17 or 18 wherein the foodstuff is a salad.

23. A foodstuff as in claim 13, 14, 15, 16, 17 or 18 wherein the foodstuff is a εeafood salad.

34. A foodstuff as in claim 13, 14, 15, 16, 17 or 18 wherein the foodstuff contains meat.