CA2040425A1 - Method of enhancing microbial stability of partially prepared refrigerated foods - Google Patents

Abstract

Method of inhibiting the growth of pathogenic bacteria in refrigerated foods utilizing a hydrolysis mixture of an aldonic acid and its lactones or a precursor thereof and the foods produced thereby.

Description

W O 91/02465 PCT/~ ~ ~ ~ 2 a "METHOD OF ENHANCING MICROBIAL STABILITY OF PARTIALLY
PREPAReD REFRIG~RATED FOODS"
BACKG~OUND OF THE INVENTION

~ecently, 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 qeneration of new types of foods preserved primarily through refrigeration. These new foods, which ~ay 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 weeXs.
: ' The long established methods of food preser~ation 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 .~ . . .- ,. . . .
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WO 91/02465 PCTIUS90/~M86 ~
20 4o 425 2 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 35F to 45F was adequate to prevent the growth of ~5 pathogenic bacteria and would provide shelf life for partially prepared foods (as well as leftovers) of a few days up to several wee~s, depending on the type of product and the amount of bacterial contamination present.

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WO 91/02465 3 p~/Us90~ ~ 2 Typically, serious cases of food poisoning have been associated mainly with bacteria like AtaPhYlococcus aureus and Salmonella tYphimurium. 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 45F, 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 32F
and 32.9F exhibited generation times of 30.2 and 6.7 hours ,.' - ~ : . . . :
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W~ 9t/0246~ ~ 5 PCT/US90/04486 ` 2 ~ 40 4 ~ 4 -respectively. Thus, a temperature increase of only o.s 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 botulinum type E, and Aeromonas hvdro~hila 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.

: . , ,. ; : -.:` ', ' . ~ ` ' ': : , W O 91/0246~ 5 PCT/US90~ ~6~ 4 2 Since 1985, the status of Listeria monoCYtoqenes has 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 sodlum 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.

10Bacteria 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.
Enterotoxiqenic strains of E. coli produce heat labile and/or heat stable enterotoxins. The organis~ is belie~ed 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 : . : -: , -WO 91/02465 ~ ~ 4~ 4~5 6 PCTt~S90/04486 the rather obvious factors of good sanitation, lowering refrigeration temperatures, avoiding temperature abuse, and the addition of microbiological "hurdles" such as acldification, reduction of water activity, addition of preservatives, and modified atmosphere pacXaging.

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 fla~or 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 achieYe the same pH in a given food, and that different genera of bacteria react differently to an acid environment. ~owever, 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 im entiOn deals with the finding that hydrolysis mixtures of aldonic acids and their lactones are exceptionally effective in preYenting 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.

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WO 91/0246~ PCT/US90/04486 The need for providing such additional barrier~ or hu 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 qarden 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 mar~etplace, be it a large supermarket, a small convenience store, a delicatessen, or other food shop, i5 often under conditions which expose the food to temperature abuse. The food which ~ay initially have been frozen or refrigerated is often displayed in refrigerated ca~inets or salad bars where it is subjected to intermittent or continuous temperatures well above temperatures which are - . .
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,',~ . . , : ' : , " ~ ' - ' ' ' WO 91/0246~ 2 o 4 0 4 2 ~ PCT/US90tO4486 _ regarded as refrigeration condit~ons. The food ~ay reach temperatures as hiqh as 50F or even higher durin~ 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 .

9 20~a~2~
procedure of directly adding acids ~uch 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 srow 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 . . .: ., . . :
, . . -~091/024~5 2040425 10 PCr/US90/04486 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 40F 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.

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,1 Moreover, many bacteria can contaminate food and cause illness without causing any change in the outward appearance or flavor cf 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, adherinq conscientiously to all good manufacturinq 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 ris~ for damages because their name is on the label of the product.

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l2 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 taXes 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 ., ...... . - ., ~ . , ~. .
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invention is particularly useful with salads, particularly pasta, meat, potato, poultry, fruit, vegetable, and seafood or other salads containing these f~odstuffs. 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 pathoqenic microorganisms to multiply.

It has been discovered that utilization of hydrolysis mixtur~s of aldonic acids and their lactones, or precursors thereof can be employed to create an en~ironment 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 so 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 p~ reducers used in the invention.

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W0 9l/0246~ 40425 PCT/US90/04486 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 ob~ectionable acid taste to the treated food such as results from utilizing even s~all 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 p~ and bacteria vary in their nutritional and pH requirements under refrigeration conditions. For example, foods with a natural pH above 4.6 are generally resarded 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 p~ 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, ..
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'5 ~04~25 particularly psychrotropic bacterial growt~. It is surprising to find that with the acidification agents of this i m ention, 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 Sor human consumption. The natural pH of a foodstuff may be determined by measuring t~e pH of the food prepared without added GDL.

Clearly there are advantages to reducing the pH as ~uch 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 ti~e 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.

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2~ 40 4~ 16 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 deriYed 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 . , - , . -. .. .. . . . . . .

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WO 9l/0246~ l7 PCT/US90/04486 pH and regarding lack of an objectionable acid taste in the treated foodstuff, would be within the scope of this invention.
For example, aldaric 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 i8 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 ~o 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 .-, :: : ~ :, : . ~ :,.
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WO 91/02465 ~8 PCT/US90/04486 2 ~ ~t~ls invention is g~ucono-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 com~i~ed 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 60S (by weight) gluconic acid and from about 45S 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 for~ the desired aldonic type, manner and/or amount of strong acid(s) - : : . , -,:: : .

Wo 91/02465 19 2 0 ~ 0 4 2 5 pCT/U590/04486 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 50 that no such acid would remain, only sone derived salt.

Any suitable method of material can be employed to bring the aldonic acid and its lactones into combination with the foodstuff, includinq sprayinq, dippinq or the use of the dry acid or its Frecursor. 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 ~orm 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 agueous solutions of the acid are equilibrium mixtures of gluconic acid and its lactones, glucono-delta lactone and glucono-gamma lactone.

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WO gl/02465 PCT/US90/04486 The most important aspect of this invention is that sufficient GDL be mixed with the foodstuff at some staqe in its preparation so that while the foodstuff is under refrigerated storaqe, its pH is sufficiently low to inhibit the growth of S 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 sXilled in the art.
See 21 CF~ 114.90, revised April 1, 1987. A pH meter is, perhaps, the most suitable method. With liquid foods, the pH is .. . . . ............................... . : : , , .
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readily determined by placing t~e 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 ~ixt~re with a plurality of components, it is essential that the sample selected for blending be large enough so that all c~mponents 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.

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WO 91/024~ 2 ~ 4 0 4 2 5 Z2 PCT/US90/04486 Several of the examples were conducted with a ground meat mixture such as is commonly e~ployed for the preparation of stuffed peppers. ~he 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.q., 1 to 10 days before purchase.
During that period, the stuffed peppers are often subject to temperature abuse or to contamination by handling.

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~ ~ - . : ., ~ 23 204~25 The meat mixture was prepared from the following components:

Component% bY Weiqht Weiqht Ground Beef41.1 32 oz.
Chopped Onion 5.~ 4 oz, Chopped Celery 10.3 8 oz.
Tomato Sauce 20.6 16 oz.
Salt 1.0 0.8 oz.
Garlic Salt0.3 0.2 oz.
Worcestershire 1.0 0.8 oz.
Minute Rice10.3 8 oz.
Water or Water Solution of GDL 10.3 8 oz.

100.0% 77.8 oz.

The celery and onion were pre-acidified by soaking overnight in a solution of GDL at the 5ame 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 . : . ~ - . . , . , . --..

WO 9]/02465 PCTtUS90/04486 2~4042~ 24 stirred in and the mixture cooked for about 7 minutes at about 190F to 212F until the liquid components were absorbed by the solid components, principally the rice and the entire mixture became workable, i.e., formable into balls by hand. The pH of the meat mixture was taken after the cooXed mixture had cooled to 25C. It was found that use of a 2% solution of GDL in the recipe yielded a pH of 4.6. Use of a l.1% colution 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.

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WO 91/0246~ PCT/US90/04486 - 5 204~2~
EXAMPT ~ 1 -GROWTH OF ESCHERICHIA COLI IN A PREPARED STUFFED PEPPER MEAT
MIXTU~E AT NORMAL pH AND ACIDIFIED TO pH 4.6 WIT~ GDL.

Procedure A. Preparation of stuffed pepper meat mixture l. The meat mixture was prepared as described above.
Ingredients were purchased at local foodstores.

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

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3. Meat mixture pH's: Normal pH - 5.5 Acidified pH = 4.6

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

5. Inoculum: The inoculum was a suspension of E. coli diluted to approximately 1.0 x 106 cells/ml.

6. Inoculation: Each thawed sample in a Whirl-PaX bag was inoculated with 0.1 ml of the suspension to provide approximately 2.2 x 105 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 42F or an abuse temperature of 58F.
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8. Growth determination: Duplicate samples were tested at each sa~ple time. 88 ~1. 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 81ender 400 for one minute. Appropriate decimal dilutions from the blend were surface plated in duplicate on Eosin Methylene-Blue Agar ~E~B) for selective recovery of the test strain at gOF. 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 90F 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 contro~s were used to determine the numbers of naturally occurring organisms in this product under the test preparation methods and conditions.

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, ~0.91/0246~ 2 ~ 9 28 PCT/US90~04486 Results As illustrated in Figure 1, there was no detectable growth of the E. coli test strain in ths ~eat mixture at normal pH or acidified to pH 4.6 during incubation at a normal refrigeration S 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 also remained constant.

During incubation at 58F, representative of a storage or handling abuse temperature, the numbers of the test organism increased from 6.50 x 103 per gram to 1.04 x 109 per gram in seven days in the unacidified product variable (Table l 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.

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GROWTH OF ESCHERIC~IA COLI AT 58 F. IN PREPARED STUFFED PEPPER
NEAT MIXTURE AT NORMAL PH (5.5l AND ACIDIFIED
TO pH 4.6 WIT:H GDL

Sample Averaqe Number E. Coli ~er Gram Meat Mixture Time pH 5.5 PH 4.6 (Davs~ EMB* APCt* EMB* APC**

0 6.5x103 9.0x103 6.9x103 8.3x103 1 2.4x104 2.1x104 6.6x103 7.3x103 2 5.5x105 5.7x105 6.6x103 8.1x103 3 9.2x106 l.lx107 7.3x103 7.5x103 4 1.8x108 1.8x108 6.5x103 7.Ox103 7 1.0x109 l.lx109 5.5x103 7.2x103 1.0x109 1.2x109 5.7x103 6.8x103 * EMB - Eosin Methylene-Blue Agar at 90F
** APC = Aerobic Plate Count in Plate Count Agar at 90F.

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W09l/0246~ PCT/VS90/04486 2 ~ 4 ~ 4 2 ~ EXAMPLE 2 GROWTH OF ESCHERICHIA COLI IN A PREPARED STUFFED PEPPER MEAT
MIXTURE AT NORMAL pK 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 = 43F
Abuse temperature = 58F

.
~: - : . ,. - ,. . . : .. ~ : .

. . . ~ , :

,' " ' ' ~

, WO 91/0246~ 2 ~ 4 ~ ~ 2 ~pCT/~S9o/o4486 4. Aim inoculum: A suspension of E. coli diluted to provide approximately 2.2 x 105 cells per bag or 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 43F 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 ~ariation. In addition, the samples were incubated in the "40F" cold room (which was running at 43F) in an effort to use a slightly higher "normal"
refrigeration temperature to facilitate bacterial grovth. 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.).

..

.. ,~. ~ . .

WO 91/02465 2 ~ ~ ~ 4 2 5 PCI/lJS90/04486 32 -~
The numbers of the E. coli test straln increased from 2.8 x 103 to 9.O x 108 per gram during incubation at the abuse temperature in the product at its normal pH (Table 2 and Figure 4J. The increase in numbers in this product variable is similar to that noted in Example 1 in the unacidified product. Growth was inhibited at 58F 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 103 to approximately 2.6 x 107 per gram of product. The acidification 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 t43F) for 20 days. At an abuse temperature (58F), the numbers of E. coli increased from 2.8 x 103 to 9.O x 108 per .
.
.

~04~4~5 gram in lO 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 103 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 107 per gram in meat mixture at pH 5.4 at the abuse temperature in 10 days.

.

WO 91/02465 PCr/US90/04486 204~425 - 34 .
GROWTH OF ESCHERICHIA COLI AT 58 F IN A P~EPARED
STUFEED PE~PER MEAT MIXTURE AT NORMAL pH (5.4) AND ACIDIFIED TO pH 4.9 WITH GDL ;

Sample Averaqe Number E. Coli Der Gram Meat Mixture Times PH 5.4 ~ H 4.9 tDavs) EMB~* APC** EMB*_ APC*~

o 2.8x103 2 . 2x103 2.6x103 2.7x103 1 2.6x104 3.8x104 2.9X103 2.6x103 2 8.sx105 8.9x105 3.~x103 3.5x103 3 3.6x107 3.1x10~ 3.1x103 ; 3.9x103 6 8.1xl08 6. 2X108 9 . 6x104 7.7x104 ;
9.0X108 7.6x108 est.l 2.6x107 est.l 2.8x107 ~,~

;', ' , .: , , -, - .

2 ~ PCT/US90/04486 WO 91/0246~
*EM8 = Eo-~in Methylene - Blue Agar at 90F.
**APC = Aerobic Plate Count in Plate Count Agar at 90F.
1 = Estimated number from the highest sample dilution plated.

WO 91/0246~ PCT/US90/04486 20~42~ 36 GROWT~ OF YERSINIA ENTEROCOLITICA IN A PREPARED STUFFED PEPPER
MEAT MIXTVRE ADJUSTED TO pH'S 4.3, 4.4, 4.6 and 4.7.

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. Varying concentrations of GDL solùtions ~shown below) were used in the recipe for the acidified . 10 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.

--, . : . - :
~ -,:

~ V 4 S GDL Meat Mixture PH
.

3.4 4.3 3.~ 4.4 :
2.2 4.6 1.6 4.7 --2. Incubation temperature: 46F.

3. The inoculum was a suspension of Y. enterocolitica ATCC
. 27739 diluted to provide approximately 2.2 x 105 cells per bag or l.0 x 104 cells per gram of meat mixture.

4. Growth determination: Duplicate sample bags were tested at each sample time as described in Example l.
Appropriate serial dilutions of the samples were surface plated in duplicate on Cefsulodin-Iragasan-Novobiocin (CIN) Agar for selective recover,v of the test strains at 90F. Colonies were counted after 24 hours. Decimal dilutions of the samples were also plated in duplicate `, . ' ' ' . ' .' ' ' ,' ' . ' . . ` .

WO ~l/0246~ 2 ~ 4 0 ~ ~ ~ 38 PCT/US90/04486 in Plate Count Agar (PCA) for an aerobic plate count determination. PCA plates were incubated at 90F and counted after 48 hours.

5. 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 4 6F (Table 3 and Figure s). 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 p~
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 days, growth was observed with numbers increasing from 6. 9 X 103 to 2.0 x 105 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 103 per gram followed by an increase to 1.8 x 10 per gram ~PCA) at 14 days.

, ,. . , , : - :- ~

- - ~ v ~ f~ ~
WO 91/0246~ PCT/US90/04486 It was also observed that acidification of the meat mixture to pH 4.7 was more effective in extending the ti~e for growth to occur at 46F than acidification to pH 5Ø 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-stressed cells and reduce their development under these conditions. Since there was no interference from the background flora during the study, the ' 15 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.

.

.
:: ..... . . .

204042~ TA8LE 3 GROUT~ Of ~ERSINIA EUTERCOLITICA AT 46 F IU ~ PREPARED STUFFED -~
PEPPER uE_T ~IXTURE ACIDIFIED TO PH'S 4.3, 4.4, 4.6 AND 4.7 SampleAverase Uumber ~ersinia entercolitica Der gram Meat Mixture S Time OaYs DH 4.3 DH 4.4 DH 4.6 eH 4.7 CINAPC CIN~ APC Cl N~ APC Cl N APC

0 1 4x104 1 6x104 1 1 104 1 S 104 4 4 1.3x10 1.8x10 c~,, .. - ~....... 8 7x103 1 7 104 1 1 1 4 4 1 0 2 est. 330 4.3x10 est.200 8.5x10 2.1x10 1.5x10 6.5xlO 1.5x10 3 --- --- --- - 5.1x10 1.3x10 2.7x10 1.3X104 4 ,,, ,,, -- --- 3.8x102 9.7x103 est.1.9x103 6.9x10 5 ~100 est. 180 ~100 est.750 --- -- --- --- ' -7 <50 est.88 ~SO est.190 est.1.4x10 est.1.6x10 1.5x10 2.0x10 1 5 9 <50 ~10 <SOest. 20 -- --- --- -.

10 ... . . <50 est.2.4xlO 1.8x10 2.7x103 12 ~SO <10 <50 <10 --- --- --- - -14 - --- --- --- ~50 est. 38 1.4x10 1.8x10 ~50 est. 10 ~50 <10 --- --- --- --SUBSTITUTE SHEET

. ., ~ .
` . . . - , .. ~ - . . . .
,. ~ `

~v~

CIN: Cefsulodin-Iragasan-NoVObiOCin Agar at 90F.
b APC: Aerobic plate count in plate count agar at 90F.
c Not tested.
est.: Estimated count from the dilutions plated.
e < Less than ,. .

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.

WO 91/0246~ PCT/US90/0~486 2040425 42 _ , EFFECT OF pH ON CLOSTRIDIUM BOIULINUM TYPE E GROWTH AND TOXIN
FORMATION IN A PREPARED STUFFED PEPPER MEAT MIXIVRE AT 54F.

Procedure A. preD-aration of Stuffed PeDDer Meat Mixture l. The frozen stuffed pepper meat mixture was prepared as described in Example l with the following exceptions:

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

- : .: :

, : . : , : , ~ - ~, :; , .- : ~ .

~u~
WO 91/0246~ 43 PCT/US90/04486 - b. ~he pH of the unacidified variable was adjusted to pH 5.7 with 10N NaOH to increase the pH to a more favorable range for growth.
~he unacidified meat mixture pH normally ranged from 5.4 to 5.5.

c. A 1.2% and 0.6~ G~L ~olution 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 t~ioglycollate to enhance growth of the test strain.

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

, ., , , : , :. ,, . - .

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WO 91/0246~ 44 PCT/US90/04486 2. Inoculum: The inoculum was a heat-shocXed (13 min. at 140F) suspension of Clostridium botulinum type E Beluga strain 070 spores diluted to provide approxi~ately l.O x 104 spores per gram of meat mixture.

3. Incubation ~emperature: 54F (represents an abuse 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 baq containinq 1.2 ml of a sterile 10% Triton X-100 solution (to facilitate fat break-up) with 132 ml of diluent containinq O.9% Brain-Heart infusion, O.OS% L-cysteine hydrochloride and 0.06% agar. The samples were blended in the baqs in a Stomacher Lab Blender 400 for one minute. After blending, an additional 132 mi of diluent was added to each bag to form thè prlmary dilution.

Appropriate decimal di7utions from the blend were cultured 'n i6 x 125 mm tubes witn ~ee~-Heart Proteose Peptone (BHPP) agar supplemented with 0.1% sodium thioglycollate and 0.14% sodium bicarbonate for recovery - - - :. .,. - ~ : : : : : - :

:: . - - : :: : :-~ u ~ u ~ ~ ~
WO 91/02465 ~5 PCT/US90/04486 of the test strain at 90F. Decimal dilutions of the blend were also plated in duplicate in Plate Count Agar (PCA) at 90F 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 samples.

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 90F for one hour. Each sample was tested for botulinal toxin by injecting each of two 20-25 gram mice intraperitoneally with O.S ml of the trypsinized samples. Mice were observed for symptomatic death for 72 hours.

. - . . -: . :,. , - : . : .; - .~ . ~ :.

. . - - . . , . ~

WO 91/0246~ 2 0 4 0 ~2~ 46 PCT/US90/04486 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 54F. When the meat was acidified with GDL to pH 5.3, S growth and toxin formation occurred at some point in tlme between 7 and lO days. Further acidification of the meat mixture to pH
5.0 prevented growth and toxigenesis at 54F during the 21-day storage period. It was assumed that 21 days would be the maximum shelf life for non-sterile, refrigerated foods of this type. The results demonstrate that acidification of this product with GDL
to pH 5.0-5.3 ca~ effectively delay or inhibit growth and toxin formation from spores of C. botulinum Type E Beluga during abuse storage at 54F.

. , .'. ~ , ~ . '' .: .
'. ' '`' ' ' ~' `''`, ` ` ' ' ~; , ' ` , . . ~

~ u ~

_ 47 - T~BLE 4 G20UTH ND FORMATION OF TOXlN B~ CLOSTRIDIUM 80TULINUM TTPE E
AT 54 F IN ~ PREPARED STUFFED PEPPER MEAT ~IXTURE I~ITHOUT
GDL ~pH 5.7) OR ~CIDIF~ED TO pH S.3 OR 5.0 ~ITH GDL

Sample :
Time DH 5.7 pH 5.3 pH 5.0 ~Days) CFU/gram ToxicitY CFU/aram Toxicity CFU/qram ToxicitY
Od 1.5x104 . 1.7x10 1.4x10 .
1d 9.6x10 ~T NT NT NT
; 1 0 2d 1.1x10 - ~T ~T 1.6X1o4 3d 2.4x10 1.0x10 - 1.2x70 4d 4.3x10 - 7.4x103 1 5 104 5d 2.4x10 ~ 1.1x10 - 1.0x10 6d 1.1x10 1 8.7x10 - 1.3x10 - ~ :
1 57d 1.8x10 ~ 9.1x10 1.1x104 8d 1.4x10 ~ ~T NT 9.7x10 9d 1.2x10 ~ NT NT
~ .

SOBSTITUTE SHEEr `:
' .
. .

WO 91t02465 PCr/US90/04486 2~0425 - 48 -10d UT NT 1.4x10 ~ LT UT
12d HT IIT1.4x10 ~ uT NT
14d IIT NT 5.8x10 ~ 1.1X104 17d NT NT 2.8x10 ~ NT NT
18d UT NT 1.1xlO ~ NT IIT
20d NT IIT1.3xlO ~ NT NT
21d UT NT NT HT 1.4XlO

UT Uot Tested SllE~S~ll UTE SHEE~ -. ~` ., . .. -.. .

~u~v~5 WO 91/02465 PCTiUS90Jo4486 EFFECT OF pH ON THE GROWTH OF YERSINIA ENTEROCOLITICA AT 45 and 54F I?N POTATO SALAD ACIDIFIED WITH GDL.

Procedure A. Preparation o f Potato Salad l. The potatoes used to prepare the salad were diced white ?
Idaho potatoes pac~ed in 303 x 406 cans with l~ 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 220F.

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 W 0 91/0246~ 2 0 ~ 0 4 2 S 50 - PCTJUS90/O M 86 c. Acid pH (1.2% GDL) in brine of ca~ned potatoes:
4.2-4.4 3. Mayonnaise: p~ 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 105 cells per bag or l.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.

SU~STITUTE SH~E~

,, .
, ~ U ~ ~
WO 91/~2465 5I PCT/US90/04486 8. Incubation Temperatures:
Normal refrigeration, 45 F.
Abuse temperature, 54F.

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 90F. Decimal dilutions were also plated in duplicate in Trypticase Soy Agar ~TSA) at 90F
for non-selective recovery.

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

.~ . ~.. , .. ",, ... .. . , ... ...... ... . ..... , . . . ................. , : . .

. . , . . . ~ . : . ................................... . .

: . . . . . :

WO 91/0246~ PCT~US90~04486 2~042~ 52 ~_ Results - .

As shown in Table 5 and Figure 7, the numbers of the Y.
enterocolitlca test strain increased approximately 3 logs (8.4 x 104/g to 4.6 x 106/g) in six days in the unacid$fied (no added GDL) potato salad (pH 5.5) at 45 F. At 54 F, a comparable increase in numbers was attained in three days ~Figure 8).

When the potato salad was acidified to an intermediate pR
(5.1) using GDL acidified potatoes, growth was observed after 48 hours at 45F and numbers increased to 1.5 x 107 in six days (~able 7 and Figure 7). At 54F, growth wa~ detected after 24 hours and numbers increased from 6.3 x 103/g to 2.8 x 10~/q (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~) 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 (54F), a 2 loq 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.

WO 9l/0246~ 53 2 ~ ~ lUS90/04486 ~he ~umbers 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 S4F may be marginal for the growth of this organism.

S 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 54F. The organisms 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 i f the potato salad is temperature abused for a prolonged period. Acidification to an intermediate pH of 5.1 will not prevent growth at 45F or 54F. The organism grew readily at 45 F and 54F in unacidified potato salad. ~-. . . - . .: . . - -.: . : ..
. . ' -. . : .
. ~, .
,. . . - ~: .

WO 91~02465 54 PCT/US90/04486 GROWTH OF YERSINIA ENTEROCOLITICA IN UNACIDIFIED POTATO SALAD
~PH 5.5) AT_45 F AND 54 F
Sample Colonv Formin~ Units Y. enterocolitica per Gram Time 45 F 54F
(DaYs) CIN* TSA** CIN* TSA*~
. _ 0 8.4x103 l.lx1048.4x103 1.2x104 1 8.9x103 l.lx1042.7x104 2.9x104 2 1.4x104 2.1x1046.9x105 1.0x106 3 9.1x104 l.lx1054.9x106 8.2x107 6 4.6x106 8.8x1061.7x109 9.1x109 7 4.2x~06 1.6x1074.5x109 7.1x109 8 1.4x107 6.4x1079.1x109 1.0x10 9 1.8x108 7.4xl085.2XlO9 8.3x109 2.5x108 2.5x109 NT NT
* CIN = Cefsulodin-Irgasan-No~obiocin Agar at 90 F
** TSA = Trypticase Soy Agar at 90F
NT = Not Tested .

-:
. , , . .. ~ . ,- . . ~:
.. . . .
~. "~' ' ~ ~ ~ U 4 WO 91~0246~ 55 PCT/US90/04486 GROWTH OF YERSINIA ENTEROCOLITICA IN POTATO SALAD ACIDIFIED
TO pH 5.1 WITH GDL AT 45F AND 54F
Sample ColonY Forminq Units Y. enterocolitica ~er Gram Time 45F 54F
(DaYs) CIN* TSA~* CIN* TSA**
0 5.8x103 l.lx104 6.3x103 1.4x104 1 4.8x103 1.2x104 9.2x103 1~4x104 2 7.5x103 l.lx104 l.lx105 1.4x105 3 1.3x105 1.7x105 NT NT
s NT NT 2.8x108 3.4x108 6 l.sx107 5.9x107 1.5x109 3.4x109 7 4.7x105 1.2xl06 2.1x109 5.0x109 8 2.6x107est.d 9.5x10 NT NT
9 NT NT 4 . 4x109 5.9x109 7.1x103 2.6x109 NT NT
13 4.7x10 6.5x10 NT NT
*CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90F
**TSA = Trypticase Soy Agar at 90F ~:
NT z Not Tested estd. = Estimated from the dilutions plated.

. : ': : - ~ . ' , WO 91~0246~ PCT/VS90/04486 2 0 ~ 0 ~ 2 ~ 56 GROWTH OF YERSINIA ENTEROCOLITICA IN POTATO SALAD ACIDIFIED
TO pH 4.3 WITH GD~ AT 45F AND 54F

Sample ColonY Forminq Units Y. enterocolitica ~er Gram Time 45 F 54 F
~DaYs) CIN* TSA** _ CIN* TSA**

0 1.3x104 2.0x104 1.2x104 1.8x104 6 1.2x104 1.5x104 1.5x104 2.0x104 l.sx104 1.5x104 1.2x106 6.2x106 14 1.4x104 1.4x104 1.2x104 1.5x104 17 1.2x104 1.5x104 l.lx104 1.3X104 21 1.2x104 1.5x104 2.8x104 5.6x104 23 l.lx104 1.4x104 2.2x107 2.8x107 28 l.lx104 l.lx104 8.7x103 1.6x104 *CIN = Cefsulodin-Irgasan-Novo~iocin Agar at 90F
**TSA ~ Trypticase Soy Ag~r at 90F ~ :

WO 91/02465 2 ~ 2 5 PCr~US90/04486 EFFECT OF pH ON GROWTH OF YERSINIA ENTEROCOLITICA AT 45F AND
54F IN SURIMI PASTA SA~AD ACIDIFIED WITH GD~. .

Procedure: .

5 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 OS
Intermediate pH 0. 40S
Acid pH û. 65%

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

,, . . . ~, ', '''. ' ' ' ~ ' ' . ', ' ' - .
" ' ' : ' ', ' , ' ~ ' '; :

, .: - ' '' ' ' ' ' - . : . ~

o 40 42~ Control 6.33 ~nter~ediate 4.80 Acid 4 . 29 2. Surimi: 8-ounce packages of frozen Xibun ~rand Sea Tails - Salad Style purchased at local food store.

a. Pasteurization: Suri~i was thawed in the package at 42F. overnight, repackaged in 6 1/2 x 8 inch Kapak (3M) plastic pouches and heat sealed. The pouches were pasteurized 15 minutes at 180F. 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-determined concentration of GDL solution for 24 hours at 42F.

.. . . . . , ., . .. . ~ . .

WO 91/0246~ 59 PcT/us9o/o ~B~ 4 2 5 Aim Surimi pH % GDL Soak 5.0-5.2 0.2 4.7-4.9 0.4 4.1-4.3 0-5 ~. Surimi Pasta Salad - target pH ~alues:
~:.. ..
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 ; g. of surimi, and 2.4 q. of mayonnai~e per bag.
Each bag was mixed by kneading to coat the pasta and surimi with mayonnaise.

5 C. Inoculum: The inoculum was a suspension of Y. enterocolitica ATCC 27739 diluted to provide approximately 2.2 x 105 cells per bag or 1.0 x 104 cells per gram of salad.

,.

: WO 91/02465 2 0 4 0 4 2 ~ PCT/US90/04486 : 60 D. Inoculation: 0.1 ~1 of worki~g suspension was added to each bag and mixed with the macaroni and surimi before adding the mayonnaise.

E. Product storage temperature:
1. Normal refrigeration: 45F.
2. Abuse temperature: 54F. -:

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.

lO Results Growth of the Y. enterocolitica test strain in the unacidified salad (pH 6.3) was noted in two days at 4SF. and at: one day at 54F. (Table 8 and Figures 9 and 10). Growth increased by approximately 5 logs in 7 days at both temperatures.

.;i When the salad was acidifiet to pH 4.9, growth increased slightly(0.06 log) somewhere between two and six days, and then markedly after six days reachinq a maximum of 3.4 x 105~g. (1.4 logs) by nine days at 45 F. (Table 8 and Figure 9). At 54F. (Table 9 .

'``` . ?

,: . . : . ~ : : -20~25 and Figure lO), growth occurred after 24 hours, the number of cells increasing from 1.4 x 104 to 3.0 x 107/g. (3.3 logs) in six days.

In salad acidified to pH 4.7 and held at 45F., 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 remalning 17 day storage period, suggesting retardation of growth rather than complete suppression. At 54 F. growth at pH 4.7 was retarded until day eight after which it increased sharply to 6.8 x 105/g.
(1.7 logs) and then declined (Table 10 and Figure 10).

As shown in ~able 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 45F. At the abuse temperat~re (~4F.), slight growth (0.2 log) was noted at nine days, but, in general,`qrowth was held in check by the acid (~able 11 and Figure 10).

The results show that acidification of refrigerated (45F.) surimi pasta salad with GDL provides an additional barrier to growth of this psychrotroph, the degree of inhibition or 2S retardation being a function of GD~ level as measured by pR. At .

.:: . . .
'' ''"'' . ' ~ ' : ~ ., WO 91/0246~ - ~ ~ ~ ~ ~ 62 PCT/US90/04486 abuse temperatures as high dS 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 ;.
S proliferation of this organism.

, : . . . :

~040425 GROWTH OF YERSIN IA ENTEROCOLITICA IN UN~CIDIFIED
SURIMI PASTA SALAD
(p~ 6.3) AT 45F AN 54F
5 Sample Colonv Forminq Units Y. enterocolitica ~er Gra~
Time 45 F _ _ 54F
(DaYs~ CIN* TSA*~ CIN* TSA**
0 1.7x104 1.8x104 1.4x104 1.8x104 1 2.0x104 2.1x104 4.7x104 4.7x104 10 2 2.2x105 2.3x105 3.2x106 1.8x107 3 2.3x106 2.5xlO~ 4.5x108 4.8x108 4 3.0x107 1.6x108 7.3x108 1.2x109 7 9.9x108 1.6x109 1.6xlO9 2.5x109 9 1.2xlo9 2.0x109 N~l NTl ~ CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90F
*~ TSA = Trypticase Soy Agar NT1 = Not Tested : ' ' ~ ' "~
, :

WO9l/0246~ ~ 2 ~ 64 PCT/~S90/04486 ~ABLE 9 GROWTH OF YERSINIA ENTE~OCOLITICA IN S~RIMI PAS~A SAL~D
ACIDIFIED _O pH 4.9 WITH GDL AT 45F AND 54F
Sample Colonv Forminq Unlts Y. enterocolitica per Gram S Time 45F _ _ 54F
rDaYs~ CIN* TSA* * CIN* ~SA**
0 1.4x104 1.6x104 1.4x104 1.6x104 1 1.2x104 l~sxlo4 1.2x104 1.6x104 2 1.4x104 1.6x104 1.8x104 2.1x104 3 1.3x104 l.sx104 2.3x104 2.2x104 6 1.6x104 l.9x104 3.0x107 4.0x107 8 1.6x104 1.8x104 4.9x106 2.1x107 9 3.4x105 4.2x105 2.1x105 2.1x105 1.2x105 1.2x105 3.9x105 3.6x106 15 13 3.2x104 3.2x104 1.6x104 1.8x104 * CIN = Cefsulodin-Irgasan-Novobi~cin Agar at 90F
** TSA = Trypticase Soy Agar : . . .. - .: ..

: . . : : :::
:: . ~ : . : : : . . . . ..... . . . . . ,. . . :
: .

2~ ~V~5 WO 9l/02465 PCT/US90/04486 GROWTH OF YERSINIA ENTEROCOLITICA IN SURIMI PASTA
SALAD ACID~FIED
TO pH 4.7 WITH GOL-l AT 45F AND 54F
SampleColonv Forminq Units Y. enterocolitica Per Gram Time 45 F 54F
(Days) CIN* TSA** CIN* TSA**
0 1.5x104 1.6x104 1.5x104 1.6x104 1 1.6x104 1.8x104 1.6x104 l.9x104 2 1.5x104 1.8x104 1.6x104 1.8x104 3 l.sxlo4 1.7x104 1.4x104 1.8x104 6 4.9x104 5.2x104 1.8x104 1.6x104 8 1.6x194 1.8x104 1.8x104 2.8x104 1.3x104 1.7x104 6.8x105 9.2xlo5 15 13 1.4x104 l.9x104 8.2x104 8.8X104 17 1.5x104 1.6x104 NTl NTl * CIN = Cefsulodin-Irgasan-Novobiocin Agar at 90F
** TSA = Trypticase Soy Agar NT1 = Not Tested ,." ' : ~ -":

WO 91/02465 PCr/US90/04486 2 0 4 0 4 2 5 $AE~I,E l l - GROWT~ OF YERSINIA ENTEROCOLITICA IN SURIMI PASTA
SAI~D ACIDIFIED
To PH 4 . l WITH GOL-l A'r 45F AND 54F
Sample Colonv Formina Units Y. enterocolitica per Gra~
Time 45F 54~F
(DaYs) CIN* TSA** CIN* TSA**
01.5x104 1.7x104 1.5x104 1.7x104 31.3x104 1.7x104 1.2x104 1.5x104 61.3x104 1.6x104 1.3x104 1.7x104 91.3x104 1.6x104 2.6x104 3.3x104 141.3x104 1.4X104 l.lx104 1.3x104 211.2x~o4 1.4x104 l.lx104 1.2x104 28~.5x103 1.3x104 6.9x103 1.2x104 * CIN = Cefsu}odin-Irgasan-Novobiocin Agar at 90F
* * 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 hydrolysis 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 claim 13, 14, 15, 16, 17 or 18 wherein the pathogenic bacteria is a psychrotropic 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 seafood salad.

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