# Patent

## Title
Composition and methods to control the outgrowth of pathogens and spoilage microorganisms in high moisture and low sodium systems

## Abstract

The invention describes a method of inhibiting the outgrowth of pathogens and spoilage microorganisms in high moisture (65-80% by weight) and low salt (<2.0% by weight) nutrient dense environments with a pH range of 5.5 to 8.5. The application of an organic acid or its salt with a fermentation derived antimicrobial peptide offers a robust solution to curtail growth of spores and vegetative cells without the need for chemicals such as sodium nitrite or nitrate.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This patent application claims the benefit of U.S. Provisional Patent Application No. 62/149,365, filed Apr. 17, 2015, the entire teachings and disclosure of which are incorporated herein by reference thereto.


FIELD OF THE INVENTION
This invention generally relates to composition and methods to inhibit pathogens and spoilage microorganisms.
BACKGROUND OF THE INVENTION
An increasing number of consumers believe foods that are free of synthetic or chemical additives are healthier. In response to these consumer trends and preferences, the food industry has focused efforts on offering various alternatives such as clean label and/or natural products that are free from artificial preservatives while retaining similar microbial safety characteristics as compared to conventionally prepared products.
Curing agents such as salts of sodium nitrate and sodium nitrite (“cured”) have a long history of preserving the microbial safety of processed meat formulations as they provide functional benefits of antimicrobial and antioxidant activities in addition to delivering desirable color and flavor attributes characteristic of such products (See e.g. Pegg, R. B., and F. Shahidi. 2000. Nitrite curing of meat: the N-nitrosamine problem and nitrite alternatives. Food & Nutrition Press, Inc., Trumbull, Conn., incorporated by reference herein in its entirety).
However, the consumption of processed meats formulated with such curing agents has recently been linked with an increased risk of colorectal cancer due to the formation of cancer causing N-nitroso compounds and poly cyclic aromatic hydrocarbons (See e.g. Santarelli, R. L., Pierre, F., & Corpet, D. E. 2008. Processed meat and colorectal cancer: a review of epidemiologic and experimental evidence. Nutrition and Cancer, 60(2), 131-144., incorporated by reference herein in its entirety). Moreover, the International Agency for Research on Cancer (IARC, a subsidiary of WHO) and American Institute of Cancer Research (AICR) recently classified processed meats as Group 1 carcinogenic agents to humans (See e.g. Bouvard, et al. 2015, on behalf of the International Agency for Research on Cancer Monograph Working Group, The Carcinogenicity of consumption of red and processed meat. The Lancet Oncology. Published Online: 26 Oct. 2015., incorporated by reference herein in its entirety).
Meat products prepared without a curing agents either from synthetic or naturally occurring sources (“uncured” or “nitrate or nitrite free”) are more susceptible to the growth of pathogens due to their antimicrobial nature. Listeria monocytogenes and Clostridium species are two pathogens that are of particular concern in “uncured” or “nitrate or nitrite-free” products. Listeria monocytogenes is a psychrotroph that can grow even at refrigeration temperatures and thus pose food safety risk in extended shelf life ready to eat (RTE) meat and poultry products. Spore forming enterotoxigenic species of Clostridia such as Clostridium botulinum and Clostridium perfringens associated with processed meat and poultry are also of particular concern. While the heat applied in manufacturing RTE processed meat products is sufficient to inhibit vegetative cells, spores will not be inactivated but rather may germinate and develop into vegetative cells.
Spoilage organisms also play an important role in reducing the shelf-life of both raw (fresh) and uncured RTE refrigerated meat and poultry. For example, species of Pseudomonas and Lactobacillus are predominantly responsible for undesirable defects such as off-flavors, discoloration, gas and slime etc.
Additionally, in recent times there has been a movement to reduce the sodium content in food (See e.g. Scientific Report of the 2015 Dietary Guidelines Advisory Committee. Advisory Report of the Secretary of Health and Human Services and the Secretary of the Agriculture). Sodium is an effective preservative and its reduction makes formulations more vulnerable to a higher risk of pathogen and spoilage growth and thus results in shorter product shelf life (See e.g. Desmond, E. 2006. Reducing salt: A challenge for the meat industry, Meat Science, 74 (2006), pp. 188-196, incorporated by reference herein in its entirety).
In cured products, low levels of sodium nitrite, approx. 50 ppm, are sufficient for the inhibition of Clostridium species in processed meat formulations (See e.g. Hustad, G. O., J. G. Cerveny, H. Trenk, R. H. Deibel, D. A. Kautter, T. Fazio, R. W. Johnston, and O. E. Kolari. 1973. Effect of sodium nitrite and sodium nitrate on botulinal toxin production and nitrosamine formation in wieners. Appl. Microbiol. 26:22-26. incorporated by reference herein in its entirety).
Nevertheless, the maximum allowed level of 156 ppm sodium nitrite when used without the addition of adjunct antimicrobials is insufficient for the inhibition of Listeria monocytogenes (See e.g. Farber, J. M., R. C. McKellar, and W. H. Ross. 1995. Modelling the effects of various parameters on the growth of Listeria monocytogenes on liver pate. Food Microbiol. 12:447-453., incorporated by reference herein in its entirety).
Similar or comparable results are expected in inhibition of Listeria and Clostridia sps. when alternative sources of nitrate or nitrite (derived either by synthetic or fermentation methods) used to deliver similar concentrations equivalent to sodium nitrite as described in the examples listed above.
Previous studies have investigated organic acids or their salts for the inhibition of these pathogens in RTE processed meat applications. In particular, studies suggest that acetic acid or its salt alone when used at concentrations (<1%) that are expected to provide acceptable sensory attributes in RTE meat and poultry products, failed to inhibit C. perfringens in turkey breast meat (See e.g. Juneja, V. K., and H. Thippareddi. 2004. Inhibitory effects of organic acid salts on growth of Clostridium perfringens from spore inocula during chilling of marinated ground turkey breast. Int. J. Food Microbiol. 93:155-163., incorporated by reference herein in its entirety).
Additional studies demonstrated that 0.3-0.5% sodium diacetate when used alone or in combination with additional antimicrobials were effective in controlling Listeria monocytogenes in turkey slurries formulated with and without sodium nitrite (See e.g. Schlyter, J. H., Glass, K. A., Loeffelholz, J., Degnan, A. J., Luchansky, J. B., 1993. The effects of diacetate with nitrite, lactate, or pediocin on the viability of Listeria monocytogenes in turkey slurries. Int. J. Food Microbiol. 19, 271-281., incorporated by reference herein in its entirety). However, the suggested levels were higher than the maximum allowed levels (0.25% of the product formulation; FSIS 7120 list) in meat and poultry products in the U.S. and is expected to contribute an unacceptable flavor to the finished product. Additionally, other attempts have demonstrated the use of propionic acid or its salt in combination with pediocin to control Listeria monocytogenes. Nevertheless, to date known methods have failed to address control of Clostridia species, one of the predominant pathogen risks in uncured meat and poultry products.
While it is known to utilize nisin in combination with organic acids, the efficacy of these systems required emulsifiers and were dependent on the sequential addition of these individual components. In addition, these compositions did not demonstrated efficacy to inhibit pathogens and spoilage of concern under the conditions specified herein that represent uncured, high moisture and low-sodium processed meats (See e.g. U.S. Patent Application Publication no. 2013/0012428 A1 to Jacobus et al.; incorporated by reference herein in its entirety).
U.S. Pat. No. 6,509,050 B1 to Henson et al., which is incorporated by reference herein in its entirety, demonstrated the use of polyphosphates in combination with an organic acid or its salts in controlling Listeria monocytogenes in a broth model and spoilage microorganisms in a cured meat system. As is known in the art, phosphates are typically used in meat applications to retain moisture and to improve the yield. However, there was no evidence of the efficacy of this approach for the inhibition of pathogens in a low sodium uncured meat system. Moreover, the levels of phosphates described therein are higher than currently allowed in the U.S. (0.5%; FSIS 7120 list).
To date, the simultaneous inhibition of Listeria and Clostridia species in RTE refrigerated meats formulated without sodium nitrite has not been reported. It is desirable to have a method that can demonstrate efficacy against foodborne pathogens and spoilage microorganisms in an “uncured” or “nitrite-free” systems with acceptable flavor that are high in moisture (65-80% by weight), low in salt (<2% by weight), and in a pH range of 5.5-8.5.
Therefore, the cumulative effects of replacing chemical preservatives with clean label options in addition to lowering the sodium levels in foods has obligated food manufacturers to compromise shelf life. Particularly, at risk are food products with high moisture and low sodium which favors microbial growth such as the uncured meat application provided above. While there are several ways (methods and antimicrobials) to control the foodborne pathogens and spoilage in traditional processed meat and poultry products formulated using sodium nitrite, there is a need in the art for methods to eliminate such compromise and enhance the safety of clean label products formulated without sodium nitrite (uncured or sodium nitrite-free). It is also preferred to demonstrate a method of inhibiting the pathogens and spoilage with one solution that has broad antimicrobial properties in diverse matrices and applications.
The current invention provides such a method of inhibition. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The invention described herein relates to a method of inhibiting the growth of pathogens and spoilage organisms in a medium of characteristically high moisture of 65-80% by weight, low salt (<2.0% by weight) that is in a pH range of 5.5-8.5, by the application of an effective amount of an antimicrobial composition and offers a robust alternative to conventional preservatives. The antimicrobial composition comprises an organic acid or its salt and a fermentation derived antimicrobial peptide and is free of any emulsifying and or chelating agents. The antimicrobial composition can be applied at all stages of processing but not limited to pre-mixing and pre-cooking when applied to processed meats. It can also applied by spraying, direct addition, injection, pumping tumbling, massaging etc.
The proposed method can suppress the growth of pathogens and spoilage microorganisms in systems including but not limited to ready-to-eat food products particularly meat and poultry, as well as cleaning agents, animal feedstuffs, cosmetics, and pharmaceuticals.
The organic acid is selected from acetic, citric or propionic acid, or the salt thereof. By a salt of an organic acid, it is meant generally a monovalent or divalent metal salt of the organic acid including but not limited to sodium, potassium, calcium and magnesium salt of the organic acid. The fermentation derived antimicrobial is comprised of a bacteriocin or its analogues or derivatives, whereby the bacteriocin is a ribosomally synthesized antimicrobial peptide produced by certain bacteria which kill or inhibit the growth of closely related bacteria.
This antimicrobial intervention applies to food and non-food systems including various packaging conditions such as vacuum, non-vacuum, and modified atmospheric conditions.
In one exemplary embodiment of the invention, an antimicrobial composition to control the outgrowth of pathogens and spoilage microorganisms in food or beverage products having a moisture content of about 65% by weight to about 80% by weight, a salt content of less than about 2.0% by weight, and having a pH range of about 5.5 to about 8.5 is provided. The composition includes an organic acid or its salt and fermentation derived antimicrobial peptide. The aforementioned pathogens may be the species of Listeria, and/or may be the species of a class of spore formers comprising species of Clostridia. The spoilage microorganisms may be the species of Lactobacilli, Leuconostoc, Pseudomonas, and Brochothrix. 
The food product may be selected from the group consisting of animal meat, beverages, feed stuffs, or agricultural produce. The packaging conditions of the food or beverage products may be one of vacuum, non-vacuum and modified atmospheric conditions.
In a subsidiary embodiment according to this aspect, the organic acid is selected from the group consisting of acetic acid, lactic acid, propionic acid, citric acid, or a salt thereof. As one specific non-limiting example, the organic acid is acetic acid or its salt at a concentration of at least about 0.275% by weight. The pH of the acetic acid or its salt is from about 5.0 to about 8.0.
In another subsidiary embodiment according to this aspect, the fermentation derived antimicrobial is a bacteriocin. The bacteriocin is a ribosomally synthesized antimicrobial peptide produced by certain bacteria which kills or inhibits the growth of closely related bacteria, for example, nisin, sakacin, pediocin, lactocin, and derivatives or analogues thereof. As one specific non-limiting example, the bacteriocin is nisin in the range of about 5 ppm to about 50 ppm. The pH of nisin is from about 3.0 to about 6.5.
In another subsidiary aspect according to this invention, an antimicrobial activity of the composition is bacteriostatic or bacteriocidal. The composition may be in powder or liquid format. When in solution, the composition has a pH from about 5 to about 8.
In another exemplary embodiment of the invention, a method for controlling the outgrowth of pathogens and spoilage microorganisms in food or beverage products is provided. The method includes providing a food or beverage product having a moisture content of about 65% by weight to about 80% by weight, pH in the range of about 5.5 to about 8.5, and salt content less than about 2.0% by weight. The method also includes contacting the food or beverage product with an antimicrobial composition comprising an organic acid or its salt and a fermentation derived antimicrobial peptide to control growth of pathogens and growth of spoilage microorganisms.
In a subsidiary embodiment, the step of providing the food or beverage product includes providing a food or beverage product that is free of nitrate and nitrite that is derived either from synthetic or fermentation process. The pathogens may be the species of Listeria. The pathogens may also be the species of a class of spore formers comprising species of Clostridia. The spoilage microorganisms are any of the species of Lactobacilli, Leuconostoc, Pseudomonas, and Brochothrix. The food or beverage product is selected from the group consisting of animal meat, beverages, feed stuffs, or agricultural produce. The packaging conditions of the food or beverage products are one of vacuum, non-vacuum and modified atmospheric conditions.
In yet another exemplary embodiment, the invention provides an antimicrobial system comprising a food or beverage product comprising the following conditions: 1) a moisture content of about 65% by weight to about 80% by weight, 2) pH in the range of about 5.5 to about 8.5, and 3) a salt content of less than about 2.0% by weight, the system also including an organic acid or its salt, a fermentation derived peptide, wherein, the organic acid or its salt and the fermentation derived peptide are applied to control microbial growth the food or beverage product at said conditions.
Other aspects, objectives and advantages of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings incorporated in and forming a part of the specification illustrate several aspects of the present invention and, together with the description, serve to explain the principles of the invention. In the drawings:
FIG. 1 illustrates the inhibition of L. monocytogenes outgrowth on surface inoculated uncured deli-style turkey slices stored in vacuum packaging at 4° C. for 14 weeks;
FIG. 2 demonstrates the antimicrobial efficacy of the peptide component alone and in combination with organic acid or its salt against L. monocytogenes following surface inoculation on uncured deli-style turkey slices, under vacuum packaging conditions at 4° C. for 13 weeks;
FIG. 3 illustrates the growth of L. monocytogenes on surface inoculated uncured deli-style turkey slices stored in non-vacuum packaging conditions at 4° C. for 11 weeks;
FIG. 4 reveals the efficacy of the antimicrobial application against lactic acid bacteria in uncured deli-style turkey slices stored in vacuum packaging conditions at 4° C. for 7 weeks;
FIG. 5 demonstrates the efficacy of antimicrobial application against L. monocytogenes following surface inoculation on cured deli-style turkey slice in vacuum packaging conditions at 4° C. for 8 weeks;
FIG. 6 demonstrates the antimicrobial efficacy of the organic acid or its salt and antimicrobial peptide against spoilage microorganisms (total plate counts) in fresh chicken breast fillets at 4° C. for 35 days;
FIG. 7 demonstrates the antimicrobial efficacy of the organic acid or its salt and antimicrobial peptide against the growth of Pseudomonas species in fresh chicken breast fillets at 4° C. for 35 days;



While the invention will be described in connection with certain preferred embodiments, there is no intent to limit it to those embodiments. On the contrary, the intent is to cover all alternatives, modifications and equivalents as included within the spirit and scope of the invention as defined by the appended claims.
DETAILED DESCRIPTION OF THE INVENTION
Uncured, high moisture and reduced sodium systems are more favorable substrates for the outgrowth of pathogens and spoilage bacteria and therefore should be formulated with efficient antimicrobial(s) to minimize the public health risks as well as economic losses to the processors.
The antimicrobial formulation in the methods described are comprised of an organic acid or its salt and antimicrobial peptide, whereby the organic acid is preferably, acetic acid at an inclusion level of at least 0.275% by weight and the antimicrobial peptide is nisin, used at a quantity to deliver activity in the range of 1-50 ppm, preferably 7-30 ppm. In addition to delaying the toxin production of spore formers, the antimicrobial composition is bacteriostatic and in some cases bactericidal for controlling vegetative pathogens as well as spoilage bacteria. Consequently, it can enhance the product safety and extend shelf life.
Nisin levels required to achieve antimicrobial efficacy were calculated by performing a modification of the agar diffusion assay previously described with the use of Pediococcus pentosaceus FBB63 as the indicator strain (See e.g. Jozala, A. F., Silva, D. P., Vicente, A, A, Teixeira, J. A., Junior, A. P., and Penna, T. C. V. 2011. Processing of byproducts to improve nisin production by Lactococcus lactis. Afr. J Biotech 10:14920-14925) The activity of the fermentation derived nisin was compared with a commercially known standard sample of Nisaplin. A conversion factor thus derived [1 Arbitrary Unit (AU)/g=1.04×International Unit (IU)/g] was used in calculating the levels in part per million (ppm) required for the antimicrobial effects (1 ppm=40 IU).
Compositions comprising various ratios of each of the components within the preferred ranges outlined are referred to as compositions A-J going forward. In those compositions, reference to percent by weight means the percent by weight taking into account the food product which the compositions are introduced in.
Example 1—Methods to Inhibit L. monocytogenes Outgrowth in an Uncured Meat with High Moisture and Low Sodium in the System
This embodiment describes the antimicrobial composition to control the outgrowth of pathogens such as L. monocytogenes in high moisture and reduced sodium systems, for example, a ready to eat uncured deli-style turkey product.
Uncured deli-style turkey (70% turkey breast, 25.6% water, 2% starch, 1% sugar, 1% salt, 0.4% sodium phosphate, 0% sodium nitrite) was prepared under Good Manufacturing Practices. Appropriate levels of antimicrobials for each treatment were added along with non-meat ingredients, stuffed in to chubs and cooked to a final temperature of 73.8° C. The moisture of the finished product compositions were in the range of 72%-76%, with reduced sodium levels of 350-450 mg per 56 g of serving and a pH value of 6.1-6.4.
The product was sliced (22-28 g/slice using a sanitized slicer to prevent contamination with spoilage microbes) and stored at 4° C. until use in the studies mentioned herein. Cooked slices were surface inoculated with 3 log CFU/g of a five-strain mixture of L. monocytogenes including strains FSL-C1-109 (serotype 4b), LM101M (4b), LM310 (4b), LM132 (1/2 a), and LM108M (1/2b), vacuum packed (100 g/package), and stored at 4° C. during the study. Populations of L. monocytogenes were enumerated from inoculated samples in triplicate. At each time point, inoculated treatments were homogenized in sterile Butterfield's buffer and plated on Modified Oxford agar (35° C., 48 h). Treatments that supported more than 2.0 log CFU/ml from day zero were deemed as spoiled and were discontinued from the study.
In a preferred embodiment, the application of the antimicrobial demonstrated the inhibition of L. monocytogenes growth over 14 weeks of storage at 4° C. Treatments included: (i) control without antimicrobials, (ii) composition B at 2.0% by weight, (iii) composition C at 2.7% by weight, and (iv) a control formulated with 156 ppm ingoing sodium nitrite and 3.8% potassium lactate-diacetate by weight, a blend that is typically used in the industry. The results are presented in FIG. 1.
Un-inoculated turkey deli slices were subjected to sensory evaluation to determine the overall acceptability as perceived by five trained panelists. Test samples were compared with a control which did not contain antimicrobials or sodium nitrite and were deemed acceptable by the panelists with descriptors that are similar to the control (salt, sweet, sour, turkey flavor).
In another preferred embodiment, buffered vinegar and antimicrobial peptide demonstrated greater efficacy than antimicrobial peptide alone in controlling the outgrowth of L. monocytogenes in uncured deli-style turkey slices, under vacuum packaging at 4° C. for 13 weeks. Treatments included: (i) control without antimicrobials, (ii) composition A at 1.2% by weight containing antimicrobial peptide alone, (iii) composition B at 3.2% by weight, and (iv) composition C at 3.9% by weight. The results are presented in FIG. 2.
In another preferred embodiment, an uncured deli-style turkey formulation did not support the outgrowth of L. monocytogenes in slices packaged in non-vacuum conditions and stored at 4° C. for 11 weeks. Treatments included: (i) control without antimicrobials, (ii) composition D at 1.85% by weight, (iii) composition E at 2.95% by weight, and (iv) composition F at 3.15% by weight. The results are presented in FIG. 3.
Example 2—Methods to Inhibit Spoilage Microorganisms Such as Lactic Acid Bacteria Growth in an Uncured Meat with High Moisture and Low Sodium in the System
This embodiment describes the efficacy of the method of allying the antimicrobial composition for controlling spoilage bacteria especially lactic acid bacteria. This experiment was conducted in an uncured meat model with high moisture and low sodium conditions as described in example 1 and subjected to a shelf-life study at 4° C. for 7 weeks. Three formulations of the uncured deli-style turkey were prepared as per the recipe mentioned in example 1. Treatments included: (i) control without antimicrobials, (ii) composition B at 2.0% by weight, and (iii) composition G at 2.36% by weight. Initial counts of background microflora in the product post cooking described herein reflects the contamination scenario during the handling and slicing. Lactic acid bacteria plate counts were determined by plating in duplicate un-inoculated samples on APT agar with bromocresol purple indicator. Plates were incubated at 25° C. for 48 h. The results are presented in FIG. 4.
These results indicate that a combination of buffered vinegar and antimicrobial peptide is more effective in controlling the spoilage bacteria under the specific conditions challenged than a combination of lactic acid and antimicrobial peptide.
Example 3—Methods to Control the Growth of Clostridium sporogenes 
Antimicrobial activity against C. sporogenes PA 3679 was demonstrated in a broth study using modified cooked meat medium as the former has proven to be anon-toxigenic surrogate for C. botulinum. Treatments included: (i) control without antimicrobials, (ii) composition B at 2.0% by weight, and (iii) composition C at 2.7% by weight. All the variables were inoculated with spores that had been heat shocked at 85° C. for 5 min at a target of 2.0 log CFU/g and incubated anaerobically at 25° C. for 3-4 days. Growth of C. sporogenes was monitored by plating appropriate dilutions on modified Mc Lung's agar and incubation at 35-37° C. for 3 days. Each treatment was assayed in duplicate. The results are shown in Table 1.







TABLE 1







Method of inhibition of C. sporogenes by the antimicrobial composition


in modified cooked meat medium at 25° C.










Initial Log CFU/ml
Final Log CFU/ml


Treatment
(Time zero)
(After 72 hours)












Control
2.0
7.23


Composition B 2.0%
2.0
0


by weight


Composition C 2.7%
2.0
0


by weight









As will be easily appreciated by those of skill in the art based on the data presented in Table 1, the application of buffered vinegar in combination with antimicrobial peptide is effective in preventing the outgrowth of C. sporogenes. 
Example 4—Methods of Inhibiting the Outgrowth of L. monocytogenes Growth in a Cured Meat Model (with Low Levels of Curing Agents than Traditional Usage Levels) with High Moisture and Low Sodium in the System
This embodiment describes the method of using the antimicrobial composition to control the outgrowth of pathogens such as L. monocytogenes in a meat model formulated with the minimum amount of curing agent required for contributing color and flavor attributes in systems. For example, in commercial processed meat formulation, a maximum of 156 ppm of ingoing sodium nitrite is used in conjunction with an antimicrobial to achieve a typical shelf-life of 90 days at refrigerated storage. In a preferred embodiment, the level of ingoing sodium nitrite is significantly reduced to as low as 20 ppm sodium nitrite in combination with the antimicrobial composition described and achieved the same shelf-life extension.
Deli-style cured turkey product (70% turkey breast, 25.6% water, 2% starch, 1% sugar, 1% salt, and 0.4% sodium phosphate) was prepared under Good Manufacturing Practices. Appropriate levels of antimicrobials for each treatment were added along with non-meat ingredients, stuffed in to chubs and cooked to a final temperature of 73.8° C. The composition of the finished product was found to be high in moisture (76% moisture), reduced sodium (340 mg of sodium/56 g of serving) and at a nearly neutral pH (6.1-6.3). Cooked slices were inoculated, vacuum packed, and stored at 4° C. to evaluate the efficacy for the control of L. monocytogenes as described in example 1. Treatments included: (i) 80 ppm sodium nitrite by weight, (ii) 40 ppm sodium nitrite by weight+composition H at 2.0% by weight, and (iii) 20 ppm sodium nitrite by weight+composition I at 2.7% by weight. The results are presented in FIG. 5.
Un-inoculated turkey deli slices were subjected to sensory evaluation to determine the overall acceptability as perceived by five trained panelists. Samples were compared to a control sample containing 80 ppm ingoing sodium nitrite (by weight) without additional antimicrobials and were deemed as acceptable by the panelists with descriptors that were similar to the control (cured, savory, sweet, sour, turkey flavor).
The results shown in FIG. 5 demonstrate that a blend of buffered vinegar and antimicrobial peptide in combination with cure (sodium nitrite) is more effective than cure alone. Furthermore, the antimicrobial composition has the potential to reduce the cure (sodium nitrite) levels in meat formulations without compromising the microbial quality.
Similar benefits are expected in inhibiting pathogens and spoilage organisms when sodium nitrates or nitrites either synthetic or natural source are used in the formulation.
Example 5—Method of Preventing or Delaying the Toxin Production by Clostridium botulinum in Uncured Chicken Batter
This embodiment describes the method of preventing or delaying the toxin production by Clostridium botulinum in an inoculated uncured chicken meat batter (100 cfu/g). Uncured (sodium nitrite-free) chicken meat batter was prepared under Good Manufacturing Practices. The formulation was prepared with chicken meat (70%), water (23%), modified corn starch (2.1%), salt (1.5%), carrageenan (0.2%), and sodium phosphate (0.4%). Treatments included in this study (i) control without antimicrobials, (ii), composition B at 2.0% by weight, (iii) composition C at 2.7% by weight. Pre-grinded meat (1⁄8′′) was mixed with non-meat ingredients in a bowl chopper to prepare a meat batter, bagged, flattened, and kept frozen until use.
For testing, frozen batter is thawed and inoculated with C. botulinum spores which had been heat shocked at 80° C. for 10 min. Two individual batches of meat batter were inoculated with either proteolytic (33A, 36A, 62A, 77A, 53B, 113B, 213B, ACC1B) or non-proteolytic (K85, K86, K87, K88, K89) strains, cooked in bag using a water bath to an internal temperature of 73.8° C. The samples were cooled, and incubated for 2 days at 26.6° C. To examine toxin production, samples were pulled at 24 and 48 hours, extracts taken and administered to mice to verify the presence of toxin. Another batch of meat batter inoculated with non-proteolytic strains only was incubated for up to 8 weeks at 7° C. At weekly intervals, samples were taken, tryspsinized for toxin activation, and extracts were administered into mice for toxin bioassay.
Standard protocols were followed in growing and harvesting Clostridia cultures, and performing mouse toxin bioassay (see FDA Bacteriological Analytical Manual for Foods, chapter 17, 2015). Briefly, at each observation inoculated samples were weighed and an equal volume of gel-phopsphate buffer added (adjusting to pH 6.2), centrifuged under refrigeration to collect the aqueous supernatant fluid for toxin assay. This mixture was filtered through a millipore filter to avoid the nonspecific death of the mice. For non-proteolytic inoculated samples, trypsinization was performed after filtration to activate the toxin. The meat extract filtrate thus collected per each test sample at each observation point was diluted and administered (0.5 ml) to a pair of mice via intraperitoneal injection. Mice were observed for 48 hours and examined for symptoms and death characteristic of C. botulinum intoxication. Deaths following meat extract administration are presumptive evidence of toxin production. Further confirmation was achieved by challenging two additional mice with a pre-incubated (37° C. for 30 min.) antitoxin preparation (protected control). Death with non-specific reasons such as chemicals present in injected fluid or trauma was dis-regarded and the challenge was repeated to confirm the toxin presence in the meat samples. The results of the study are shown in Tables 2 and 3. The results demonstrate that formulations prepared with the antimicrobial composition were effective in delaying the toxin formation in samples inoculated with a cocktail of proteolytic or non-proteolytic C. botulinum strains until 24 h of in incubation at 30° C. Furthermore, the antimicrobial compositions were also effective in delaying toxin formation in samples inoculated with non-proteolytic samples incubated for 9 weeks at 7° C.







TABLE 2







Presence of Clostridium botulinum toxin in uncured meat batters inoculated


with proteolytic and non-proteolytic spore cocktails and incubated at 26.6° C. for 48 hours.










Inoculated with proteolytic cocktail and
Inoculated with non-proteolytic cocktail and



Incubated for 48 hours.
Incubated for 48 hours.















Treatment
0 hour
24 hours
36 hours
48 hours
0 hour
24 hours
36 hours
48 hours





Control (No
Negative
Positive
Positive
Positive
Negative
Positive
Positive
Positive


antimicrobials)


Composition B -
Negative
Negative
Positive
Positive
Negative
Negative
Positive
Positive


2.0% by


weight


Composition C -
Negative
Negative
Negative
Positive
Negative
Negative
Positive
Positive


2.7% by


weight
















TABLE 3







Presence of Clostridium botulinum toxin in uncured meat batters inoculated


with non-proteolytic spore cocktails and incubated at 7° C. for 9 weeks.









Non-proteolytic cocktail Incubated for 9 weeks.
















Treatment
Week-1
Week-2
Week-3
Week-4
Week-5
Week-6
Week-7
Week-8
Week-9





Control (No antimicrobials)
Negative
Negative
Negative
Positive
Positive
Not tested*
Not tested
Not tested
Not tested


Composition B - 2.0% by
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative


weight


Composition C - 2.7% by
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative


weight





*Subsequent samples were not tested as results were positive in 2 prior consecutive time points.






Example 6—Efficacy of Antimicrobial Composition Against Spoilage Microorganisms in Fresh Chicken Breast Fillets
Boneless, skinless, uncured chicken breast fillets were vacuum tumbled to achieve a target of 12% marinade pick-up based on the meat block. Marinated chicken breast fillets were stored in plastic bags (sealed without vacuum) at 4° C. until spoilage (≧6.0 log cfu/g). Samples were plated in duplicate on days 0, 7, 14, 21, 28, and 35. Twenty-five grams of sample was taken from each treatment bag under aseptic conditions and diluted (1:2) in 0.1% peptone water and homogenized for 1 min. Samples were plated on tryptic soy agar and Pseudomonas agar base. Treatments included: (i) control without antimicrobials, (ii) composition B at 2.0% by weight, and (iii) composition J at 1.6% by weight.
Results presented in FIGS. 6 and 7 demonstrate that marinated chicken breast fillets without antimicrobials spoiled by day 14 (total plate counts>6.0 log cfu/g), while the chicken breast fillets formulated with composition B (1.6% by weight) or J (2.0% by weight) extended the shelf life to 35 days at refrigerated storage.
All references, including publications, patent applications, and patents cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) is to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. A method using an antimicrobial composition to control the outgrowth of pathogens and spoilage microorganisms in food products, comprising the steps of:
providing a food product having a moisture content of 65% by weight to 80% by weight, pH in the range of 5.5 to 8.5, and salt content less than 2.0% by weight, wherein the food product is free of nitrate and nitrite that is derived from either synthetic or fermentation processes;
contacting the food product with the antimicrobial composition comprising acetic acid or its salt and a fermentation derived antimicrobial peptide to control growth of pathogens and growth of spoilage microorganisms, wherein the acetic acid or its salt has a concentration in the food product of at least 0.275% by weight; and
packaging the food product, wherein the packaged food product is free of nitrate and nitrite.

2. The method of claim 1, wherein the pathogens are species of Listeria.

3. The method of claim 1, wherein the pathogens are species of a class of spore formers comprising species of Clostridia.

4. The method of claim 1, wherein the spoilage microorganisms are any of the species of Lactobacilli, Leuconostoc, Pseudomonas, and Brochothrix.

5. The method of claim 1, wherein the food product is selected from the group consisting of animal meat, feed stuffs, and agricultural produce.

6. The method of claim 1, wherein packaging conditions of the food products are one of vacuum and modified atmospheric conditions.

7. An antimicrobial system comprising a food product, said food product comprising the following conditions: 1) a moisture content of 65% by weight to 80% by weight, 2) pH in the range of 5.5 to 8.5, 3) a salt content of less than 2.0% by weight, and 4) the food product is free of nitrate and nitrite that is derived from either synthetic or fermentation processes, the system also comprising acetic acid or its salt at a concentration in the food product of at least 0.275% by weight and a fermentation derived peptide, wherein the acetic acid or its salt and the fermentation derived peptide are operable in combination to control microbial growth in the food product at said conditions free of nitrate and nitrite that is derived from either synthetic or fermentation processes.

8. The method of claim 1, wherein the pH of the acetic acid or its salt is from 5.0 to 8.0.

9. The method of claim 1, wherein the fermentation derived antimicrobial peptide is a bacteriocin.

10. The method of claim 9, wherein the bacteriocin is a ribosomally synthesized antimicrobial peptide produced by bacteria that kills or inhibits the growth of other bacteria.

11. The method of claim 10, wherein the bacteriocin is selected from the group consisting of nisin, sakacin, pediocin, and lactocin.

12. The method of claim 11, wherein the bacteriocin is nisin in the range of 1 ppm to 50 ppm in the food product.

13. The method of claim 1, wherein an antimicrobial activity of the antimicrobial composition is bacteriostatic or bacteriocidal.

14. The method of claim 1, wherein the antimicrobial composition is in powder or liquid format.

15. The method of claim 1, wherein the antimicrobial composition in solution has a pH from 5 to 8.

16. The method of claim 1, wherein packaging conditions of the food products are non-vacuum conditions.

17. A method using an antimicrobial composition to control the outgrowth of pathogens and spoilage microorganisms in beverage products, comprising the steps of:
providing a beverage product having a moisture content of 65% by weight to 80% by weight, pH in the range of 5.5 to 8.5, and salt content less than 2.0% by weight, wherein the beverage product is free of nitrate and nitrite that is derived from either synthetic or fermentation processes;
contacting the beverage product with the antimicrobial composition comprising acetic acid or its salt and a fermentation derived antimicrobial peptide to control growth of pathogens and growth of spoilage microorganisms, wherein the acetic acid or its salt has a concentration in the beverage product of at least 0.275% by weight; and
packaging the beverage product, wherein the packaged beverage product is free of nitrate and nitrite.

18. An antimicrobial system comprising a beverage product, said beverage product comprising the following conditions: 1) a moisture content of 65% by weight to 80% by weight, 2) pH in the range of 5.5 to 8.5, 3) a salt content of less than 2.0% by weight, and 4) the beverage product is free of nitrate and nitrite that is derived from either synthetic or fermentation processes, the system also comprising acetic acid or its salt at a concentration of at least 0.275% by weight and a fermentation derived peptide, wherein the acetic acid or its salt and the fermentation derived peptide are operable in combination to control microbial growth in the beverage product at said conditions free of nitrate and nitrite that is derived from either synthetic or fermentation processes.