Fermented food products comprising antibiotic-susceptible bacteria

Abstract

Disclosed herein is a method of making an antibiotic-resistant bacteria-free fermented food product to treat gut microbiota dysbiosis. Also disclosed is a fermented food product comprising a combination of unique starter strains of bacteria of Leuconostoc sp., Lactobacillus sp., Pediococcus sp., and / or other genera of lactic acid bacteria, such as Leuconostoc K6-Leu13 (WL-1) Leuconostoc mesenteroides Pediococcus K6-Pedio (WL-2), Pediococcus inopinatus, Lacticaseibacillus paracasei, Leuconostoc citreum, Lactobacillus SH16 (WL-3), Lactobacillus brevis, Pediococcus acidilactici, Latilactobacillus sakei, etc., for successful fermented products, with or without microbial succession.

Description

FERMENTED FOOD PRODUCTS COMPRISING ANTIBIOTIC-SUSCEPTIBLEBACTERIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Serial No. 63 / 609,537, filed December 13, 2023, the disclosure of which is expressly incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The sequence listing submitted on April 22, 2024, as an .XML file entitled “103361- 619W01_ST26.xml” created on April 11, 2024, and having a file size of 12,288 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUND

[0003] Food fermentation has an ancient history, initiated by spontaneous and random outgrowth of natural microbiota associated with raw food materials, the environment, and the handlers. The products with preferred features are consumed as fermented foods, and those with detrimental features were considered as spoilage or unsafe products. With artisan fermentation, there is inconsistency in safety and quality, due to the randomness of the microbiota involved. The modernization of dairy fermentation was achieved by pasteurization at 68°C or above followed by using well-characterized fermentation starter cultures. However, pasteurization is not preferred for vegetables and fruits, as heat treatment changes the texture and potentially flavor of the raw materials. Fermented vegetables are popular, but almost all are still made with artisan processes, such as kimchi and sauerkraut.

[0004] Artisan fermented products have been a critical carrier transmitting antibiotic resistant (AR) bacteria to consumers. For instance, with limited assessment conditions, Li et al. (2021, 2022) found that approximately 90% of retail kimchi products sampled contained antibiotic resistant (AR) bacteria, ranging from multidrug resistant pathogens, lactic acid bacteria driving fermentation, to commensals and plant pathogens. It was further illustrated that AR bacteria are inevitable during conventional fermentation. Introducing these AR bacteria can lead to seriouspublic health consequences such as bacteremia and sepsis resistant to antibiotic treatment. Targeted mitigation has become a must for the whole industry.

[0005] Thus, there is a need to address the aforementioned problems and other shortcomings associated with traditional food fermentation processes and the products thereof.SUMMARY

[0006] The present invention relates to the method of making an antibiotic-resistant (AR) bacteria other unfavorable microbial or chemical risks free or significantly mitigated fermented food product. The present disclosure addresses at least a portion of the problems described above through the consumption of traditionally fermented food products, and methods of making and using an inventive two-step process to make an antibiotic-resistant bacteria-and pathogen-free or mitigated fermented food product.

[0007] In one aspect, as disclosed herein, is a fermented food product, comprising a combination of two or more strains of bacteria, wherein the strains comprise: 1) Leuconostoc sp, and 2) Lactobacillus sp. or / and 3) Pediococcus sp.. This can further include other genera of lactic acid bacteria able to drive fermentation, that are susceptible to major antibiotics of clinical significance, which do not consistently and naturally co-exist and dormant in artisan fermented fruits and vegetables. Some examples are K6-Leul3 (WL-1) (SEQ ID NO: 1) which has 98.3% identity to Leuconostoc mesenteroides (NCBI ID HF562942.1), Pediococcus K6-Pedio (WL-2) (SEQ ID NO: 2), which has 97.96% identity to Pediococcus inopinatus (ID JQ612701.1), and Lactobacillus SH16 (WL-3) (SEQ ID NO: 3), which has 98.84% identity to Lacticaseibacillus paracasei (ID MH393 109.1), that do not naturally existing together and in a food product. In one embodiment, the Leuconostoc, Lactobacillus and Pediococcus bacteria can comprise 95% or more identity to the aforementioned bacterial sequences, or with probiotic Lactobacillus crispatus WZ12.

[0008] In some embodiments the food product is a fruit. In some embodiments the food product is a vegetable. In some embodiments the food product has dairy ingredients. In some embodiments the food product is a meat. In some embodiments the food product can be seafood or other aquaculture products or can consist of juices or puree of fruits and vegetables. In some embodiments the vegetable is a napa cabbage. In some embodiments the fermented food product is kimchi. As further described herein the fermented food product comprises other ingredients including but not limited to, seasoning, such as, salt, sugar, a chili-based product or a combination thereof. In another embodiment, the vegetable is a cabbage, and the fermented food product is sauerkraut. In additional embodiments the vegetables or fruits can be tomato, cucumber, peppers,radish, carrot, other greens, legumes, ginger, olive, etc., and their corresponding fermented food products.

[0009] In another aspect, also disclosed herein, is a method of fermenting a food product, wherein the method comprises: sanitizing the food item to be fermented, comprising microbial inactivation of natural microbiota including but not limited to antibiotic-resistant (AR) bacteria, food-borne pathogens, spoilage microbes and other random commensal bacteria, and then fermenting the cleaned food item(s) comprising inoculation with a starter culture cocktail for at least 24 hours.

[0010] In some embodiments, the method of sanitizing the food product is by using a Microcide®, an organic sanitizer, high pressure, washing in electrolyzed water or vinegar, sanitation gases, or blanching in hot water for short period of time, etc. In some embodiments, the sanitation gas is chlorine dioxide or ozone. In some embodiments sanitation is validated by the plate counting method for bacteria.

[0011] In some embodiments, the starter culture comprises at least one or a combination of unique strains of bacteria, that all are susceptible to major antibiotics of clinical significance, for example, Penicillins (such as Ampicillin), Macrolides, Tetracyclines etc. (except the genus naturally with innate resistance), not naturally existing together in a food product. In some embodiments the starter culture comprises one or more Leuconostoc sp., such as Leuconostoc K6-Leul3 (WL-1), Leuconostoc me senter oides, Leuconostoc citreum, and at least one or more of Lactobacillus or pediococcus, such as Lactobacillus SHI 6 (WL-3), Pediococcus K6-Pedio (WL-2), Pediococcus inopinalus. Lacticaseibacillus paracasei. Lactobacillus brevis, Pediococcus acidilactici, Latilactobacillus sakei, and / or probiotic Lactobacillus crispatus WZ 12. In further embodiments, the inoculated food product is going through anaerobic fermentation. In some embodiments the fermented food product is validated by sensory quality evaluation of taste and odor. In some embodiments the fermented food product quality is tested by rapid volatile compounds assessment using gas chromatography and mass spectrometry. In some embodiments the microbial safety and quality of the fermented food product is assessed by plate counting, Minimum Inhibitory Concentration (MIC), and / or DNA sequencing.

[0012] Further disclosed herein, is a method of treating gut microbiota dysbiosis, by administering the fermented food product of claim 1 to the subject, daily for 1, 2, 3, 4 or more weeks. In some embodiments the subject is a human.

[0013] Additional aspects and advantages of the disclosure will be set forth, in part, in the detailed description and any claims which follow, and in part will be derived from the detailed description or can be learned by practice of the various aspects of the disclosure. The advantages describedbelow will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.BRIEF DESCRIPTION OF THE FIGURES

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and together with the description, serve to explain, without limitation, the principles of the disclosure. Like numbers represent the same elements throughout the figures.

[0015] FIG. 1 shows the impact of 10-week food intervention by conventional fermented foods on the change of antibiotic resistome of fecal microbiota of human subjects in percentage comparing to the baseline. Green: by foods rich in traditionally including artisan fermented products (without antibiotic resistance mitigation); orange: by foods rich in plant fibers.

[0016] FIGS. 2A-2B show violin-boxplots with Student’ s t-test p-values illustrating the impact of 10-week food intervention on the change of antibiotic resistome of fecal microbiota of human subjects comparing to the baseline by diets rich in (FIG. 4A) traditionally including artisan fermented foods (without antibiotic resistance mitigation); (FIG. 4B) plant fibers.

[0017] FIGS. 3A-3B show an antibiotic resistome of pooled traditionally fermented food microbiota recovered from agar plates. FIG. 3 A) 4 kimchi samples (K1-K4) purchased in 2021. FIG. 3B) 4 artisan cheeses (C1-C4) purchased in 2021.

[0018] FIGS. 4A-4C show the resistome assessment of individual fermented foods purchased in 2022. FIG. 4A) direct resistome of kimchi samples; FIG. 4B) resistome of kimchi microbiota (left bar) and recovered BHI microbiota (right bar) of kimchi sample 7; FIG. 4C). resistome of BHI recovered cheese microbiota.

[0019] FIG. 5 shows the detected cheese microbiota recovered from BHI agar plates with and without antibiotics.

[0020] FIG. 6 shows traditional (left) and Clean -fermented (right) kimchi.DETAILED DESCRIPTIONDefinitions

[0021] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0022] Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0023] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0024] The term “administer,” “administering”, or derivatives thereof refer to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

[0025] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0026] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0027] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., gut microbiota dysbiosis). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to beunderstood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. In some aspects, the composition disclosed herein comprises four unique strains of bacteria, not naturally found together or in a food product.

[0028] A "decrease" or “mitigate” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. For example, the fermented food product disclosed herein can have a decreased level of AR bacteria compared to a control or to others where the specific combination of microbes hasn’t been used. This is also true for the method of making the fermented food products disclosed herein. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0029] An "effective amount" is an amount sufficient to affect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. As disclosed herein, an “effective amount” is the amount of microbes, or types of microbes, needed to provide an AR-free fermented food. An “effective amount of probiotics or fermented food intake” can also refer to an amount of probiotics or fermented food intake sufficient to reduce or prevent gut microbiota dysbiosis and associated symptoms.

[0030] As used herein, “enhance”, “enhanced”, “enhancement”, “enhancing”, and any grammatical variations thereof as used herein, refers to an act of intensifying, increasing, or further improving the quality, value, or shelf-life of a food product.

[0031] " Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity compared to a control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. An example is that the fermented food product disclosed herein can inhibit the amount of AR bacteria present in the food product.

[0032] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could beprevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0033] Used herein, the term “probiotics” refers to live microorganisms promoted with claims that they provide health benefits when consumed, generally by improving or restoring the gut flora.

[0034] By “reduce” or other forms of the word, such as “reducing” or “reduction,”, “mitigate”, “mitigation” is meant lowering of an event or characteristic (e.g., symptoms of gut microbiota dysbiosis). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces dysbiosis” means reducing inflammation but increasing the microbial diversity relative to a standard or a control. “Mitigate antibiotic resistance” in fermented foods or host gut microbiota can also mean increase microbial diversity and improve host overall health relative to a standard or a control.

[0035] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. The “subject” can also be birds such as poultry and turkey, or aquaculture such as fish, shrimp, and other shellfish, etc.

[0036] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0037] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportivetreatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0038] The terms “treat,” “treating,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating, or reducing the intensity of one or more attendant symptoms of a disorder or condition and / or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the disclosure may be applied preventively, prophylactically, palliatively, or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of gut microbiota dysbiosis), during early onset (e.g., upon initial signs and symptoms of acid reflux, flatulence, indigestion, or food intolerance or after an established development of gut microbiota dysbiosis.

[0039] By “fermentation” is meant a biochemical reaction which involves releasing energy from an organic substrate, under the action of microorganisms. It is a conversion process of a raw material by the microorganisms, this conversion then produces biomass and metabolites. In particular, lactic fermentation is an anaerobic process of the consumption of lactose or other carbohydrates by the bacteria in the ferments, which causes the formation of lactic acid, with or without involving other metabolites (such as alcohol, acidic acid, CO2, etc.), and a lowering of the pH.

[0040] The term “probiotics,” as used herein, refers to a live microorganism or a culture thereof, which beneficially affects host animals including humans by improving their intestinal microbial balance in the gastrointestinal tract of host animals, and may further mean a microorganism or a culture thereof, which is capable of improving a microbial balance in vivo or in vitro as well as the gastrointestinal tract.

[0041] The meaning of “microorganisms” and “microbes” includes, but is not limited to, bacteria, fungi, algae, protozoans, and viruses. The microbes can be either aerobic, anaerobic, or facultative with respect to oxygen use. It is also understood that the microbes within water or aqueous systems can be located or suspended within the fluid (planktonic) or localized on a surface in contact with the aqueous system (biofilms).

[0042] “Antibiotic resistance” as used herein refers to when microbes evolve mechanisms that protect them from the effects of antimicrobials. This specifically refers to bacteria that become resistant to antibiotics. Some examples of antibiotic-resistant bacteria are Klebsiella pneumoniae and Serratia marcescens. Some examples of disease-causing bacteria are Clostridium difficile and Enterococcus.

[0043] The words and phrases “control”, “microbial control”, “controlling”, and “antimicrobial efficacy” should be broadly construed to include within their meaning, without being limited to, inhibiting the growth of microbes, killing microbes, disinfection, preservation, sanitization, or preventing the re-growth of microbes.General DescriptionFermented food product

[0044] Disclosed herein is a fermented food product, wherein the food product is a fruit, vegetable, dairy products for example, cheese, meat or seafood, wherein the food product is fermented. In one example, the vegetable is napa cabbage. As disclosed herein, the food product is fermented by multiple unique strains of antibiotic resistant mitigated bacteria, such as a combination of at least two or more strains of bacteria, wherein the strains comprise: 1) Leuconostoc sp., and 2) Lactobacillus sp. or / and 3) Pediococcus sp. or alike, that are susceptible to major antibiotics of clinical significance, which do not naturally co-exist in artisan fermented fruits and vegetables. Some examples are Leuconostoc sp., such s Leuconostoc K6-Leul3 (WL- 1), Leuconostoc mesenteroides, Leuconostoc citreum, and at least one or more of Lactobacillus or pediococcus, such as Lactobacillus SHI 6 (WL-3), Pediococcus K6-Pedio (WL-2), Pediococcus inopinatus, Lacticaseibacillus paracasei, Lactobacillus brevis, Pediococcus acidilactici, Latilactobacillus sakei, and / or probiotic Lactobacillus crispatus WZ12. These antibiotic susceptible strains do not naturally exist together, particularly in naturally fermented food products.

[0045] A bacterial strain is said to be susceptible to a given antibiotic when it is inhibited in vitro by a concentration of a drug that is associated with a high likelihood of therapeutic success. The standard for susceptibility is outlined in Rodolf et al. (Susceptible, intermediate, and resistant - the intensity of antibiotic action. Dtsch Arztebl Int. 2008 Sep;105(39):657-62, hereby incorporated by reference in its entirety for its teaching concerning antibiotic resistant / susceptible bacteria). If a given microbial strain is susceptible to antibiotics, a bacterial suspension with the antibiotic is reduced compared with a bacterial suspension without antibiotics, whereas with a resistant strain the amount of bacteria with or without antibiotics will be comparable. By “reduced” is meant by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any amount in between or below these values.

[0046] The terms “antibiotic” and “antimicrobial compound” are used interchangeably herein and are used herein to describe a compound or composition which decreases the viability of amicroorganism, or which inhibits the growth or reproduction of a microorganism. “Inhibits the growth or reproduction” means increasing the generation cycle time by at least 2-fold, preferably at least 10-fold, more preferably at least 100-fold, and most preferably indefinitely, as in total cell death. As used in this disclosure, an antibiotic is further intended to include an antibacterial, bacteriostatic, or bactericidal agent. Non-limiting examples of antibiotics useful in aspect of the invention include penicillins, cephalosporins, aminoglycosides, sulfonamides, macrolides, tetracyclines, lincosamides, quinolones, chloramphenicol, glycopeptides, metronidazole, rifampin, isoniazid, spectinomycin, folate inhibitors, sulfamethoxazole, and others.

[0047] In one example, lactic acid bacterial strains susceptible to major antibiotics, such as Leuconostoc K6-Leul3 (WL-1) which has 98.3% identity to Leuconostoc mesenteroides (NCBI ID HF562942.1), Lactobacillus SH16 (WL-3), which has 98.84% identity to Lacticaseibacillus paracasei (ID MH393109.1), or Pediococcus K6-Pedio (WL-2), which has 97.96% identity to Pediococcus inopinatus (ID JQ612701.1), that not naturally existing together and in a food product wherein the Leuconostoc, Lactobacillus and Pediococcus bacteria comprises 95% or more identity to the aforementioned bacterial sequences, or with probiotic Lactobacillus crispatus WZ12, these strains do not exist together in a traditionally or artisan fermented food product. Wherein, not naturally existing together means that these antibiotic susceptible and resistant mitigating bacterial strains mentioned above are not found together, especially in an artisan or traditionally or standard fermented food product.

[0048] In some embodiments, the Leuconostoc sp., Pediacoccus sp., and Lactobacillus sp. bacteria comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identities similar to the bacteria with aforementioned sequences. An example of a vegetable to be fermented is napa cabbage. The fermented napa cabbage is mixed with other ingredients wherein an example of other ingredients is seasoning to enhance the flavor of the fermented food product, and wherein examples of seasoning are salt, sugar, fish sauce, soy sauce, red chili powder, red chili paste, garlic, ginger, red pepper flakes, vinegar, onions and a combination thereof. The result of the fermentation of napa cabbage in the presence of seasoning is a fermented food product, such as kimchi.Method of Fermentation

[0049] Disclosed herein, is an innovative method of fermenting a food product, wherein, fermentation is the chemical breakdown of a substance by bacteria, yeasts, or other microorganisms, typically involving effervescence and the giving off heat. Some foodproducts for example, fruit, vegetable, dairy products for example, cheese, seafood or meat, cannot be subjected to high heat as done during pasteurization to reduce pathogenic microbial load, as heat alters the properties of flavor, texture and appearance of the food product and therefore the method of fermentation disclosed herein, is used.

[0050] The innovative fermentation method comprises two steps, wherein the first step is a pretreatment step, wherein the food product to be fermented is sanitized, wherein sanitizing means treating with a sanitizer to reduce the amount of or to inactivate microbes. This is done so that the food product won’t require pasteurization or ultra-pasteurization, wherein heat is used to inactivate, kill, or reduce microbes. Pre-treatment can involve treating the food to be fermented with an organic sanitizer, for example, Microcide®, or high pressure, washing in electrolyzed water or vinegar, sanitation gases, or blanching in hot water for short period of time. Some examples of sanitation gases are chlorine dioxide and ozone. The microbes that are present on the food to be fermented can be antibiotic-resistant (AR) bacteria, disease-causing bacteria, or spoilage bacteria causing quality defects. Antibiotic resistant bacteria are defined above.

[0051] As disclosed herein, for fermenting the cleaned food product, the said food product is inoculated with a starter culture, wherein the starter culture comprises at least one or a combination of unique strains of bacteria, not naturally existing together in a food product, wherein the starter culture comprises a combination of two or more strains of bacteria, wherein the strains comprise: 1) Leuconostoc sp., 2) Lactobacillus sp. or / and 3) Pediococcus sp. Effectiveness of the sanitization process is validated by the plate counting method for bacteria, wherein a sample of the sanitized food is spread on an agar plate and incubated to allow any viable microbes present to grow and divide many times to form visible colonies. At the end of the incubation period, the microbial colonies from bacterial agar media with and without antibiotics are counted and compared between treatment groups and with control and / or by Minimum Inhibition Concentration (MIC) assessment for the survival of microbes in media with clinically important antibiotics, or / and by antibiotic resistome assessment of food or fecal samples for the prevalence and abundance of antibiotic resistance gene pools, and compared between treatment groups and with control.

[0052] The second step in the method of fermentation comprises of treating the food product to be fermented in a process of microbial succession using a starter culture comprising a combination of unique antibiotic susceptible strains of bacteria, not naturally found together in a food product. These microbial strains provide the unexpected effect of limiting the outgrowth of antibiotic resistant pathogens, spoilage and commensal bacteria via microbial succession. Microbialsuccession is a process wherein the bacteria in the starter culture are succeeded by other bacteria such that the safety and qualities of the fermented food product enhances per day, wherein the safety and qualities can be the lowering of pH and in some embodiments can be the increased alcohol concentration. In other cases, can be the accumulation of other metabolites. In other cases, unwanted metabolites impacting unpleasant odor or host health, such as sulfur compounds or acetaldehyde are mitigated.

[0053] As described earlier, the starter culture comprises at least one a combination of two or more strains of bacteria, wherein the strains comprise: 1) Leuconostoc sp., 2) Lactobacillus sp. or / and 3) Pediococcus sp., or other lactic acid bacteria found in vegetable fermentation such as Wesissella wherein the method of fermenting a food product comprises rotating for aeration of the starter culture, other ingredients, and the food product to be fermented. The food product fermentation process takes at least 24 hours to be completed. In some embodiments, it takes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days or more, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 years or more. Thereafter, the quality of the fermented food product is validated by sensory evaluation for taste and odor and by testing the volatile compounds by rapid or standard assessment using gas chromatography and mass spectrometry etc. Volatile compounds are chemical compounds for example, (-)-alpha-pinene, acetaldehyde or dimethyl trisulfide, that are detected in fermented food products. Furthermore, representative sulfa-compounds contributing to offensive odors, likely produced by the broad spectrum of microbiota associated with raw plant materials during fermentation. The disclosed method of fermentation resulted in fermented food products with major differences in chemical compounds compared to food products of traditional fermentation.Method of treatment

[0054] Further disclosed herein is a method of treating gut microbiota dysbiosis, by administering the improved fermented food product to the subject, wherein the fermented food product can be administered to the subject hourly, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 hours, daily once, twice or three times, weekly, for up to 1, 2, 3, 4 week(s), monthly for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 month(s), 1, 2, or 3 years and wherein the subject is a human. As described above, “microbiota” refers to the range of microorganisms that may be commensal, symbiotic, or pathogenic found in and on all multicellular organisms, including plants and animals. These include bacteria, archaea, protists, fungi, and viruses and have been found to be crucial for immunologic, hormonal, and metabolic homeostasis of the host. Gut microbiotadysbiosis is characterized by a decrease in microbial diversity and increase in proinflammatory species, as well as the unfavorable change of certain metabolites. This imbalanced microbiota is unable to protect from pathogenic organisms, that can trigger inflammation and produce genotoxins or carcinogenic metabolites. The administered amount will be an effective or therapeutic amount to reduce or prevent gut microbiota dysbiosis.EXAMPLES

[0055] To further illustrate the principles of the present disclosure, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compositions, articles, and methods claimed herein are made and evaluated. They are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.); however, some errors and deviations should be accounted for. Unless indicated otherwise, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of process conditions that can be used to optimize product quality and performance. Only reasonable and routine experimentation will be required to optimize such process conditions.EXAMPLE 1

[0056] Disrupted gut microbiota as a critical risk factor for many noncommunicable diseases is largely driven by gut microbiota-impacting drugs, especially orally administrated as well as biliary excreted antibiotics. Fermented food consumption has been encouraged to replenish disrupted gut microbiota, but its overall impact on host gut health remains to be elucidated. This study examined traditionally fermented foods and gut microbiota of consumers of fermented foods for antibiotic resistome. Dietary intervention by fermented foods was found leading to a surge of the antibiotic resistome in gut microbiota of most human subj ects. Antibiotic resistome was further illustrated in traditionally fermented food samples, and viable antibiotic resistant (AR) bacteria were recovered and highly prevalent in retail kimchi and artisan cheeses assessed in this pilot screening. Identified AR isolates included pathogens of importance in nosocomial infections such as Klebsiella pneumoniae, Enterococcus, etc., as well as commensals and lactic acid bacteria, some exhibited extremely high minimum inhibitory concentration (MIC) against antibiotics of clinical significance. Exposing fermented food microbiota to representative antibiotics further led to a boost of the corresponding antibiotic and multidrug-resistance gene pools and disturbed microbiota. These results revealed an underestimated public health risk associated with fermentedfoods intervention, particularly to susceptible population with gastrointestinal tract symptoms and compromised immune functions seeking gut microbiota rescue. The findings call for more comprehensive investigation and investment on the benefits and potential safety challenges associated with traditionally fermented foods, productive intervention of foodborne antibiotic resistance, and strategic movements to mitigate unnecessary damages to the host gut microbiota.Introduction

[0057] The rapid rise of antibiotic resistance (AR) negates effective treatment of bacterial infections, shaking the foundation of modem medicine. AR bacteria in host gastrointestinal tract further indirectly contribute to gut microbiota dysbiosis resulting from even short-term antibiotic treatment (Zhou et al., 2020). Gut microbiota destruction has been recognized as a critical, shared risk factor for a growing list of noncommunicable “modern” diseases, ranging from malfunction of the host immune system, type-II diabetes, brain / neurological disorders, Clostridium difficile infections, to certain cardiovascular diseases and cancers (Sekirov et al., 2010; Chung et al., 2012; Foster et al., 2013; Blaser, 2016; Lynch & Pedersen, 2016; Smits et al., 2016; Meng et al., 2018; Gurung et al., 2020; Witkowski et al., 2020). Both AR and noncommunicable diseases are among the top global public health threats in the 21stcentury (World Health Organization (WHO), 2019).

[0058] For decades, the broad applications of antibiotics have been blamed for the rapid surge of AR, and more recently, for disrupted host gut microbiota and associated “modem” diseases. But despite limiting the uses of antibiotics has been the primary control strategy worldwide, even essential antibiotic applications for infection prevention and treatment still cause irreversible damages in host gut microbiota (Zimmermann & Curtis, 2019; Wang, 2022). AR is a complicated issue with multiple risk factors (Wang, 2009; Wang et al., 2019). Particularly, gut impacting antibiotics, i.e., the mainstream oral administration of antibiotics, and using drugs with primary biliary instead of renal excretion, rather than the application of antibiotics, have been the key and shared driver for the rapid surge of the antibiotic resistance gene (ARG) pool, massively disrupted gut microbiota, and the rise of pathogens in gut microbiota (Zhang et al., 2013; Zhou et al., 2020). This paradigm-changing discovery is further supported by clinical observations on the trends of AR worldwide (Luber et al., 1996; Zhou et al., 2020). The finding is also applicable to nonantibiotic drugs with impact on gut microbiota (Weersma et al., 2020). According to CDC, over 210 million outpatient oral antibiotic prescriptions were given in the US annually (U.S. Centers for Disease Control and Prevention (CDC), 2021). Assuming even distribution, this is equivalent to over 60% of the U.S. population alone affected every year, contributing to the global rising trends of the aforementioned diseases.

[0059] Given its impact on host health, various approaches to replenish damaged gut microbiota have been attempted. Fecal microbiota transplant (FMT) is getting approval for treating C. difficile infections (U.S. Food and Drug Administration (FDA), 2019; Therapeutic Goods Administration, 2021), but the procedure was associated with acquired infections including a death by ESBL E. coli (FDA, 2019). It further led to a surge in resistome in both human and gnotobiotic pig recipients post FMT procedures (Liu and Wang, 2020). Autologous fecal microbiota transplantation (auto-FMT) facilitated the restoration of the gut microbiota and immune functions in patients (Schluter et al., 2020). Yet, reassessing the gut microbiota profiles and resistome il lustrated that auto-FMT using own fecal microbiota banked before disruptive drug therapy, with the intention to reduce introduction of infectious agents from other donor(s), still resulted in the rise of resistome and opportunistic pathogens in multiple recipients even without further antibiotic treatment (Wang et al., unpublished data. Communicated with authors of MKSCC and agreed on the above conclusion in January 2021).

[0060] Fermented foods have emerged in recent years as a popular alternative to repair disrupted gut microbiota (Taylor et al., 2020; Wastyk et al., 2021; O’Connor, 2022). Wastyk et al. (2021) reported increased diversity of the gut microbiota of the human recipients by consuming diets high in fermented foods, but not by diets high in plant fibers. Fermented vegetables, followed by kombucha and fermented dairy products (yogurt and kefir) were among the most consumed categories of fermented foods by this group of subjects (Table 6). However, fermented foods rich in viable microbes are susceptible to AR. Before 2007, fermented dairy foods had been the most significant foodbome avenue transmitting AR to consumers (Wang et al., 2006). A gram of retail cheese contained up to 108copies of AR genes (Manuzon et al., 2007), and the Bifidobacterium strain supplemented to yogurt products worldwide contained a tetracycline (Tet) resistanceencoding gene (personal communication, 2007 ASM General Meeting). Various bacteria isolated from fermented foods with mobile AR genes were able to cause acquired resistance in human commensals and pathogens via horizontal gene transfer mechanisms (Wang et al., 2006; Li et al, 2011a; Jahan et al., 2016). Although successful mitigation of the AR gene pool was quickly achieved in mainstream fermented dairy products by 2011, primarily by removal of problematic fermentation starter cultures and probiotic strains from the product lines (Li et al, 2011b; Wang et al., 2019), other traditionally fermented products may still be prone to AR (Belletti et al., 2009; Comunian et al. 2010; Karasu et al., 2010; Nawaz et al., 2011; Pan et al., 2011; Zhou et al., 2012; Rebecchi et al., 2015; Zielinska et al., 2015; Nunes et al., 2016; Park et al., 2016; Guo et al. 2017; Erginkaya et al., 2018; Touret et al., 2018; Wang et al., 2018; Li et al., 2019; Leech et al., 2020;Yasir et al., 2022). Unlike mainstream modern dairy fermentation, which uses pasteurized milk and commercial starter cultures carefully screened by major culture companies, traditionally fermented vegetables and artisan cheeses, for instance, still rely on microbiota associated with raw materials, environment, or “mother” cultures other than carefully screened commercial starters (Belletti et al., 2009; Comunian et al. 2010; Pan et al., 2011; Wang & Schaffner, 2011; Zielinska et al., 2015; Nunes et al., 2016; Park et al., 2016; Touret et al., 2018; Leech et al., 2020; Yasir et al., 2022).

[0061] The fermented food and beverage market is expected to grow by 5.6% in the next 10 years and will exceed USS 989.2 billion by 2032 amid escalating demand for healthy and nutritious foods (Future Market Insights, 2022). The COVID-19 pandemic has further facilitated the explosive growth of sales of fermented products such as sauerkraut and kimchi (Manskar & Raskin, 2020). Given the booming market and increased consumption, re-assessing potential AR risks associated with traditionally fermented food products has become an urgent food safety and public health need, especially for susceptible populations with compromised digestive tract and immune systems. Therefore, this study investigated the incidences of antibiotic resistome in representative fermented foods and the impact of fermented food intervention on human gut resistome.Methods and Materials

[0062] Host gut resistome assessment. A total of 39 healthy adult participants were recruited, with 18 randomly assigned to the dietary intervention group by fermented foods and the remaining 21 assigned to the plant fiber intervention group. Three participants in the fiber intervention group dropped out of the study, thus both groups had 18 participants who finished the whole study (25 female, 11 male, with an average age of 52 ± 11 years). Fecal shotgun metagenomics data of these participants were collected at four checkpoints, i.e., Week -2, Week 0, Week 8, and Week 10, throughout the study and deposited at the NCBI BioProject database (ID number: PRJNA743361). After initial screening for data availability, 3 participants in the fiber group and 1 participant in the fermented food group were dropped from the study due to missing Week 10 endpoint shotgun sequencing data in the database provided by the original research team. The raw sequencing data were retrieved from the database in Sequence Read Archive (.sra) format, converted to .fastq using the NCBI SRA Toolkit (version 3.0.0), and further processed on a high-performance supercomputer at the Ohio Supercomputer Center.

[0063] For quality control, FASTP tool (version 0.22.0) was used (parameters: -q 20 -u 20 -n 2 -180) to clean and trim the raw sequences before further annotation and analysis, as described by Chen et al. (2018). The cleaned sequences were then processed using the ARGs-OAP pipeline (Online Analysis Pipeline for Antibiotic Resistance Genes, version 2.5) (Yin et al., 2018). The resistome analysis criteria were set as hit length of 50%, e-value of le-07 and identity of 80%. The generated data were further analyzed and plotted using Tidyverse packages including ggplot2 (version 3.3.6) and dplyr (version 1.0.10), ggstatsplot package (version 0.10.0) and car package(Companion to Applied Regression, version 3.1-1) on R version 4.2.1 (Wickham et al., 2016; Fox& Weisberg, 2018; Wickham et al., 2019; Patil, 2021).

[0064] Data transformation and analysis. The sums of the total ARG were calculated by adding up the relative abundances of all types of ARG annotated by ARGs-OAP. The percentage of total resistome change for each subject was calculated by the equation .resistome reads after dietary intervention- ^resistome reads before dietary intervention) / ^resistome reads before dietary intervention x 100%. The resistome data from Week -2 and Week 0 were averaged as the baseline to make sure all subjects have baseline data before dietary intervention and to reduce natural variation in gut microbiota. Normality and variance of data sets were verified using Shapiro-Wilk normality test and Levene's test. Violin-boxplots with Student’s t-test p-values (a<0.05) were generated using ggstatsplot package (Patil, 2021). Other plots were constructed using the ggplot2 package (Wickham et al., 2016). Summary statistics such as mean, median and standard deviation and significance of dietary intervention were calculated using the dplyr package (Wickham et al., 2022).

[0065] Source of fermented food samples. Representative kimchi samples of different brands were purchased from 7 retail stores including independent operations and national chains, 1 chain restaurant, and 3 local Japanese and Korean restaurants in Columbus, OH. The artisan cheese products were purchased from a national grocery chain store in Columbus, OH. Sample designation and brief description were illustrated in Table 7.

[8066] Recovery and assessment of viable bacteria from fermented food samples. Five grams of each food sample were stomached in 45 ml 0.1% peptone water using a Seward stomacher 80 (Seward, UK). The juice was assessed for total bacteria and AR bacteria using Brain Heart Infusion (BHI), Luria-Bertani (LB) or De Man, Rogosa and Sharpe (MRS) agar plates with cycloheximide or nystatin as mold inhibitor, with or without the corresponding antibiotic (16 or 32 pg / mL ampicillin; 32 or 64 pg / mL tetracycline; 2 pg / mL erythromycin for BHI or MRS). Acontrol sample with only peptone water was processed under the exact same condition and plated to make sure no contamination was introduced during the sample processing. The plates were incubated at 30°C, aerobically or anaerobically. Single colonies recovered from antibioticcontaining agar plates were picked based on representation in morphology and identified by Sanger sequencing of 16S rRNA gene PCR amplicons (Wang et al., 2006). The MIC of recovered AR isolates against 4 commonly used antibiotics were determined by a microdilution procedure (Stock et al., 2003), but using the corresponding recovery media instead. MIC of some representative AR isolates against a broader spectrum of antibiotics was further determined using commercial antimicrobial susceptibility kits Sensititre® (Thermo Scientific, USA, MA) GPN3F for Gram positive isolates and GN3F for Gram negative isolates, following the manufacturer's instructions, with MRS or BHI broth replacing the Sensititre Mueller Hinton broth for better growth of each microbe. CLSI control strains Escherichia coli ATCC 25922 and Enterococcus faecalis 29212 (ATCC, Manassas, VA) were used as the MIC control standards.

[0067] Antibiotic resistome of representative fermented food items. Twenty-five grams of food sample were mixed with 25 ml 0.1% peptone water and stomached as above. The large particles of food debris were removed with a sterile sieve. The liquid phase was centrifuged at 2500 relative centrifugal force by Multifuge X1R (Thermo Scientific, USA, MA) for 15 min. The pellet was washed once with 0.1% peptone water and subjected to total DNA extraction with a QIAamp PowerFecal Pro DNA Kit (Qiagen, Germany) following manufacturer’ s instruction. Alternatively, the culture-recovered microbiota was scraped from agar plates with 3mL 0.1% peptone water and subjected to DNA extraction using the aforementioned QIAamp kit.

[0068] The DNA samples were subjected to shotgun metagenomic sequencing (2X155bp) with average depth of 16.7 to 20 million paired-end reads for each sample, using an Illumina NextSeq2000 Sequencing System. The raw sequences were cleaned (parameters: -q 20 -u 40 -n 2 - 1 80) and analyzed with the same metagenomic tools and parameters as described above to obtain relative abundance of AR genes normalized to the number of 16S rRNA gene. The sequencing data were further assessed for microbiota profiles through the Kraken2 pipeline with standard database and minimum-base-quality 20, as described previously (Wood et al., 2019). Food microbiota profile of Kimchi #7 was also assessed by 16S rRNA sequencing by an Illumina MiSeq system.

[0069] All local metagenomic data analyses were conducted on a high-performance supercomputer at the Ohio Supercomputer Center.Results

[0070] Impact of food intervention on fecal microbiota resistome. Ten-week dietary interventions by diets high in fermented foods or plant fibers led to 11 increased and 6 decreased, as well as 10 increased and 5 decreased gut antibiotic resistome in human subjects, respectively, but the impact by the two types of diets was distinctive (Fig 1 & Fig 2). Fermented foods (traditional including artisan fermented products, without antibiotic resistant mitigation) intervention led to significant changes in fecal antibiotic resistome of subjects with a p-value of 0.03, and the mean of resistome increased from 0.36 to 0.42 copies / 16S rRNA gene (Fig 2A). Meanwhile, intervention by high plant fiber diets caused insignificant change in host gut resistome, with a p-value of 0.94. The mean of resistome remained as 0.38 copies / 16S rRNA gene in subjects before and after dietary intervention (Fig 2B). The summarized resistome data used are illustrated in Table 8. For details of the resistome data see Table 15 & 16.

[0071] Antibiotic resistome of traditionally fermented foods. This pilot screening of retail fermented foods included various brands distributed nationwide through national, regional grocery chains and independent retailers. Multidrug resistance, bacitracin and macrolide-lincosamide- streptogramin (MLS) genes were most abundant in pooled retail kimchi samples (K1-K4) recovered from BHI plates (Fig 3 A and Table 1), and they were also among the most abundant AR genes of all the 5 individual kimchi microbiota (K7-K11) assessed directly, despite the exact abundance of the top AR genes varied among these samples (Table 1 & Fig 4A). Likewise, even though AR isolates were identified in 9 out of 10 kimchi samples illustrated (K5, K7-K14, Table 2), the overall abundance of resistome of assessed samples also varied, ranging from 0.372 (K9) to 0.029 (KI 1) copies of AR genes / 16S rRNA (Fig 4A).

[0072] Figure 4B further compares the resistome outcomes of kimchi sample K7 using total DNA extracted from kimchi microbiota directly (0.048 copies of AR genes / 16S) and total DNA of kimchi microbiota recovered from BHI agar plate (2.909 copies of AR genes / 16S). The results indicated that with specific medium and incubation condition, even without antibiotic selective pressure, certain bacteria of the kimchi microbiota might have been selectively enriched, leading to the drastic difference in detected antibiotic resistome. Likewise, the top genera of pooled kimchi microbiota recovered from BHI plates (Table 9) were affected by cultivation conditions too, with and without antibiotics, as the illustrated resistome (Fig 4B).

[0073] Indeed, Table 3 illustrates that the top genera of each individual kimchi microbiota (K7 to Kl l) assessed using total DNA extracted directly were dominated by lactic acid bacteria well-recognized for driving kimchi fermentation. The data further demonstrated that antibiotic resistome and AR bacteria were still prevalent in these successfully fermented kimchi samples.

[0074] The resistome results of pooled artisan cheese samples Cl to C4 (Fig 3B) and individual cheese samples of C5 to C8 (Fig 4C), all using microbiota recovered from BHI agar plates, further illustrated the prevalence of various AR genes in these products. Multidrug, MLS and bacitracin were among the top AR genes of the pooled samples C1-C4, while multi drug, bacitracin, tetracycline and MLS were most abundant among individual samples C5 to C8 (Table 4). It is worth noting that the presented abundance of resistome of cheese microbiota recovered from BHI plates may also be affected by the medium and culture conditions, deviated from those of the original samples. Table 11 further illustrated that Staphylococcus (including Mammaliicoccus, also known as Staphylococcus) was the top dominant genus of individual artisan cheese microbiota recovered from BHI agar plates.

[0075] Representative AR bacteria from traditionally fermented foods. Various AR bacteria were recovered from traditionally fermented products. Under very limited cultivation conditions applied in this study, AR bacteria were identified from 9 out of 10 kimchi and 4 out of 4 cheese samples assessed by culture recovery (Table 2 & 5).

[0076] Identified AR isolates from kimchi products ranged from important human pathogens to organisms still considered as commensals, as well as lactic acid bacteria driving food fermentation. For instance, AR opportunistic pathogens such as Klebsiella pneumoniae and Serratia marcescens are recognized agents for nosocomial infections (Farmer, 2003; Mahlen, 2011). Rahnella aquatilis, a human pathogen can cause bacteremia, sepsis, urinary tract infection, etc. As illustrated in Table 2 and Table 12, kimchi isolates Klebsiella pneumoniae-6 and Rahnella aquatilisKl l-3, which are resistant to multiple antibiotics. Particularly, Serratia marcescens \<A - \ is highly resistantto almost all key antibiotics (21 out of 22), including the 4thgeneration of cephalosporin antibiotic as well as carbapenem antibiotics, with MICs exceeded the highest detection limit of 12 antibiotics by the Sensititre assessment (Table 12). In addition, isolates of several species of Latilactobacillus, Leuconostoc and Weissella. commonly involved in driving kimchi fermentation, were among those highly resistant to tetracycline, erythromycin, ampicillin and vancomycin, and some isolates exhibited multi drug resistance (Table 2). Weissella K5-3 further exceeded the highest MIC levels of all 18 antibiotics examined using the Sensititre panel (Table 13).

[0077] Identified AR isolates from cheese products were mostly Staphylococcus spp. resistant to tetracycline or erythromycin, with one isolate also resistant to vancomycin. An isolate ofEnterococcus sp. was highly resistant to vancomycin and could be classified as vancomycin resistant Enterococcus (Rengaraj, 2016). It is also multidrug-resistant (Table 13).

[0078] Antibiotic exposure further shaped the profiles of fermented food originated microbiota.As illustrated in Fig. 5 and Table 11, cheese microbiota harvested from BHI plates without antibiotics were dominated by Mammaliicoccus spp. (formerly Staphylococcus'), Staphyloccocus, Camobacterium, Psychrobacter and Glutamicibacter (belonging to Micrococcaceae) for cheese sample C5. But at the presence of Tet or Amp, the dominant bacteria switched to Stenotrophomonas, Pseudomonas, Burkholderia, Alcanivorax and Streptococcus. Likewise, while the dominant cultures for cheese C7 recovered on BHI were Staphylococcus and Lactococcus, the top ranked bacteria of cheese originated microbiota shifted to Pseudomonas, Burkholderia, Staphylococcus, Streptococcus and Leuconostoc on BHI- Amp, and Staphylococcus on BHLTet. In the case of C7 and pooled sample C1-C4, the cheese starter culture Lactococcus was essentially eliminated when the cheese microbiota was exposed to either of the two antibiotics.

[0079] Although antibiotic-containing agar plates were used to screen for AR bacteria, the above results also indicated how the food-originated microbiota might impact gut microbiota when the hosts received antibiotic treatment. Stenotrophomonas, Pseudomonas, Burkholderia, and Staphylococcus are recognized opportunistic pathogens associated with antibiotic resistant hospital acquired infections, and most Streptococcus species are pathogens.

[0080] Microbial profiling by metagenomics of dominant cheese bacteria recovered on BHI plates included Staphylococcus, Enterococcus and Lactocaseibacillus (formerly Lactobacillus) for cheese sample C6, as well as Enterococcus, Tetragenococcus, Staphylococcus, Lactococcus and Camobacterium for sample C8, respectively (Fig. 5 and Table 11). In agreement, some of the mentioned antibiotic resistant bacteria such as multidrug resistant Enterococcus sp. and Staphylococcus were also confirmed in these samples (Table 5).Discussion and Conclusion

[0081] Although unexpected for many, the susceptibility of fermented foods to AR has been well- recognized at least in the food microbiology community. Since the first systematic demonstration of the problem in the early 2000s with a broad spectrum of AR isolates, including starter cultures, opportunistic pathogens and commensals of mainstream fermented dairy products being identified and characterized (Wang et al., 2006), AR bacteria have further been isolated from various fermented foods worldwide (Munoz et aS., 2014; Fraqueza, 2015; Kim et al., 2021). For instance, Pantoea agglomerans (formerly Enterobacter agglomerans, or Erwinia herbicola), an opportunistic pathogen causative to a wide range of opportunistic infections, especially inimmunocompromised patients, was isolated from kimchi in South Korea. The genome of P. agglomerans isolate KM1 contained 13 genes conferring resistance to clinically important antibiotics, and the strain exhibited immunostimulatory' properties in vitro, including the production of pro-inflammatory and anti-inflammatory' cytokines in stimulated cells (Guevarra et al., 2021).

[0082] This study, however, using a combination of approaches, illustrated the high prevalence and the abundance of antibiotic resistome in popular traditionally fermented foods. Although the retail products were purchased in Columbus Ohio, the products were mostly made in the US and distributed nationwide through independent stores and grocery chains. Therefore, they serve as a good indication for the prevalence of AR in similar products nationwide. Further studies have been conducted traditional kimchi fermentation carefully in microbial controlled setting and have concluded that AR is inevitable in the final products due to the AR bacteria associated with the raw vegetable materials. This is consistent with the principle of fermentation that the natural microbiota from raw materials drive microbial succession in natural fermentation.

[0083] The original human study on dietary intervention by Wastyk et al. (2021) had a total of 36 healthy adult subjects completed the study, half received fermented foods and the other half dietary fiber intervention. Among those, the fecal microbiome outcomes of 32 subjects with required data points, including 17 subj ects with fermented foods intervention and 15 subj ects with diet high in plant fiber for comparison, were assessed and reported for the impact of the dietary intervention on host gut resistome in this study.

[0084] The illustration of antibiotic resistome in kimchi and artisan cheese microbiome, along with the high prevalence of confirmed AR bacteria in kimchi and artisan cheeses assessed in this study, support the conclusion that consuming these traditionally fermented products can result in the rise of gut antibiotic resistome in consumers. The recovered isolates in fact only represent a small percentage of the AR microbiota of the fermented foods. The pivotal impact of foodborne AR on host gut antibiotic resistome has previously been illustrated using the mice model by Zhang et al. (2013), as oral antibiotic treatment only led to the surge of the targeted AR gene pool in host gut microbiota when the mice had prior oral seeding of AR bacteria. Results from this pilot study thus call for more extensive investigations on the food safety risks associated with traditionally fermented foods, and for effective mitigation strategies to protect public health.

[0085] It is further worth noting that even in subjects who consumed only yogurt and kombucha (Subject 8020) or yogurt, kombucha and kefir (Subject 8014) (Table 3), the gut resistome of the subjects still increased 11% and 17%, respectively, after the 10-week intervention. The datasuggests that consuming these products at least did not reduce the gut antibiotic resistome in the subjects.

[0086] The identification of AR bacteria from traditionally fermented products, ranging from human and plant pathogens to lactic acid bacteria driving food fermentation, especially those exhibited multidrug resistance and with extremely high MICs against antibiotics of clinical significance, is particularly concerning. Multidrug resistant Klebsiella pneumoniae, Serratia marcescens, as well as Stenotrophomonas, Pseudomonas, Burkholderia, Staphylococcus and Streptococcus are also known for causing hospital-acquired infections, including some of the most troublesome cases in healthcare facilities. Data from this study thus also provides an alternative interpretation on the route of dissemination and potential origins of infections in patients. For instance, Serratia marcescens K13-1 is extremely resistant to most antibiotics assessed by the Sensititre panel for Gram-negative bacteria, including the carbapenem antibiotics Meropenem and Ertapenem that are usually reserved for treating multidrug resistant bacterial infections (Table 12). Weissella s . K5-3 is further highly resistant to all 18 antibiotics assessed by the Sensititre panel for Gram positive bacteria including the 3rdgeneration of cephalosporin antibiotic Ceftriaxone (Table 13). As lactic acid bacteria, Weissella spp. are known for driving vegetable fermentation and thus likely survive well even in successfully fermented products. Revealing the genetic elements responsible for the unusual AR profiles and their potential involvement in horizontal gene transfer will provide further insights on the food safety and public health risks associated with these AR bacteria.

[0087] Balanced gut microbiota as well as effective mucosal, intestinal epithelial and gut vascular barriers as part of an integral and functional gastrointestinal tract system are essential to host health (Gao et al., 2022). Physical damage to the intestinal barriers further has various health consequences. Intestinal tract problems such as inflammatory bowel diseases, enteric infections or overgrowth by pathogens such as EHEC and Shiga toxin-producing E. coli, Shigella, Salmonella, Campylobacter, C. difficile, enteric viruses, certain drug treatments such as the antibiotic clindamycin etc., even alcohol consumption can all cause acute or chronic intestinal barrier damages. Once the intestinal barriers collapse, intestinal microorganisms and their metabolites can get into the bloodstream, reach other organs and cause further health damage. Once entering the bloodstream, even commensals and probiotics can be problematic. For instance, the most popular probiotic strains Bifidobacterium and L. rhamnosus GG have still been found causative to bacteremia and sepsis in high-risk populations, with strain confirmation by metagenomic sequencing (Land et al., 2005; Boyle et al., 2006; Ohishi et al., 2010; Kochan et al., 2011; Bertelliet al., 2015; Angurana et al., 2018; Chiang et al., 2021; Aydogan et al., 2022; Kulkami et al., 2022; Colautti et al., 2022). While bacteremia and sepsis by commercial probiotic strains absence of AR genes are still controllable by mainstream antibiotics such as ampicillin, introducing the broad spectrum of AR bacteria highly resi slant to clinically important antibiotics associated with traditionally fermented foods presents an underestimated and potentially serious public health risk, especially to targeted populations suffering from various gut symptoms associated with “leaky gut” as well as compromised immune functions. Bacteremia and sepsis risk, antibiotic therapy failures, and further gut microbiota destruction post-antibiotic treatment may significantly increase in the targeted susceptible consumer popul tions who intend to repair damaged gut microbiome by enhancing fermented food consumption.

[0088] With the recipients of over 210 million oral antibiotic prescriptions added to the susceptible population suffering from broad gut microbiota destruction annually in the US alone (CDC, 2021), the impacted patient population worldwide is astonishing. While various gut microbiota-replenishing approaches including fermented food intervention have been practiced desperately, they all have introduced additional public health risks to the patients. Results from this study thus further call for strategic efforts to tackle the key and shared driver of the antibiotic resistome surge and gut microbiota disruption in human and animals, by mitigating the applications of gut-impacting antibiotics (oral administration and biliary excretion) and offering alternatives, to significantly minimize unnecessary damages to gut microbiome for productive outcomes.EXAMPLE 2

[0089] In a second example, vegetable fermentations, which primarily rely on spontaneous fermentation by microbiota associated with the raw food materials, are vulnerable to AR. Plant materials are generally exposed to various environmental factors including AR microbes in soil, water, wild animals, manure and even in the air. During the process from field to table, various cross-contamination by environmental and human microbiota may further contribute to the prevalence of AR bacteria in these products. While the prevalence of AR in raw plant materials is still inevitable, effective mitigation of AR in the final products through proper processing intervention can be a productive option to reduce the transmission of AR through food consumption.

[0090] Multiple sets of AR mitigated starter cultures were prepared, consisting of lactic acid bacteria formulated based on desirable microbial succession for successful vegetable fermentation.After comparing a number of microbial inactivation approaches for the raw ingredients, such as high pressure, blanching and sanitizer treatment, it was found that treatment using an organic sanitizer by Microcide® retained the texture of napa cabbage and had sufficient microbial inactivation outcome (3 logs reduction). In the test round of the prototype clean kimchi fermentation products made with Microcide®-treated raw materials and AR mitigated starter cultures had pleasant and clean kimchi aroma, pH 3.7-3.8 in 48 hours, and no AR bacteria observed by plate counting method. On the other hand, control kimchi made by traditional fermentation using the same batch of raw materials but without Microcide® treatment and the AR mitigated stater cultures, had 103- 106CFU / g product of Tetrand Amprbacteria by plate counting, and pH of 4.8 in 48 hrs. The data illustrated that clean kimchi fermentation is achievable.

[0091] Rapid volatile compounds assessment was also conducted, and Table 14 clearly illustrated that there were major differences in chemical compounds detected in kimchi products of traditional fermentation and the fermentation process disclosed herein, even though they used the same batch of raw materials. For instance, representative sulfa-compounds contributing to offensive odors, and acetaldehyde, likely produced by the broad spectrum of microbiota associated with raw plant materials during fermentation, were significantly reduced in products by the method of fermentation disclosed herein. Table 14 illustrated that originalade without either or both elements of the method of fermentation disclosed herein (#1, sanitization of the raw materials, and #2, inoculation of the dedicated starter cultures), for instance KOS, being kimchi made with non-sanitized napa cabbage but inoculated with starter OCS I, as well as Original kimchi made by natural fermentation (no sanitation of raw materials, nor starter inoculation) had tremendously higher concentration of offensive compounds (bolded, Table 14), while the same compounds were much lower in kimchi made with sanitized raw materials and inoculated with the OSU formulated starter cultures dedicated for successful microbial succession.

[0092] The data matched well with the sensory feature that kimchi made by method of fermentation disclosed herein process had clean and pleasant aroma, comparing to those by traditional fermentation.

[0093] Offensive odors limited the broad offering of sauerkraut products in restaurant settings. For instance, it was removed from the menu of Bob Evans® for this particular issue. Thus, the success of the method of fermentation disclosed herein in producing products of clean aroma with significantly reduced offensive volatile compounds and minimized risk of soilage and food safety problems is useful, and benefits the fermented fruits and vegetables industry, previously relying on natural fermentation, in general.

[0094] Lastly, it should be understood that while the present disclosure has been provided in detail with respect to certain illustrative and specific aspects thereof, it should not be considered limited to such, as numerous modifications are possible without departing from the broad spirit and scope of the present disclosure as defined in the appended claims.

[0095] Data: Using modernized fermentation process, kimchi products made with Microcide- treated raw materials and AR mitigated starter cultures had pleasant kimchi aroma, pH 3.7-3.8 in 48 hours, and no AR bacteria observed by plate counting method. On the other hand, control kimchi made by traditional fermentation using the same batch of raw materials but without Microcide treatment and the AR mitigated starter cultures, had 10A3 - 10A6 CFU / g product of Tet- resistant and Amp-resistant bacteria by plate counting, and pH of 4.8 in 48 hrs. The data illustrated that clean kimchi fermentation is achievable. Because this modernized process actually inactivates not only AR bacteria but also many other pathogens and spoilage microbes and relies on starter cultures with known features for fermentation, it also addresses other quality and safety challenges associated with products of natural / artisan fermentation, therefore with broad industrial applications.

[0096] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.SEQUENCES

[0097] SEQ ID NO 1: Leuconostoc K6-Leul3 (WL-1)CCACTCTGTCTGCTTAGACGGCTCTTCCTAAAGGTTAGGCCACCGGCTTTGGGCATTACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGTCGAGTTGCAGACTACAATCCGAACTGAGACGTACTTTAAGAGATTAGCTCACCCTCGCGGGTTGGCAACTCGTTGTATACGCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCGCCTTCCTCCGGTTTGTCACCGGCAGTCTCGCTAGAGTGCCCATCTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCACTTTGTCTCCGAAGAGAACACTTCTATCTCTAAAAGCTTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAACACTTAATGCGTTAGCTTCGGCACTAAGAGGCGGAAACCTCCTAACACCTAGTGTTCATCGTTTACGGTGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACACTTTCGAGCCTCAACGTCAGTTGCAGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTTACCTCTACTGCACTCAAGTTAACCAGTTTCCAATGCCATTCCGGAGTTGAGCTCCGGGCTTTCACATCAGACTTAATAAACCGTCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTCGGGACATACGTATTACCGCGGCTGCTGGCACGTATTTAGCCGTCCCTTTCTGGTATGGTACCGTCAAACTAAAATCATTTCCTATTCTAGCTGTTCTTCCCATACAACAGTGCTTTACGACCCGAAAGCCTTCATCACACACGCGGCGTTGCTCCATCAGGCTTTCGCCCATTGTGAAGATCCCTACTGCAGCCTCCCGTAGAGTTTGGGCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCACTCGGCTATGCATCATTGTCTTGGTAGGCTTTACCCACACTACTATGCACCGCGGATCATCTCTAGTGACGCGAGCGCTTTTACTTTGTGTCATGCGACACTAGTTTATTCGGATAGCATCTGTTTCCAATGTTATCCCCAGCCTTGGAGGC A

[0098] SEQ ID NO 2: Pediococcus K6-Pedio (WL-2)ATTGTCACTTAGACGGCTAGCTCCCGAGGGTTACTCCACCGGCTTTGGGTGTTACAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTAGCTTAACCTCGCGGTCTCGCAACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAATAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTAGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCTTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCAATGCACTTCTTCGGTTAAGCCGAAGGCTTTCACATTAGACTTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACTGCATGAGCAGTTACTCTCACACACGTTCTTCTCCAACAACAGAGTTTTACGACCCTAAAGCTTCATCACTCACGCGGCGTTGCTCATCAGACTTTCGTCATGTGAGATCCCTACTGCTGCTCCCGTAGAGTCTGGGCGTGTCTCAGTCCCATGTGCGATACCTCTCAGTCGCTACGTATCACAGCTGGTGGGCTTATCTCACACTAGCTATACGCGCGGTCATCGGATGATAGCGAGGCATCTTTAAGAAAACATGCGTACTTATATGCGGATAGCATCGTCCAGGTATCCCACTCTGGGCAGTACCAGTATACCGTGCCACTCTAGTAATCTCAGCGAACTACCTACGAGGTTGCAATCAGTAGACTAG

[0099] SEQ ID NO 3: Lactobacillus SHI 6 (WL-3)CTTGGTCCACCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATCTATTCCGGATATATAGCCATCTTGTTTTCCAAAATGTTTAATCCCCCCACCTTAAAGGGCAAGGTTTACCCCAGCGTGTGTTTATCACACACCCGGCTCGCCCACACTCCGCTCCCCACATGTTGTTTATAAATTCTTCGGTGTCGCAGAGAGCACAACACCGACATATCTACATACACAACACACAGAGAAAAAACACATTCTCTTTCTTCTGCATAAAATTTABLES

[0100] Table 1. Most abundant AR genes in kimchi samples*.*Numerical readings of AR: in total ARG copies per 16S gene.**MLS: Macrolide-Lincosamide-Streptogramin.

[0101] Table 2. Summary of identified AR colonies from kimchi products purchased in 2022 and 2023*.*Tested MIC for representative antibiotics including Van: vancomycin; Amp: ampicillin; Ery: Erythromycin; Tet: Tetracycline.**Likely due to natural resistance of most gram-negative bacteria against vancomycin and erythromycin (Soares, 2012; Antonoplis, 2019).***MIC of this isolate against a broader spectrum of antibiotics determined with Sensititre® kit available in Supplement Table 7 and 8.

[0102] Table 3. Top 5 genera classified of microbiota directly of 5 kimchi samples purchased in 2022.

[0103] Table 4. Most abundant AR genes in BHI plates recovered microbiota of artisan cheese samples.*Numerical readings in total ARG copies per 16S gene.**MLS: Macrolide-Lincosamide-Streptogramin.

[0104] Table 5. Summary of identified AR colonies from artisan cheese products purchased in 2022*.*Tested MIC for representative antibiotics including Van: vancomycin; Amp: ampicillin; Ery: Erythromycin; Tet: Tetracycline.**MIC of this isolate against a broader spectrum of antibiotics determined with Sensititre® kit available in Supplement Table 7 or 8.

[0105] Table 6. Categories of foods consumed by subjects for dietary intervention.

[0106] Table 7. Fermented food samples assessed in this study.

[0107] Table 8. Gut antibiotic resistome of subjects with dietary intervention.'Numerical readings in total ARG copies per 16S gene.

[0108] Table 9. Top 5 genera classified of microbiota recovered from BHI agar plates of pooled 4 kimchi samples purchased in 2021.

[0109] Table 10: Top 5 genera classified of microbiota recovered from BHI agar plates of pooled 4 cheese samples purchased in 2021.

[0110] Table 11 : Top 5 genera classified of BHI-recovered microbiota of individual cheese samples purchased in 2022.

[0111] Table 12. Sensititre MIC for representative Gram-negative isolates from kimchi*.*Bold MIC numbers: strongly indicative of the isolates being resistant to the corresponding antibiotics based on references (CLSI, 2020; Marchaim, 2014; Papich & Lindeman, 2018). Italicized antibiotics: penicillin and derivatives. Italicized and highlighted antibiotics: 4thgeneration of cephalosporin antibiotic Cefepime) or Carbapenem antibiotics (Meropenem, Ertapenem).

[0112] Table 13. Sensititre MIC for representative Gram-positive isolates from kimchi and artisan cheeses*.*Bold MIC numbers: strongly indicative of the isolates being resistant to the corresponding antibiotics based on references (CLSI, 2020). Italicized antibiotics: penicillin and derivatives.

[0113] Table 14. Kimchi volatiles by SIFT-MS. *The numbers are concentrations in ppb. KOS: Non-sanitized napa cabbage with starter OCS I; KS: MFP with starter OCS I; KSNG: MFP with starter OCSI no ginger; Original: natural fermentation (Li, Kaur, Barringer, Wang, 2023, unpublished data).

[0114] Table 15. Fecal antibiotic resistome of subjects by (traditional / standard / artisan included) fermented foods intervention.

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Claims

CLAIMSWhat is claimed is1. A fermented food product comprising a combination of 1) Leuconostoc sp., and 2) Lactobacillus sp. or / and Pediococcus sp. bacteria, wherein said bacteria is susceptible to antibiotics of clinical significance.

2. The fermented food product of claim 1, wherein the: Leuconostoc sp. is selected from Leuconostoc K6-Leul3 (WL-1), Leuconostoc mesenteroides ^^ Leuconostoc citreum.

3. The fermented food product of claim 1, wherein the Lactobacillus sp. is selected from Lactobacillus SHI 6 (WL-3), Lacticaseibacillus paracasei. Lactobacillus brevis, and Latilactobacillus sakei.

4. The fermented food product of claim 1, wherein the Pediococcus sp. is selected from Pediococcus K6-Pedio (WL-2), Pediococcus inopinatus and Pediococcus acidilactici.

5. The fermented food product of any one of claims 1-4, wherein the food item is a fruit, vegetable, or meat, seafood, their juices, with or without dairy ingredients,6. The fermented food product of claim 5, wherein the vegetable is a napa cabbage.

7. The fermented food product of any one of claims 5 or 6, wherein the food product is kimchi.

8. The fermented food product of any one of claims 1-7 wherein the Leuconostoc bacteria comprises 95% or more identity to SEQ ID NO 1.

9. The fermented food product of any one of claims 1-7, wherein the Lactobacillus bacteria comprises 95% or more identity to SEQ ID NO 2.

10. The fermented food product of any one of claims 1-7, wherein the Pediococcus bacteria comprises 95% or more identity to SEQ ID NO 3.

11. The fermented food product of any one of claims 1-10, wherein the fermented food product comprises other ingredients including, but not limited to, seasoning.

12. The fermented food product of claim 11, wherein the seasoning includes, but is not limited to, salt, sugar, a chili-based product or a combination thereof.

13. A method of fermenting a food product, wherein the method comprises:a. sanitizing the food item to be fermented, comprising microbial inactivation of antibiotic-resistant (AR) bacteria and food-borne pathogens, using methods other than standard pasteurization, and b. fermenting the cleaned food item of step a) comprising inoculation with a starter culture, wherein the starter culture comprises at least one a combination of 1) Leuconostoc sp., and 2) Lactobacillus sp. or / and 3) Pediococcus sp, bacteria, wherein said bacteria is susceptible to antibiotics of clinical significance.

14. The fermented food product of claim 13, wherein the: Leuconostoc sp. is selected from Leuconostoc K6-Leul3 (WL-1), Leuconostoc mesenteroides ^^ Leuconostoc citreum.

15. The fermented food product of claim 13, wherein the Lactobacillus sp. is selected from Lactobacillus SHI 6 (WL-3), Lacticaseibacillus paracasei. Lactobacillus brevis, and Latilactobacillus sakei.

16. The fermented food product of claim 13, wherein the Pediococcus sp. is selected from Pediococcus K6-Pedio (WL-2), Pediococcus inopinatus and Pediococcus acidilactici.

17. The fermented food product of any one of claims 13-16, wherein the Leuconostoc bacteria comprises 95% or more identity to SEQ ID NO 1.

18. The fermented food product of any one of claims 13-17, wherein the Lactobacillus bacteria comprises 95% or more identity to SEQ ID NO 2.

19. The fermented food product of any one of claims 13-18, wherein the Pediococcus bacteria comprises 95% or more identity to SEQ ID NO 3.

20. The method of claim 13, wherein sanitizing the food product to be fermented comprises using a Microcide® sanitizer, high pressure, washing or blanching in electrolyzed water or vinegar, and / or sanitation gases.

21. The method of claim 20, wherein the sanitation gas is chlorine dioxide or ozone.

22. The method of any one of claims 13-21, wherein sanitization is validated by the plate counting method for bacteria.

23. The method of claims 13-22, wherein the food product to be fermented is treated in a process of microbial succession using a starter culture comprising a combination of uniquestrains of bacteria susceptible to major antibiotics of clinical significance, not naturally found together in a food product.

24. The method of fermenting a food product of any one of claims 13-23, comprising rotating of the starter culture, other ingredients, and the food product to be fermented.

25. The method of fermenting a food product of any of claims 13-24, wherein said fermentation takes at least 24 hours.

26. The method of any one of claims 13-25, wherein the fermented food product is validated by sensory evaluation of taste and odor.

27. The method of any one of claims 13-26, wherein the quality of the fermented food product is tested by rapid volatile compounds assessment using gas chromatography and mass spectrometry.

28. The method of any one of claims 13-27, wherein the food item does not require pasteurization or ultra-pasteurization.

29. A method of treating gut microbiota dysbiosis, the method comprising administering a therapeutically effective amount of the fermented food product of any of claims 1-12 to the subject.

30. The method of claim 29, wherein the product is given to the subject daily, or multiple times a day.

31. The method of claim 29, wherein the product is given weekly, monthly, or only once.

32. The method of nay one of claims 29-31, wherein the product is given orally.

33. The method of claim 30, wherein the fermented food product or derivative(s) is administered to the subject daily for at least 1, 2, 3, 4, 5, 6,7, 8 weeks.

34. The method of any one of claims 29-33, wherein the subject is a human.

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