Organic natural antimicrobial preservative for meat products and its production process

Abstract

An organic natural antimicrobial preservative for meat products. The present invention relates to an organic natural antimicrobial preservative for meat products comprising natural organic salts (mainly lactates and acetates) which are completely safe, organic and natural ingredients to prevent the deterioration of fresh meats and sausages under refrigeration and to impart an increased shelf life to meat products. The present organic preservative is produced by a two-stage microbial fermentation of reducing sugars extracted from sweet potato and cassava starches using Lactobacillus plantarum for the first anaerobic fermentation stage, followed by a second aerobic fermentation stage by a bacterial association comprising Saccharomyces cerevisiae and Acetobacter aceti, followed by subsequent purification steps.It provides long-term preservation and a shelf life for meat products exceeding 20 to 25 days for fresh salted meat and 6 to 8 months for processed, cured, and refrigerated meats.

Description

1 / 12 “ORGANIC NATURAL ANTIMICROBIAL PRESERVATIVE FOR MEAT PRODUCTS AND THEIR PRODUCTION PROCESS” FIELD OF THE INVENTION

[001] The present invention relates to an organic product with antimicrobial properties, produced naturally through a two-stage microbial fermentation of carbohydrates from sweet potato and cassava sources using bacterial strains of Lactobacillus plantarum, Saccharomyces cerevisiae and Acetobacter aceti. This product prevents the spoilage of fresh meats and sausages under refrigeration and confers an increased shelf life to meat products. It is a safe and natural preservative that is good for intestinal health and is able to exert antimicrobial activity for a long time in meat products treated with it. FUNDAMENTALS OF THE INVENTION

[002] Preventing spoilage during the storage and transport of meat products, sausages, and fish products has always been a major concern for professionals in the meat industry. Food spoilage can be caused by a wide range of physical, chemical, enzymatic, and microbial reactions. Examples of the most widely used chemical food preservatives that inhibit microbes include benzoates, nitrites, ascorbates, and sorbates (01). But, according to food safety regulations, these chemical preservatives cannot be used above the recommended dosage level of 0.05 to 0.1%. Furthermore, there is always some health risk associated with the use of synthetic chemical additives, and consequently, natural preservatives are preferred by both sellers and consumers.

[003] Plant essential oils, as well as similar compounds, have been considered as promising natural antimicrobials (02). The presence of fat, carbohydrate, protein, salt and pH generally influences the effectiveness of these agents in food. Their antimicrobial strength is also reduced in food. Petition 870260011082, dated 05 / 02 / 2026, page 27 / 41 2 / 12 with low water activity (02).

[004] Originally added to alter or enhance flavor, spices and herbs can also be used to enhance the shelf life of food products by inhibiting foodborne pathogenic bacterial and fungal species. Usually, compounds with phenolic groups are considered to be effective antimicrobials, but they are generally more inhibitory against Gram-positive bacteria than Gram-negative bacteria (03, 04, 05).

[005] The applicant’s “Organic Natural Antimicrobial Preservative for Meat Products” is an effective antimicrobial product with proven benefits in controlling microbial growth and proliferation for a wide range of harmful food spoilage microbes, and helps extend the shelf life of all types of meat products without affecting the tenderness, textural properties and flavor of the meat products.

[006] It may be a better alternative to chemical preservatives, as it has no toxic effects on intestinal health and is a safe natural alternative while maintaining the advantages of increased shelf life and imparting desired aromas and flavors. SUMMARY OF THE INVENTION

[007] The present invention relates to a “Natural Organic Antimicrobial Preservative for Meat Products”, which is a completely organic natural ingredient for controlling unwanted microbial load in meat products. This product prevents the spoilage of fresh meats and sausages under refrigeration and confers an increased shelf life to meat products. It is a safe and natural preservative that is good for intestinal health and is able to exert antimicrobial activity for a long time in meat products treated with it.

[008] In one embodiment of the present invention, the active ingredient in the “Organic Natural Antimicrobial Preservative for Meat Products” is a Petition 870260011082, dated 05 / 02 / 2026, page 28 / 41 3 / 12 combination of naturally produced organic salts, including lactates and acetates that have antimicrobial properties along with the efficiency of improving the flavor of the preserved meat product.

[009] Another embodiment of the present invention relates to a process for producing “Organic Natural Antimicrobial Preservative for Meat Products”, produced by means of a two-stage microbial fermentation of carbohydrates from sweet potato and cassava sources to obtain natural organic salts (mainly lactates and acetates) as preservative components using bacterial strains of Lactobacillus plantarum, Saccharomyces cerevisiae and Acetobacter aceti by subsequent processing steps including filtration and concentration.

[010] In one embodiment, the “microbial source” used for the fermentative production of this product includes three bacterial strains, namely, NCIM 3594 of Saccharomyces cerevisiae, NCIM 2094 of Acetobacter aceti and NCIM 2084 of Lactobacillus plantarum, which were modified through strain improvement by means of medium optimization experiments for increasing product yield in the 'internal R&D section' of Prathista Industries Limited.

[011] In one embodiment of the present invention, “Lactobacillus plantarum” is the effector microbe for the first anaerobic fermentation step for the production of lactic acid, followed by the second “aerobic fermentation” step for the production of acetic acid by a bacterial association comprising Saccharomyces cerevisiae and Acetobacter aceti.

[012] In another embodiment of the present invention, a diluted solution [10.0% (v / v) in demineralized water] of “Organic Natural Antimicrobial Preservative” is applied at 10 mL per Kg of meat weight during the brining process of fresh ground meat before vacuum packaging and refrigeration of fresh ground meat / beef / pork preparations.

[013] In another embodiment of the present invention, during the preparation of Petition 870260011082, dated 05 / 02 / 2026, page 29 / 41 4 / 12 cured meats, such as sliced, cooked and boneless ham; sliced ​​cooked meatloaf; snack sticks; jerky; grilled meats (pulled pork or beef); poultry rolls and breasts; For roasted beef, etc., the “Organic Natural Antimicrobial Preservative” [10.0% (v / v) in demineralized water] applied at 0.5% (v / w) of meat weight during the hydration process, has the potential to replace chemical flavorings and curing agents such as nitrates / nitrites without significantly impacting the expected meat texture, appearance, and flavor of the cured meat from the controls, while conferring long-term preservation and shelf life to meat products by retaining the bacteriostatic effect and effectively controlling microbial spoilage for a period longer than 20 to 25 days for fresh salted meat and longer than 6 to 8 months for processed, cured, and refrigerated meats. BRIEF DESCRIPTION OF THE DRAWINGS

[014] Figure 1: Comparative profile of Listeria sp. growth in fresh ground beef after application of different treatments. Control: Ground beef treated with 0.9% (v / w) saline solution; T1: “Antimicrobial Preservative Natural Organic” at 0.5% (v / w) of meat weight; T2: “Antimicrobial Preservative” Natural Organic” at 1.0% (v / w) of meat weight; T3: “Antimicrobial Preservative Natural Organic” at 1.5% (v / w) of meat weight. T4: “Antimicrobial Preservative "Natural Organic" at 2.0% (v / w) by weight of meat.

[015] Figure 2: Aerobic plate count in fresh chicken meat after application of different treatments. Control: Chicken meat treated with 0.9% (v / w) saline solution;

[016] T 1: “Organic Natural Antimicrobial Preservative” at 0.5% (v / w) of meat weight; T2: “Organic Natural Antimicrobial Preservative” at 1.0% (v / w) of meat weight; T3: “Organic Natural Antimicrobial Preservative” at 1.5% (v / w) of meat weight. Petition 870260011082, dated 05 / 02 / 2026, pages 30 / 41 5 / 12

[017] Figure 3: Flowchart of the process for the production of Organic Antifungal Baking Additive. DETAILED DESCRIPTION OF THE INVENTION (A) Overview

[018] The present invention relates to a “Natural Organic Antimicrobial Preservative for Meat Products” which is a completely organic natural ingredient for controlling unwanted microbial load in meat products. This product prevents the spoilage of fresh meats and sausages under refrigeration and confers an increased shelf life to meat products. It is a safe and natural preservative that is good for intestinal health and is able to exert antimicrobial activity for a long time in meat products treated with it.

[019] In one embodiment of the present invention, the active ingredient in the “Organic Natural Antimicrobial Preservative for Meat Products” is a combination of naturally produced organic salts, including lactates and acetates, which have antimicrobial properties along with the efficiency of improving the flavor of the preserved meat product. In one of its preferred embodiments, this product comprises naturally produced lactic and acetic acids that impart antimicrobial properties, thereby inhibiting microbial growth in the treated food product (especially meat products), thus preventing spoilage without affecting its inherent flavor and texture.

[020] The other embodiment of the present invention relates to a process for producing an “Organic Natural Antimicrobial Preservative for Meat Products” produced by means of microbial fermentation of natural carbohydrate sources followed by further processing steps including filtration and concentration.

[021] In this embodiment of the invention, the applicant's product is produced by microbial fermentation of reducing sugars extracted from sweet potato starches and Petition 870260011082, dated 05 / 02 / 2026, pages 31 / 41 6 / 12 cassava to obtain natural organic salts (mainly lactates and acetates) as preservative components. This product is produced by a two-stage microbial fermentation process followed by subsequent purification steps. “Lactobacillus plantarum” is the effector microbe for the first anaerobic fermentation step for the production of lactic acid, followed by the second “aerobic fermentation” step for the production of acetic acid by a bacterial association comprising Saccharomyces cerevisiae and Acetobacter aceti.

[022] In one embodiment, the “microbial source” used for the fermentative production of this product includes three bacterial strains, namely, NCIM 3594 of Saccharomyces cerevisiae, NCIM 2094 of Acetobacter aceti and NCIM 2084 of Lactobacillus plantarum, which were modified through strain improvement by means of medium optimization experiments for increasing product yield in the 'internal R&D section' of Prathista Industries Limited.

[023] In another embodiment of the present invention, a diluted solution [10.0% (v / v) in demineralized water] of the “Organic Natural Antimicrobial Preservative” is applied at 10 mL per Kg of meat weight during the brining process of fresh ground meat before vacuum packaging and refrigeration of fresh ground meat / beef / pork preparations.

[024] In another embodiment of the present invention, during the preparation of cured meats, such as sliced, cooked and boneless ham; sliced ​​cooked meatloaf; snack sticks; jerky; grilled meats (shredded pork or beef); poultry rolls and breasts; roast beef, etc., the “Organic Natural Antimicrobial Preservative” [10.0% (v / v) in demineralized water] applied at 0.5% (v / w) by weight of meat during the hydration process, has the potential to replace chemical flavorings and curing agents such as nitrates / nitrites without significantly impacting the expected texture, appearance and flavor of the cured meat from the controls, while providing long-term preservation. Petition 870260011082, dated 05 / 02 / 2026, pp. 32 / 41 7 / 12 extends the shelf life of meat products by retaining the bacteriostatic effect and effectively controlling microbial spoilage for a period longer than 20 to 25 days for fresh salted meat and longer than 6 to 8 months for processed, cured, and refrigerated meats.

[025] The present invention is further explained by the following examples. However, the present invention is not limited to these examples in any way. The following examples are intended to illustrate the operation of the disclosure and are not intended to restrictively apply any limitations to the scope of the present invention. A person skilled in the art will understand that equivalent substitutes for the specific substances described herein, or corresponding improvements, are considered to be within the scope of the invention. (B) Experimental Details & Results

[026] This product is produced by a two-stage microbial fermentation process, anaerobic fermentation and aerobic fermentation. All fermentation studies were carried out in stainless steel (SS)-lined stirred tank bioreactors with capacities of 25 L and 50 L. EXAMPLE 1 (i) Previous Processing Anaerobic fermentation:

[027] The first step involves anaerobic fermentation for the production of Lactates using “Lactobacillus plantarum” as the effector microbe. The Lactobacillus strain used for this fermentation is a culturally improved bacterial strain modified by medium optimization experiments using the NCIM 2084 stock strain of Lactobacillus plantarum. The fermentation was carried out in a synthetic medium with the following composition (% (w / v)): __________________________ Components % (w / v) Glucose 11 to 15% Petition 870260011082, dated 05 / 02 / 2026, pages 33 / 41 8 / 12 Yeast extract 1% Potassium dihydrogen phosphate 0.1% Diammonium hydrogen phosphate 0.2% Manganese sulfate 0.001% Cobalt chloride 0.001% Magnesium sulfate 0.001% Sodium chloride 0.001% Ferrous sulfate 0.0005%

[028] The medium (without glucose) was heat sterilized at 121 °C and 1034 hPa (15 psi) for 25 min. The glucose was autoclaved separately at 115 °C for 15 min and added aseptically to the remaining medium.

[029] The parameters of the anaerobic fermentation process were as follows: Parameter Value pH 6.5 ± 0.2 Temperature 45 ± 2 °C Agitation (RPM) 100 Nitrogen 0.3 L / min

[030] The pH was maintained using potassium carbonate (K2CO3). Temperature and pH were monitored using temperature and pH probes, respectively (Sartorius). Sterile nitrogen gas was fluxed into the reactor headspace using a sterile PTFE filter with a 0.2 μm pore size (Axiva® 200050 RI, AXIVA Sichem Biotech Pvt. Ltd., India) to maintain anaerobic conditions throughout this fermentation step. The pre-sterilized fermentation medium in the bioreactor was inoculated with 10% static flask culture inoculum cultivated for 48 h. The seed culture was prepared in 500 mL Erlenmeyer flasks and incubated at 45 °C under anaerobic conditions in SS anaerobic jars using a Whitley Jar Gassing device. Petition 870260011082, dated 05 / 02 / 2026, pages 34 / 41 9 / 12 System (Don Whitley Scientific Limited, United Kingdom). After 84 to 90 hours of fermentation, all the glucose is consumed and a lactic acid concentration of 10 to 12% is obtained in the fermented broth. After that, the broth undergoes a second fermentation stage (aerobic fermentation) for the production of acetate. Aerobic fermentation:

[031] Acetate production is achieved using a microbial association comprising two internally improved strains, namely, Saccharomyces cerevisiae 3594 and Acetobacter aceti 2094, modified by medium optimization experiments. The fermented broth from the 25 L fermenter is transferred to a 50 L fermenter containing an equal volume of pre-sterilized medium.

[032] The composition of the aerobic fermentation medium (% (w / v)) was as follows: Ingredients % (w / v) Glucose 8 to 10% Yeast extract 0.5% Potassium dihydrogen phosphate 0.1% Diammonium hydrogen phosphate 0.2% Manganese sulfate 0.00015%

[033] The medium (without glucose) was heat sterilized at 121 °C and 1034 hPa (15 psi) for 25 min. The glucose was autoclaved separately at 115 °C for 15 min and added aseptically to the remaining medium.

[034] The parameters of the aerobic fermentation process were as follows: Parameter pH value 5.5 ± 0.2 Temperature 30 ± 2 °C Petition 870260011082, dated 05 / 02 / 2026, pp. 35 / 41 10 / 12 Agitation (RPM) 100 Nitrogen 0.5 L / min

[035] The pH was maintained using potassium carbonate (K2CO3). Temperature and pH were monitored using temperature and pH probes, respectively (Sartorius). The pre-sterilized fermentation medium in the bioreactor was inoculated with 10% shaken flask culture inoculum cultivated for 24 h. The seed culture was prepared in 500 mL Erlenmeyer flasks, incubated at 30 °C at 120 rpm. EXAMPLE 2 (ii) Ongoing Monitoring of Microbial Growth and Product Yield

[036] Microbial growth measurement during fermentation was performed in terms of optical density, in 3 mL cuvettes, at a wavelength of 600 nm using a UV-Vis spectrophotometer. To estimate the dry weight of the cells, 10 to 15 mL of fermentation broth were centrifuged at 10,000 rpm for 10 min in a pre-weighed Falcon tube and dried at 60 °C under vacuum until constant weight was obtained. EXAMPLE 3 (iii) Further Processing and Product Recovery

[037] Immediately after the increase in organic acid production, neutralization was carried out by dosing with pre-sterilized potassium carbonate paste to form a potassium-enriched organic acid-based product. As maximum organic acid production and complete glucose utilization are achieved within 70 h of the aerobic fermentation step, a typical production batch is completed between 60 and 72 h of fermentation. This is followed by filtration through fabric filters with sizes of 0.3 to 0.4 microns in a plate and frame filtration assembly. The filtered product is concentrated by evaporation at an elevated temperature of approximately 80 °C to obtain the desired product specification of the respective organic acids before packaging. The Petition 870260011082, dated 05 / 02 / 2026, pages 36 / 41 11 / 12 The preceding and subsequent process was represented as a flowchart in Figure 3. EXAMPLE 4 (iv) Shelf life test

[038] The impact of the “Organic Natural Antimicrobial Preservative” on the shelf life of fresh uncured meats was evaluated in terms of growth of the bacterial pathogen Listeria sp. in samples treated with varying concentrations of the product during a period of 45 days at a storage temperature of 4 °C. Samples treated only with 0.9% (w / v) saline solution served as a 'Control'.

[039] 10 g samples of fresh uncured meat (ground beef) and raw chicken breast were analyzed for microbial growth. It was observed that the ground beef sample containing the “Organic Natural Antimicrobial Preservative” as an additive at 2.0% (v / w) could last for more than a month without a significant increase in microbial load (Figure 1), while the raw chicken sample treated with 1.5% (v / w) of the additive prevented spoilage for up to 30 days (Figure 2). In contrast, the 'Control' meat samples showed growth of pathogenic bacteria from day 3 onwards. INDUSTRIAL APPLICABILITY OF THE INVENTION

[040] This invention has applicability as a natural clean-label antimicrobial preservative for meat and fish products during storage and transportation. REFERENCES CITED 1) Anand, S. P. e Sati, N. (2013) Artificial preservatives and their harmful effects: looking toward nature for safer alternatives. International Journal of Pharmaceutical Sciences and Research, 4(7): 2496-2501. 2) Seow, Y. X., Yeo, C.R., Chung, H. L., e Yuk, H. G. (2014) Plant Essential Oils as Active Antimicrobial Agents. Critical Reviews in Food Science and Nutrition, 54 Petição 870260011082, de 05 / 02 / 2026, pág. 37 / 41 12 / 12 (5): 625-644. 3) Hammer, K. A., Carson, C. F. e Riley, T. V. (1999) J. Appl. Microbiol., 86(6): 985. 4) Kalemba, D., Kunicka, A. (2003) Curr. Med. Chem., 10(10): 813. 5) Bagamboula C.F., Uyttendaele M., Debevere J. (2003) J. Food Prot., 6(4): 668. Petição 870260011082, de 05 / 02 / 2026, pág. 38 / 41

Claims

1 / 2 CLAIMS 1. Organic natural antimicrobial preservative CHARACTERIZED in that it comprises: a combination of fermentation products of 40% potassium lactate and 25% potassium acetate produced naturally; wherein the fermentation products are potassium-enriched products of the microbial fermentation of carbohydrates from sweet potato and cassava sources by the bacterial strains Saccharomyces cerevisiae NCIM 3594, Acetobacter aceti NCIM 2094 and Lactobacillus plantarum NCIM 2084.

2. Production process of the organic natural antimicrobial preservative, as defined in claim 1, CHARACTERIZED in that said preservative is produced naturally through a two-step microbial fermentation of carbohydrates from sweet potato and cassava sources using three bacterial strains of Saccharomyces cerevisiae NCIM 3594, Acetobacter aceti NCIM 2094 and Lactobacillus plantarum NCIM 2084, which have been modified by means of strain enhancement through medium optimization, followed by subsequent processing steps including filtration and concentration, wherein Lactobacillus plantarum is the effector microbe for the first anaerobic fermentation step for lactic acid production,followed by a second stage of aerobic fermentation for the production of acetic acid by a bacterial consortium comprising Saccharomyces cerevisiae and Acetobacter aceti; wherein the first stage involves anaerobic fermentation for the production of Lactates using Lactobacillus plantarum NCIM 2084 as the effector microbe using glucose derived from sweet potato at 11 to 15%, yeast extract at 1%, potassium dihydrogen phosphate at 0.1%, diammonium hydrogen phosphate at 0.2%, manganese sulfate at 0.001%, cobalt chloride at 0.001%, magnesium sulfate at 0.001%, sodium chloride at 0.001% and ferrous sulfate at 0.0005%, carried out at pH 6.0 ± 0.2, temperature at 45 ± 2 °C, agitation at 100 rpm and nitrogen at 0.3 L / min for 84 to 90 minutes. Petition 870260011082, dated 05 / 02 / 2026, page 1. 39 / 41 2 / 2 hours,wherein the second stage of aerobic fermentation for acetate production by a bacterial consortium comprising two improved strains of Saccharomyces cerevisiae NCIM 3594 and Acetobacter aceti 2094 is carried out using 8-10% yucca-derived glucose, 0.5% yeast extract, 0.1% potassium dihydrogen phosphate, 0.2% diammonium hydrogen phosphate, and 0.00015% manganese sulfate at pH 5.50 ± 0.2, temperature 30 ± 2 °C, agitation at 100 rpm, aeration at 0.5 L / min, carried out for 60-72 hours, wherein subsequent processing steps include neutralization of organic acids produced by fermentation using potassium carbonate paste, filtration carried out using fabric filters with sizes of 0.3-0.4 microns in a filtration assembly of plate and frame, and concentrated filtered product obtained by evaporation at 80 °C.

3. Process according to claim 2, CHARACTERIZED in that the glucose-free medium is heat-sterilized at 121 °C and 1034 hPa for 25 min; glucose is separately autoclaved at 115 °C for 15 min and aseptically added to the remainder of the medium.

4. Process, according to claim 2, CHARACTERIZED in that it is carried out in bioreactors with stirred tanks lined with SS Stainless Steel with capacities of 25 L and 50 L. Petition 870260011082, dated 05 / 02 / 2026, pp. 40 / 41

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