WO2025191562A1

WO2025191562A1 - Methods for microbial biomass production using lactic acid microbes growing in high salinity conditions and products thereof

Info

Publication number: WO2025191562A1
Application number: PCT/IL2025/050233
Authority: WO
Inventors: Nir Friedman
Original assignee: Naerobix
Current assignee: Naerobix
Priority date: 2024-03-12
Filing date: 2025-03-12
Publication date: 2025-09-18
Anticipated expiration: 2026-09-12

Abstract

The present disclosure provides methods for microbial biomass production using lactic acid microbes capable of growing in high-salinity conditions. The disclosed methods may involve culturing at least one lactic acid microbial cell, including strains of Tetragenococcus, on substrates with high salt concentrations to produce high-protein biomass. The invention further comprises microbial products, waste treatment applications, and methods for reducing carbon emissions.

Description

METHODS FOR MICROBIAL BIOMASS PRODUCTION

USING LACTIC ACID MICROBES GROWING IN HIGH SALINITY CONDITIONS AND PRODUCTS THEREOF

FIELD OF THE INVENTION

The present disclosure relates to the field of microbial biomass production and/or bioconversion of organic substrates, specifically organic substrates with high salinity, into valuable enriched biomass including for example high-protein content using specific lactic acid microbes.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

- [1] US 4,018,650;

- [2] EP1014801;

[3] Ayushi et al. (2019) International Journal of Pharmacy and Biological Sciences- IJPBSTM 9 (2): 164-168;

- [4] US 2023/0000125;

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND OF THE INVENTION

Annually, hundreds of millions of tons of organic wastes and by-products are produced from the food and agriculture industries. Most of these wastes are recalcitrant, containing high salinity and/or nitrogen contents, which require high costs for its effective treatment while conferring negative effect on the environment. Scarcity in high quality protein for animal and human consumption is also a global problem that requires innovative approaches for finding alternative sources. Microbial based products offer a consolidated approach for these challenges.

There is a need for novel microbial isolates which are able to grow on adverse conditions (e.g. high salt and/or nitrogen concentration) to transform low quality organic materials towards the production of high value products.

US 4,018,650 [1] relates to single cell protein production using Bacillus.

EP1014801 [2] relates to the bioconversion of industrial or agricultural cellulose containing wastes using lactic acid bacteria. Ayushi et al. [3] relates to the production of single cell protein from mix fruits waste using Lactobacillus.

US 2023/0000125 [4] relates to microbial consortia which may comprise Tetragenococcus for the production of single cell protein via gas fermentation.

SUMMARY OF THE INVENTION

In a first aspect, the present disclosure provides a method of producing microbial biomass from at least one substrate comprising high salt concentration, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbial cell on said at least one substrate, and/or recovering said cells and/or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by the ability of growing on high salt concentration substrate/s.

In a second aspect, the present disclosure provides a method of producing at least one product of interest, the method comprising culturing at least one lactic acid microbial cell or any population of cells comprising said microbial cell on at least one substrate comprising high salt concentration, and recovering said at least one microbial cell, any population of cells comprising said microbial cell, or any product, derivative, extract or preparation thereof from the culture, thereby producing said at least one proteinaceous product, wherein said at least one microbial cell is characterized by at least one of (a) the ability of growing on high salt concentration substrate/s; (b) naturally residing in the digestive tract of at least one ruminant; and (c) being at least one bacterium of the Tetragenococcus genus.

In a third aspect, the present disclosure provides a product comprising at least one lactic acid microbial cell and/or population of cells comprising said at least one microbial cell, or any preparation or extract thereof, wherein said at least one lactic acid microbial is characterized by at least one of: (a) the ability of growing on high salt concentration substrate/s; (b) naturally residing in the digestive tract of at least one ruminant; and (c) being at least one bacterium of the Tetragenococcus genus.

In a fourth aspect, the present disclosure provides an isolated bacterium of the Tetragenococcus genus originating from the digestive tract of at least one ruminant, wherein said bacterium is characterized by the ability of growing on high salinity conditions (or in high salt concentration).

In a fifth aspect, the present disclosure provides a method of waste treatment, by converting at least one substrate originating from said waste and comprising high salt concentration into microbial biomass, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbial cell on said at least one substrate, and/or recovering said cells and/or any product , derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by the ability of growing on high salt concentration substrate/s.

In a sixth aspect, the present disclosure provides a method for reducing carbon emissions and/or generating carbon credits, the method comprising:

(a) culturing at least one lactic acid microbial cell or a population of cells comprising said microbial cell on at least one organic substrate comprising high salt concentration;

(b) converting at least a portion of the carbon content in said substrate into microbial biomass; and

(c) quantifying the reduction in CO₂ emissions resulting from said conversion to obtain carbon credits, wherein said at least one lactic acid microbial cell is characterized by at least one of: (a) the ability of growing on high salt concentration substrate(s); (b) naturally residing in the digestive tract of at least one ruminant and (c) being at least one bacterium of the Tetragenococcus genus.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Figure 1: Growth of NbiA29 under different NaCl concentration.

Growth of NbiX29 in MH+ medium supplemented with 0.5% or 5% NaCl at 37°c with gentle shaking under anaerobic conditions. Optical density at 600nm (OD600) was measured every 24h. Each dot represents the average of triplicate measurements.

Figure 2: Concentrated samples of VbiA29 grown in MH+ supplemented with 0.5% and 5% NaCl.

Figure 3: Growth of AbiA29 and VbiA30 in anaerobic digester sludge. NbiXX29 and NbiXX30 were grown in anaerobic digester sludge (fed with agriculture organic waste), at 37°c with gentle shaking under anaerobic conditions. Microbial growth was monitored by CO₂ production by Gas chromatography. Each dot represents the average of triplicate measurements.

Figure 4: Growth of VbiA29 and VbiX30 in different types of dairy wastes. NbiX X29 and XN3b0iX were grown in 3 mixes of dairy wastes. Waste A (5.3% lactose + 3.3% NaCl), waste B (4% lactose + 5% NaCl) and waste C (2% lactose + 2.5% NaCl) at 37°c with gentle shaking under anaerobic conditions. Following 72h, microbial growth was monitored by CO₂ production by Gas chromatography. Each bar represents the average of duplicate tests.

Figure 5A-5B: Growth of NbiX29 in Brewer’s Spent Grain.

NbiX29 was grown in BSG based medium (10% w/v), at 37°c with gentle shaking under anaerobic conditions.

Fig. 5A: Microbial growth monitored by CO₂ production by Gas chromatography of BSG (alone), NbiX29 (alone), NbiX29 on BSG. Each bar represents the average of triplicate measurements.

Fig. 5B: Comparison of CO₂ production of BSG (alone) + NbiX29 (alone) with NbiX29 on BSG.

Figure 6: 7VbiX29 crude protein from dry matter under different NaCl concentration.NbiX X29 was grown in MH+ medium supplemented with 0.5% or 5% NaCl at 37°c with gentle shaking under anaerobic conditions. Each bar represents the average of duplicate tests.

Figure 7A-7B: Effect of salinity on protein content and amino acid profile of 7VbiX29.NbiX X29 was grown in MH+ medium supplemented with 0.5% or 5% NaCl at 37 °c with gentle shaking under anaerobic conditions.

Fig. 7A: Relative abundance of 17 amino acids from X29 hydrolyNsabtieX grown in MH+ medium supplemented with 0.5% and 5% NaCl.

Fig. 7B: Fold change ratios between amino acids hydrolysate from X29 grown under NbiX 5% NaCl and hydrolysate of X29N gbrioXwn under 0.5% NaCl in MH+ medium. Stripped bars represent fold change higher than 1 and black bars represent fold change lower than 1 between amino acids concentration of hydrolysate from 5% compared to hydrolysate from 0.5%. Figure 8: Changes in body weight during the study.

Data plotted as the percentage of change, relative to the initial body weight (Day 1). Each time point represents mean +SEM for all mice in each group.

DETAILED DESCRIPTION OF THE INVENTION

Novel microbial isolates from the Tetragenococcus genus were isolated, characterized and analyzed for their ability to grow and degrade high salinity and high organic load wastes. They were able to grow on dairy waste and on anaerobic digester sludge, two highly problematic wastes in terms of organic load and salt concentrations. Furthermore, genomic and metabolic analyses demonstrated that these strains possess important features as high-quality food and feed as they are able to produce and accumulate high protein concentration as confirmed by crude protein and amino acid profile analyses.

The aim of this work was to develop novel microbial isolates that are capable of growing in high salt environment, to degrade and transform low quality organic materials towards production of high value products. The present disclosure shows that novel strains from the Tetragenococcus genus, isolated from ovine rumen are capable of consuming simple sugars and proteins that are present in low quality streams, and produce high protein biomass out of them.

Thus in a first aspect, the present disclosure provides a method of producing microbial biomass from at least one substrate comprising high salt concentration, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbial cell on said at least one substrate, and/or recovering said cells and/or any product or preparation , derivative, extract thereof from the culture, wherein said at least one microbe is characterized by the ability of growing on high salt concentration substrate/s.

In a specific embodiment, the present disclosure provides a method of producing microbial biomass from at least one substrate comprising high salt concentration, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product , derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by the ability of growing on high salt concentration substrate/s and naturally residing in the digestive tract of at least one ruminant.

In a specific embodiment, the present disclosure provides a method of producing microbial biomass from at least one substrate comprising high salt concentration, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product , derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by the ability of growing on high salt concentration substrate/s, naturally residing in the digestive tract of at least one ruminant and being at least one bacterium of the Tetragenococcus genus.

In a specific embodiment, the present disclosure provides a method of producing microbial biomass from at least one substrate comprising high salt concentration, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product , derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by naturally residing in the digestive tract of at least one ruminant.

In a specific embodiment, the present disclosure provides a method of producing microbial biomass from at least one substrate comprising high salt concentration, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product , derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by being at least one bacterium of the Tetragenococcus genus.

In some specific embodiments, the present disclosure provides a method of producing microbial biomass from at least one substrate comprising high salt concentration, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product , derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by at least one of: (a) the ability of growing on high salt concentration substrate/s; (b) by naturally residing in the digestive tract of at least one ruminant; and (c) being at least one bacterium of the Tetragenococcus genus.

As used herein, the term “microbial biomass” refers to the total mass of microorganisms, such as bacteria, fungi, yeast, algae, and archaea and to any products, fragments or derivatives thereof. Thus, it includes both living microorganisms and their remnants, such as dead cells and intracellular and extracellular products. Products and derivatives thereof of microbial biomass may include but are not limited to nutritional elements or supplements, cosmetics, pharmaceuticals, fertilizers, biofuels, enzymes, bioplastics, antibiotics, vitamins, biosurfactants, biopesticides, bioherbicides, bioinsecticides, and bioactive peptides. In some embodiments, said microbial biomass is edible. In some embodiments, the microbial biomass produced according to the method of the present disclosure is used as a food. In some further embodiment, the microbial biomass produced according to the method of the present disclosure is used as feed.

As used herein, the term “high salt concentration” or “high salinity” or “high salinity conditions” may be used interchangeably and relates to a condition where the concentration of dissolved salts in a liquid, e.g. water, is elevated beyond normal levels. Salinity may be measured in parts per thousand (ppt) or as a percentage of the total weight of the solution.

In some embodiments, high salinity refers to a salt concentration of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5% or 25%.

In some embodiments, a range of high salt concentrations relates to 1-8% or about 1-10%, or about 1-5%, 1-5.5%, 1-6%, 1-6.5%, 1-7%, 1-7.5%, 1-8%, 1-8.5%, 1-9%, 1-9.5%, 1-10%, 1- 11%, 1-12%, 1-13%, 1-14%, 1-15%, 1-16%, 1-17%, 1-18%, 1-19%, 1-20%, 1-21%, 1-22%, 1- 23%, 1-24% or 1-25%.

In some other embodiments, a range of high salt concentrations relates to about 2-20%, 3- 20%, 4-20%, 5-20%, 6-20%, 7-20%, 8-20%, 9-20%, 10-20%, 11-20%, 12-20%, 13-20%, 14-20% or 15-20%.

In some further embodiments, a range of high salt concentrations relates to about 2-10%, 2.5-10%, 3-10%, 3.5-10%, 4-10%, 4.5-10%, 5-10%, 5.5-10%, 6-10%, 6.5-10%, 7-10%, 7.5-10%, 8-10%, 8.5-10%, 9-10% or 9.5-10%.

In some embodiments, high salinity refers to a salt concentration of about 10 ppt (parts per thousand), 15 ppt, 20 ppt, 25 ppt, 30 ppt, 35 ppt, 40 ppt, 45 ppt, 50 ppt, 55 ppt, 60 ppt, 65 ppt, 70 ppt, 75 ppt, 80 ppt, 85 ppt, 90 ppt, 95 ppt, 100 ppt, 105 ppt, 110 ppt, 115 ppt, 120 ppt, 125 ppt, 130 ppt, 135 ppt, 140 ppt, 145 ppt, 150 ppt, 155 ppt, 160 ppt, 165 ppt, 170 ppt, 175 ppt, 180 ppt, 185 ppt, 190 ppt, 195 ppt, 200 ppt, 205 ppt, 210 ppt, 215 ppt, 220 ppt, 225 ppt, 230 ppt, 235 ppt, 240 ppt, 245 ppt, or 250 ppt.

In some embodiments, a range of high salt concentrations relates to about 10-80 ppt (parts per thousand), 10-100 ppt, or about 10-50 ppt, 10-55 ppt, 10-60 ppt, 10-65 ppt, 10-70 ppt, 10-75 ppt, 10-80 ppt, 10-85 ppt, 10-90 ppt, 10-95 ppt, 10-100 ppt, 10-110 ppt, 10-120 ppt, 10-130 ppt, 10-140 ppt, 10-150 ppt, 10-160 ppt, 10-170 ppt, 10-180 ppt, 10-190 ppt, 10-200 ppt, 10-210 ppt, 10-220 ppt, 10-230 ppt, 10-240 ppt, or 10-250 ppt.

In some other embodiments, a range of high salt concentrations relates to about 20-200 ppt (parts per thousand), 30-200 ppt, 40-200 ppt, 50-200 ppt, 60-200 ppt, 70-200 ppt, 80-200 ppt, 90- 200 ppt, 100-200 ppt, 110-200 ppt, 120-200 ppt, 130-200 ppt, 140-200 ppt, or 150-200 ppt.

In some further embodiments, a range of high salt concentrations relates to about 20-100 ppt (parts per thousand), 25-100 ppt, 30-100 ppt, 35-100 ppt, 40-100 ppt, 45-100 ppt, 50-100 ppt, 55-100 ppt, 60-100 ppt, 65-100 ppt, 70-100 ppt, 75-100 ppt, 80-100 ppt, 85-100 ppt, 90-100 ppt, or 95-100 ppt.

In some further embodiments, said at least one substrate further comprises high nitrogen concentration.

As used herein, the term “high nitrogen concentration” refers to an elevated level of nitrogen compounds in a substance, typically in the context of environmental or chemical analysis. Nitrogen can exist in various forms, including nitrate (NO3-), nitrite (NO2-), ammonia (NH3), and organic nitrogen compounds. High nitrogen concentration can occur naturally or as a result of human activities such as agriculture, industrial processes, or wastewater discharge. In natural ecosystems, excessive nitrogen levels can lead to eutrophication, where an overabundance of nutrients promotes rapid growth of algae and aquatic plants, disrupting the balance of the ecosystem. In chemical processes or industrial settings, high nitrogen concentrations can pose safety hazards or affect the quality of products.

In some embodiments, high nitrogen concentration relates to about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5% or 25%.

In some embodiments, a range of high nitrogen concentrations relates to about 3-10%, or about 3-15% or about 3-5%, 3-5.5%, 3-6%, 3-6.5%, 3-7%, 3-7.5%, 3-8%, 3-8.5%, 3-9%, 3-9.5%, 3-10%, 3-11%, 3-12%, 3-13%, 3-14%, 3-15%, 3-16%, 3-17%, 3-18%, 3-19%, 3-20%, 3-21%, 3- 22%, 3-23%, 3-24% or 3-25%.

In some further embodiments, a range of high nitrogen concentrations relates to about 1- 5%, 1-5.5%, 1-6%, 1-6.5%, 1-7%, 1-7.5%, 1-8%, 1-8.5%, 1-9%, 1-9.5%, 1-10%, 1-11%, 1-12%, 1-13%, 1-14%, 1-15%, 1-16%, 1-17%, 1-18%, 1-19%, 1-20%, 1-21%, 1-22%, 1-23%, 1-24% or 1-25%.

In some other embodiments, , a range of high nitrogen concentrations relates to about 2- 20%, 3-20%, 4-20%, 5-20%, 6-20%, 7-20%, 8-20%, 9-20%, 10-20%, 11-20%, 12-20%, 13-20%, 14-20% or 15-20%.

In some further embodiments, a range of high nitrogen concentrations relates to about 2- 10%, 2.5-10%, 3-10%, 3.5-10%, 4-10%, 4.5-10%, 5-10%, 5.5-10%, 6-10%, 6.5-10%, 7-10%, 7.5- 10%, 8-10%, 8.5-10%, 9-10% or 9.5-10%.

As used herein “lactic acid microbial cell” or “lactic acid microbe” may be used interchangeably and refers to microorganisms or microbes capable of producing lactic acid through fermentation. In some embodiments, said microbial cell may be at least one of bacteria, fungi, yeast, algae, and archaea.

Lactic acid microbes are widely used in various industrial processes, including food fermentation (e.g., yogurt, cheese, sauerkraut), pharmaceuticals, and production of lactic acid for various applications.

These microbes convert carbohydrates, such as sugars or starches, into lactic acid (homofermentative) and also to Volatile Fatty Acids (VFAs) such as formate, acetate, propionate and butyrate, ethanol and CO₂ (heterofermentative) through anaerobic fermentation. This process is important not only for food preservation and flavor development but also for generating lactic acid, which has numerous industrial applications, including as a food additive, in biodegradable plastics, pharmaceuticals, and cosmetics.

Specifically, in some embodiments, the lactic acid microbes may be used for Food Fermentation. Lactic acid microbes play a crucial role in food fermentation processes, including the production of yogurt, cheese, sauerkraut, kimchi, and sourdough bread. In these processes, lactic acid bacteria convert carbohydrates (such as lactose in milk or sugars in vegetables) into lactic acid through fermentation. This not only helps to preserve the food by creating an acidic environment that inhibits the growth of harmful bacteria but also contributes to the characteristic flavors and textures of these fermented foods. Additionally, lactic acid fermentation can improve the digestibility and nutritional quality of certain foods.

In some embodiments, lactic acid microbes may also be used for pharmaceuticals. In some embodiments, lactic acid microbes may also be used for production of lactic acid. Lactic acid is an important industrial chemical with numerous applications. Lactic acid microbes are utilized in large-scale fermentation processes to produce lactic acid economically and sustainably. This lactic acid can be further processed into various derivatives, including lactate salts, polylactic acid (PLA), and other biodegradable plastics. PLA, in particular, is widely used in packaging materials, textiles, medical implants, and other biodegradable products, offering a renewable and environmentally friendly alternative to petroleum-based plastics.

In some embodiments, lactic acid microbes may also be used for production of bioremediation and waste valorization. Lactic acid microbes are also employed in bioremediation processes to treat organic waste and pollutants as illustrated in the present examples, specifically Examples 3, 4 and 5. Certain lactic acid bacteria have the ability to degrade organic compounds and detoxify environmental pollutants, contributing to environmental cleanup efforts. Additionally, lactic acid fermentation can be used to valorize agricultural and industrial waste streams by converting them into value-added products such as biofuels, biogas, and biofertilizers.

While bacteria are the most common microorganisms associated with lactic acid production, there are some non-bacterial microorganisms capable of producing lactic acid such as for example certain species of yeast.

In some embodiments, the lactic acid microbe may be a yeast.

In some embodiments, the lactic acid microbe may be a yeast. Some yeast species, such as certain strains of Saccharomyces cerevisiae and Candida utilis, have been found to produce lactic acid under specific conditions. Although yeast is primarily known for its role in alcoholic fermentation, certain strains can produce lactic acid as a metabolic byproduct under anaerobic conditions. This ability has been explored in various industrial applications, including bioethanol production and the production of lactic acid for industrial uses.

Lactic acid microbes comprise large and diverse groups of bacteria capable of producing lactic acid as the primary fermentation product.

Thus, in some specific embodiments, said at least one lactic acid microbial cell comprises at least one bacterium. In some embodiments, said bacteria are non-genetically modified bacteria. In some alternative embodiments, said bacteria are genetically modified bacteria.

In some further specific embodiments, said at least one bacterium (or microbe) is a non- aerobic bacteria.

As used herein, the term “non-aerobic bacteria” relates to microorganisms that do not require oxygen for their growth and metabolism. Unlike aerobic bacteria, which need oxygen to survive, non-aerobic bacteria thrive in environments where oxygen levels are low or absent. They obtain energy through processes such as fermentation or anaerobic respiration, which do not rely on oxygen.

In some embodiments, said at least one bacterium (or microbe) may be anaerobic. As used herein, “anaerobic bacteria” relate to a diverse group of microorganisms that thrive in environments devoid of oxygen. They have adapted to metabolize without the need for oxygen, utilizing alternative electron acceptors such as nitrate, sulfate, or carbon dioxide for their energy production. These bacteria are essential for various ecological processes, including decomposition and nutrient cycling. Anaerobic bacteria can be found in a wide range of environments, including: soil, sediment, intestinal tract (human and animal digestive systems), wetlands, deep-sea vents. Anaerobic bacteria are involved in the decomposition of organic matter, nitrogen fixation, bioremediation of pollutants, and the production of valuable products such as methane and hydrogen. In the human body, anaerobic bacteria in the gut microbiota contribute to digestion, vitamin synthesis, and protection against pathogenic organisms.

Examples of genera of anaerobic bacteria include but are not limited to Bacteroides, Clostridium, Prevotella, Fusobacterium, Peptostreptococcus, Actinomyces, Propionibacterium, Porphyromonas, Veillonella or Eubacterium.

In some further embodiments, said at least one lactic acid bacterium (or microbe) is facultative anaerobic. As used herein, “facultative anaerobic bacteria” relate to microorganisms capable of thriving in environments with or without oxygen. Unlike obligate anaerobes, which can only survive in the absence of oxygen, facultative anaerobes have metabolic versatility, allowing them to switch between aerobic respiration (utilizing oxygen) and anaerobic respiration or fermentation (in the absence of oxygen). This adaptability enables them to colonize a wide range of habitats and environments such as soil, water, intestinal tract (human and animal digestive systems) and fermented foods.

Examples of genera of facultative anaerobic bacteria include but are not limited to Tetragenococcus, Enterococcus, Listeria, Mycobacterium, Pseudomonas, Bacillus, Streptococcus, Lactobacillus, Klebsiella, Proteus, Enterobacter.

In some embodiments, said at least one lactic acid microbe naturally resides in the digestive tract of at least one ruminant.

As used herein, the term “digestive tract of at least one ruminant” refers to the alimentary canal or gastrointestinal (GI) tract which is responsible for the digestion and absorption of nutrients from food. The digestive tract of ruminants consists of the upper digestive tract comprising the mouth, esophagus and stomach, the small intestine, and the large intestine. The stomach of ruminants has particular structure and function and comprises the rumen, reticulum, omasum, and abomasum.

In some embodiments, said at least one lactic acid microbe naturally resides in the upper digestive tract at least one ruminant. In some specific embodiments, at least one lactic acid microbe naturally resides in the rumen.

As used herein, the term “rumen” relates to the largest compartment of the stomach in ruminant animals. It plays a crucial role in the digestive process of these animals, which rely on microbial fermentation to break down plant material. The rumen serves as a fermentation vat where ingested food, primarily cellulose-rich plant material, undergoes initial breakdown. Microorganisms, including bacteria, protozoa, and fungi, inhabit the rumen and aid in the fermentation process. Additionally, the rumen allows for the process of rumination, where partially digested food (cud) is regurgitated from the rumen to be rechewed and further broken down.

As used herein, a “ruminant” relates to a mammal that belongs to the order Artiodactyla and possesses a specialized stomach with four compartments: the rumen, reticulum, omasum, and abomasum. These compartments allow for a unique digestive process called rumination, where partially digested food (called cud) is regurgitated from the rumen and rechewed before being further digested. Ruminants include animals such as cattle, sheep, goats, deer, and giraffes. They are known for their ability to efficiently digest plant material, making them important contributors to ecosystems and agriculture. In some embodiments, a ruminant may relate to cattle (Bovidae family, Bos genus). In some specific embodiments, the ruminant may be domestic cattle (Bos taurus), Bison (Bison bison), Yak (Bos grunniens), Gaur (Bos gaurus) or Banteng (Bos javanicus).

In some embodiments, a ruminant may relate to sheep (Bovidae family, Ovis genus). In some specific embodiments, the ruminant may be a domestic sheep (Ovis aries), Bighorn sheep (Ovis canadensis), Dall sheep (Ovis dalli), Mouflon (Ovis orientalis) or Argali (Ovis ammon).

In some embodiments, a ruminant may relate to goats (Bovidae family, Capra genus). In some specific embodiments, the ruminant may be domestic goat (Capra aegagrus hircus), Mountain goat (Oreamnos americanus), Markhor (Capra falconeri), Ibex (Capra ibex) or Spanish goat (Capra aegagrus hircus).

In some embodiments, a ruminant may relate to deer (Cervidae family). In some specific embodiments, the ruminant may be White-tailed deer (Odocoileus virginianus), Red deer (Cervus elaphus), Roe deer (Capreolus capreolus), Moose (Alces alces) or Elk (Cervus canadensis).

In some specific embodiments, the ruminant may be an ovine.

The major genera of lactic acid bacteria are Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Tetragenococcus, Pediococcus, Oenococcus and Enterococcus.

In some embodiments, the lactic acid bacteria may be a lactobacillus. Lactobacillus is one of the largest and most well-known genera of lactic acid bacteria. It includes various species commonly found in dairy products, fermented vegetables, and the human gastrointestinal tract.

In some embodiments, the lactic acid bacteria may be a Streptococcus. Streptococcus is another important genus of lactic acid bacteria, although some species are pathogenic, others are used in food fermentation.

In some embodiments, the lactic acid bacteria may be a Lactococcus. Lactococcus species are commonly used in the dairy industry for the production of cheese and other fermented dairy products.

In some embodiments, the lactic acid bacteria may be a Leuconostoc. Leuconostoc species are often found in plant-based fermentations, such as sauerkraut, pickles, and sourdough bread. They contribute to the fermentation process and the development of characteristic flavors in these foods.

In some embodiments, the lactic acid bacteria may be a Oenococcus. Oenococcus oeni is primarily known for its role in wine fermentation, where it contributes to malolactic fermentation, a secondary fermentation process that helps reduce acidity and improve the flavor profile of wines. In some embodiments, the lactic acid bacteria may be a Enterococcus.

In some embodiments, the lactic acid bacteria may be a Pediococcus. Pediococcus species are commonly found in fermented foods and beverages, including beer, sauerkraut, and cured meats, where they contribute to acid production and flavor development.

In some embodiments, said lactic acid microbe is at least one bacterium of the Tetragenococcus genus.

Tetragenococcus, is another genus of gram-positive, facultatively anaerobic bacteria within the family Enterococcaceae. Its typical cell morphology is as follows: non-motile, spherical cells (about 0.5-0.8 pm), which divide in two planes at right angles to form tetrads.

Tetragenococcus species are commonly found in salty and acidic environments, such as fermented seafood products like fish sauce and soy sauce. They contribute to the fermentation process and the development of characteristic flavors in these products. Tetragenococcus species can ferment sugars into lactic acid, but they may also produce other metabolites depending on the specific conditions of the fermentation.

Examples of Tetragenococcus species include Tetragenococcus halophilus, which is commonly used in the fermentation of fish sauce.

In some embodiments, the bacterium may be Tetragenococcus halophilus. Tetragenococcus halophilus is a species of halophilic (salt-loving) bacteria within the family Enterococcaceae. These bacteria are gram-positive, facultatively anaerobic, and cocci-shaped. They are notable for their ability to thrive in high-salt environments, such as salted foods and saline habitats like salted fish, brine, and fermented products. T. halophilus plays a significant role in the fermentation process of various salted foods, particularly in the production of fermented seafood products like fish sauce and shrimp paste. In these fermentation processes, T. halophilus contributes to the development of flavor, aroma, and preservation of the food product.

In some embodiments, the bacterium may be Tetragenococcus koreensis. Tetragenococcus koreensis is an halophilic bacterium which is gram-positive, facultatively anaerobic, and typically cocci-shaped. Tetragenococcus koreensis was first isolated from traditional Korean fermented seafood known as jeotgal. Jeotgal is made by fermenting seafood, such as shrimp or fish with salt. The ability of Tetragenococcus koreensis to thrive in such high- salt environments makes it significant in the fermentation process of jeotgal and similar salted foods. Like other members of the genus Tetragenococcus, T. koreensis likely plays a role in the fermentation process by contributing to flavor development, preservation, and other biochemical changes in the food matrix.

In some embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 1. In some further embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 2.

In some other embodiments, at least one of the following substrate is relevant to the present disclosure:

(i) said substrate comprises an organic substrate;

(ii) said substrate is composed of at least about 4% to about 25% dry material; and

(iii) said substrate is a liquid substrate.

As used herein, the term “organic substrate” relates to any material of organic origin that serves as a medium or support for the growth of living organisms, typically microorganisms or plants.

In some embodiments, the organic substrate or liquid organic substrate may relate to a substance comprising about 4-15% dry material. In some further embodiments, the organic substrate or liquid organic substrate may comprise about 4.5-15%, 5-15%, 5.5-15%, 6-15%, 6.5- 15%, 7-15%, 7.5-15%, 8-15%, 8.5-15%, 9-15%, 9.5-15%, 10-15%, 10.5-15%, 11-15%, 11.5-15%, 12-15%, 12.5-15%, 13-15%, 13.5-15%, 14-15% or 14.5-15% dry material.

In some embodiments, the organic substrate or liquid organic substrate relates to a substance comprising about 4-25% dry material. In some further embodiments, the organic substrate or liquid organic substrate may comprise about 4-15%, 4-16%, 4-17%, 4-18%, 4-19%, 4-20%, 4-21%, 4-22%, 4-23%, 4-24% or 4-25% dry material.

In some embodiments, the organic substrate or liquid organic substrate relates to a substance comprising about 4-22% dry material. In some further embodiments, the organic substrate or liquid organic substrate may comprise about 4.5-22%, 5-22%, 5.5-22%, 6-22%, 6.5- 22%, 7-22%, 7.5-22%, 8-22%, 8.5-22%, 9-22%, 9.5-22%, 10-22%, 10.5-22%, 11-22%, 11.5-22%, 12-22%, 12.5-22%, 13-22%, 13.5-22%, 14-22% or 14.5-22% dry material. In some embodiments, the organic substrate or liquid organic substrate relates to a substance comprising about 4-22% dry material.

In some further embodiments, the organic substrate or liquid organic substrate may comprise about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24% or 25% dry material.

In some further embodiments, the organic substrate comprises an organic waste and/or industrial byproduct/s.

In some embodiments, the organic substrate may comprises an organic waste. As used herein, the term “organic waste” relates to any material derived from living organisms that can undergo decomposition by biological processes. This includes various types of biodegradable waste such as food scraps, yard waste, paper products, and other organic materials. Organic substrates are rich in carbon and provide a source of nutrients for microorganisms, which break them down into simpler compounds like carbon dioxide, water, and organic matter.

In some embodiments, the organic substrate may be organic waste with high salinity. Organic waste with high salinity can come from various sources, including industrial processes, agricultural activities, and natural environments. Non limiting examples include brine from food processing, saline agricultural runoff, aquaculture waste, saltwater aquarium waste or biological treatment of saline effluents.

In some embodiments, the organic substrate may be byproduct/s or industrial byproduct/s. As used herein, the term “byproduct/s” or “industrial byproduct/s” refers to secondary or incidental products that are produced alongside the main product during a manufacturing or industrial process. These additional products may have commercial value or may be considered waste depending on their characteristics and potential uses. Byproducts can arise from various processes across different industries such as for example food processing, manufacturing and energy production.

In some embodiments, the organic substrate originates from at least one of: (i) the food industry;

(ii) the agriculture industry;

(iii) municipal solid waste.

In some embodiments, the organic waste originates from the food industry. Organic waste generated in the food industry can encompass a wide range of materials, such as for example food Scraps, food processing residues, dairy waste, meat and fish processing waste, bakery waste, expired or damaged products, coffee grounds, oil and grease, vegetable and fruit processing waste or packaging materials.

In some embodiments, the organic waste originates from the agriculture industry. Organic waste generated in the agriculture industry may encompasses various materials, including but non limited to crop residues, fruit and vegetable waste, animal manure, poultry feathers and bedding, aquaculture waste, horticultural waste, dairy farm waste, processed byproducts, silage and hay spoilage or unused feed.

In some specific embodiments, the organic substrate comprises dairy waste.

In some embodiments, the organic waste originates from municipal solid waste. As used herein, the term “Municipal solid waste (MSW)” refers to the waste generated by households, businesses, institutions, and other non-industrial sources within a municipality or urban area. Non limiting examples of organic waste originating from MSW are food scraps, yard waste, paper products (e.g., napkins, paper towels), wood waste, textiles and biodegradable plastics.

In some specific embodiments, the organic substrate comprises waste derived from anaerobic fermenters facilities and/or biogas facilities.

As used herein, “waste derived from anaerobic fermenters facilities and/or biogas facilities” refers to the residual materials remaining after the anaerobic digestion process, including solid residues, liquid digestate, biogas effluent, filtration residues, unused feedstock residues, and any contaminants present in the waste stream.

In some embodiments, waste derived from biogas facilities may be residual solids such as solid residues left after the anaerobic digestion process, often referred to as digestate solids. This may include fibrous materials, undigested organic matter, and inert materials. In some embodiments, waste derived from biogas facilities may be digestate liquids such as liquid remnants of the anaerobic digestion process, containing water, nutrients, and organic compounds. It typically has a high nutrient content, including nitrogen, phosphorus, and potassium.

In some embodiments, waste derived from biogas facilities may be biogas effluent: effluent or liquid discharge from biogas production facilities, containing water and residual organic matter.

In some embodiments, waste derived from biogas facilities may be residues generated from the filtration or separation processes used to remove solids from the biogas or digestate which may include filter cakes, screenings, or other solid residues.

In some embodiments, waste derived from biogas facilities may be unused feedstock residues i.e. portions of organic waste feedstock that remain unconverted or partially digested after anaerobic digestion.

In some further specific embodiments, the organic substrate or organic waste may comprises at least one of brine, leachate, bagasse, dairy products/by-products , organic pomace, high moister husks/hulls, whey, digestate, brewer’s spent grain, high fats liquids (oilseed), high proteinaceous liquids, blood meal, fish waste, seafood shells, eggshells, coffee grounds, tea leaves, yard trimmings, grass clippings, sawdust, wood chips, paper waste, cardboard, straw, hay, algae biomass, biodegradable plastics, kitchen waste, restaurant grease, sewage sludge, food-soiled paper, sugarcane bagasse, cotton gin trash, corn cobs, nut shells, coconut husks, rice straw, wheat bran, banana peels, pineapple crowns, avocado pits, olive pomace, molasses, fruit pits (e.g., peach, plum), vegetable tops and tails, seaweed, grass clippings, flower trimmings, plant roots, shellfish shells and/or livestock bedding waste.

In some specific embodiments, , the organic substrate or organic waste comprises brewer’s spent grain.

In some further specific embodiments, the organic substrate comprises at least one of dairy waste, waste derived from anaerobic fermenters facilities (and/or biogas facilities) and brewer’s spent grain.

In some embodiments, the method of the present disclosure further comprises a pretreatment step of the at least one substrate prior to microbial culturing. In some embodiments, the pre-treatment step is applied to improve the digestibility, bioavailability, or conversion efficiency of the substrate by removing inhibitors, modifying nutrient composition, or enhancing microbial accessibility to organic components. In some embodiments, pre-treatment is necessary to reduce potential antimicrobial compounds that may hinder microbial growth or to solubilize complex organic matter into more readily metabolizable forms.

In some embodiments, the pre-treatment step comprises physical treatment, including but not limited to mechanical grinding, milling, homogenization, heating, steam explosion, ultrasonication, or filtration to alter the particle size, remove debris, or disrupt cellular structures in complex substrates. In some embodiments, the pre-treatment step comprises chemical treatment, including but not limited to acid hydrolysis, alkaline hydrolysis, oxidation, solvent extraction, or chemical neutralization, in order to modify pH, break down complex polymers, or remove undesirable contaminants from the substrate.

In some embodiments, enzymatic pre-treatment is used to break down macromolecules such as lignocellulose, starch, or proteins into smaller, more bioavailable units. In some embodiments, specific enzymes such as cellulases, amylases, proteases, or lipases are applied to degrade fibers, carbohydrates, proteins, or fats present in the substrate, thereby enhancing microbial access to nutrients.

In some embodiments, biological pre-treatment is performed using microbial cocktails comprising bacteria, fungi, or yeasts that possess enzymatic activity capable of breaking down complex organic matter. In some embodiments, these microbial cocktails are selected to reduce toxic compounds, partially ferment the substrate, or introduce beneficial microbial communities that support subsequent lactic acid microbial growth. In some embodiments, pre-fermentation or partial anaerobic digestion is employed as a biological pre-treatment step to initiate substrate degradation.

In some embodiments, e.g. following the pre-treatment step, the method further comprises a washing step. In some embodiment, the washing may enable to remove residual inhibitors, excess salts, unwanted chemical residues, or undesirable metabolic byproducts that may have accumulated during pre-treatment. In some embodiments, the washing step involves rinsing the substrate with water, buffered solutions, or specific chemical agents to ensure optimal conditions for microbial growth. In some embodiments, washing is performed by filtration, centrifugation, dialysis, or decantation to selectively remove unwanted compounds while retaining the essential nutrients required for microbial cultivation.

In certain embodiments, the cells recovered by the recovering step comprise whole cells.

In certain embodiments, the cells recovered by the recovering step may comprise intact cells.

In some further embodiments, the cells recovered by the recovering step comprise lysed cells or any preparation thereof.

As shown in Example 7, the method in accordance with the present disclosure enables to produce microbial biomass having high protein content. In some embodiments, the method of the present disclosure enables to obtain a microbial biomass having a high protein content.

In some specific embodiments, when growing the at least one lactic acid microbe at a salt concentration of 0.5%, the protein content comprised into the microbial biomass is about 50% from the total biomass dry matter. In some further embodiments, when growing at high salt concentration (e.g. 5%), the protein content comprised into the microbial biomass is more than 50%, or more than 60% or up to 69% from the total biomass dry matter.

Thus, in some further embodiments, the method of the present disclosure may comprise the step of increasing the concentration of salt. In some embodiments, the method enables to obtain microbial biomass with higher protein content.

As shown in Example 7, the amino acid profile may be modified as a consequence of salt concentration increasing. In some further embodiments, when growing at higher salt concentration (e.g. up to 5%), the concentration of at least one of the following amino acids is increased in comparison to growing at low salt concentration (e.g. 0.5%): threonine, serine, glutamic acid, proline, glycine, alanine and/or histidine.

In yet some specific embodiments, the microbial biomass comprises single cell protein biomass.

As used herein, the term “Single cell protein (SCP)” or “Single cell protein biomass” refers to protein-rich biomass derived from microbial cells, e.g. as grown in large-scale fermentation processes. These microorganisms may be such as bacteria, fungi, yeast, or algae, and may be cultivated on various substrates like agricultural waste, industrial by-products, or hydrocarbons or proteinaceous waste/by-product. SCP may serve as an alternative protein source for human and animal consumption. SCP are therefore especially useful when traditional protein sources like meat or soy may be scarce or environmentally unsustainable. SCP offers potential benefits such as high protein content, rapid growth rates, efficient resource utilization, and reduced environmental footprint compared to conventional protein sources.

The protein concentration and digestibility in single cell protein (SCP) can vary depending on the specific microorganism being used, the fermentation process, and the substrate on which the microorganism is grown.

Specifically, in some embodiments, SCP may be bacterial SCP. In some embodiments, said SCP may comprise from 40% to 80% protein concentration (e.g. dry weight basis).

In some further embodiments, SCP may be fungal SCP. In some embodiments, said SCP may comprise from 40% to 70% protein concentration (e.g. dry weight basis).

In some other embodiments, SCP may be algal SCP. In some embodiments, said SCP and may comprise from 40% to 60% protein concentration (e.g. dry weight basis).

In some additional embodiments, SCP may yeast SCP. In some embodiments, said SCP and may comprise from 40% to 70% protein concentration (e.g. dry weight basis).

These ranges are approximate and can vary depending on factors such as the specific strain of microorganism, the composition of the growth medium, and the fermentation conditions.

In some other embodiments, SCP may comprise at least 40% protein concentration (e.g. dry weight basis). In some further embodiments, SCP may comprise at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, at protein concentration (e.g. dry weight basis).

In some embodiments, the single cell protein biomass is at least 40%. In some embodiments, 40% or more of said single cell biomass is protein.

In some embodiments, the protein concentration in said single cell protein biomass may be at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% , or at least 95%, or or at least 100% (e.g. dry weight basis).

The amino acid profile of bacterial isolates was shown in Example 7. Thus in some embodiments, the most abundant amino acid(s) in the single cell protein biomass may be at least one of Glutamic acid, aspartic acid, alanine and/or lysin. In some embodiments, the microbial biomass produced according to the method of the present disclosure may provide a sustainable and high-protein alternative to conventional protein sources. Thus in some embodiments, the microbial biomass produced according to the method of the present disclosure is used as a food or feed ingredient.

In some embodiments, microbial biomass serves as a direct protein source for human nutrition, or is incorporated into functional foods, protein supplements, meat substitutes, or fermented food products. In some embodiments, microbial biomass is processed into textured protein ingredients, suitable for inclusion in plant-based meat analogs or as an alternative to soy- or animal-derived proteins.

In some embodiments, microbial biomass is utilized as an animal feed ingredient, either as a primary protein source or as a nutritional supplement for livestock, poultry, aquaculture, or pet food. In some embodiments, the high protein content of the biomass, ranging from 40% to 70% by dry weight, supports its use as a replacement for soybean meal, fishmeal, or other conventional feed ingredients. In some embodiments, microbial biomass is further processed into protein concentrates or isolates to enhance its digestibility and nutritional profile for specific dietary applications.

In some embodiments, microbial biomass contains essential amino acids and/or vitamins (e.g., B-complex vitamins, including B12). In some embodiments, the controlled microbial fermentation process ensures that the biomass is free from contaminants, pathogens, or anti- nutritional factors, making it a safe and reliable protein source.

In some embodiments, the microbial biomass can be fortified or blended with other protein sources, fibers, or functional ingredients to enhance its nutritional properties. In some embodiments, additional processing, such as drying, extrusion, fermentation, or encapsulation, is used to optimize the texture, shelf life, and functional characteristics of the microbial biomass for specific food or feed applications.

In some embodiments, the use of microbial biomass as food or feed contributes to sustainability goals, as its production requires significantly less land, water, and energy compared to traditional animal-based protein sources. In some embodiments, the method enables efficient valorization of organic waste streams, reducing environmental impact while producing high-value, protein-rich food or feed products.

In some further embodiments, the method according to the present disclosure may further comprise purifying proteins from the biomass to generate protein isolate, e.g. disrupting cells of the biomass to generate a lysate, and separating and/or concentrating proteins from the lysate.

As used herein, the term “protein isolate” refers to a composition that comprises primarily proteins isolated, extracted or purified from a microbial biomass that comprises primarily, consisting essentially of, or consisting of a biomass of the lactic acid microbe of the invention. A composition that comprises primarily proteins isolated from a microbial biomass refers to a composition of which more than 40%, or 50 % (e.g., more than 55%, 60%, 70%, 75% or 80%) by weight is proteins from the microbial biomass. Through the use of protein isolation, extraction or purification technique(s), protein isolate has higher protein content (e.g., measured by percentage crude protein) than the microbial biomass from which the protein isolate is prepared. However, protein isolation, extraction or purification does not need to be to the extent that individual proteins are separated from each other. Instead, protein isolate in general comprises a mixture of proteins isolated, extracted or purified from a bacterial biomass in which at least some of the other components (e.g., nucleic acids or lipids) in the microbial biomass are removed.

In some embodiments, the protein isolate may be produced by disrupting cells of the microbe to generate a lysate, and separating and/or concentrating proteins from the lysate.

In some other embodiments, the method may further comprise purifying proteins from said lactic acid microbe to generate protein isolate and/or protein concentrate.

As used herein, the term "protein concentrate" refers to a composition derived from microbial biomass that has undergone processing to increase its protein content relative to the original biomass. In some embodiments, a protein concentrate is obtained by removing non-protein components such as water, lipids, and carbohydrates through techniques such as centrifugation, filtration, precipitation, or solvent extraction. In some embodiments, protein concentrate retains a portion of the original microbial matrix, including minor amounts of nucleic acids, fibers, or residual carbohydrates, while exhibiting an enhanced protein content compared to the untreated biomass. In some embodiments, protein concentrate comprises at least about 40% protein by dry weight. In some further embodiments, the protein content in the concentrate is at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or higher. In some embodiments, protein concentrate is suitable for use in food, animal feed, pharmaceuticals, or industrial applications.

In some embodiments, protein concentrate differs from protein isolate, which undergoes further purification steps to achieve an even higher protein content, typically above 90% by dry weight. In some embodiments, protein concentrate serves as an intermediate product that can be used directly or further processed into protein isolate, peptides, or hydrolysates.

In a second aspect, the present disclosure provides a method of producing at least one product of interest, the method comprising culturing at least one lactic acid microbe or any population of cells comprising said microbial cell on at least one substrate comprising high salt concentration, and recovering said at least one microbial cell, any population of cells comprising said microbe, or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by at least one of: (a) the ability of growing on high salt concentration substrate(s); (b) naturally residing in the digestive tract of at least one ruminant and (c) being at least one bacterium of the Tetragenococcus genus.

In some specific embodiments, the present disclosure provides a method of producing at least one product of interest, the method comprising culturing at least one lactic acid microbe or any population of cells comprising said microbe on at least one substrate comprising high salt concentration, and recovering said at least one microbe, any population of cells comprising said microbe, or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbe is characterized by the ability of growing on high salt concentration substrate/s and naturally residing in the digestive tract of at least one ruminant.

In some specific embodiments, the present disclosure provides a method of producing at least one product of interest, the method comprising culturing at least one lactic acid microbe or any population of cells comprising said microbe on at least one substrate comprising high salt concentration, and recovering said at least one microbe, any population of cells comprising said microbe, or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbe is characterized by the ability of growing on high salt concentration substrate/s, naturally residing in the digestive tract of at least one ruminant and being at least one bacterium of the Tetragenococcus genus.

In some specific embodiments, the present disclosure provides a method of producing at least one product of interest, the method comprising culturing at least one lactic acid microbe or any population of cells comprising said microbe on at least one substrate comprising high salt concentration, and recovering said at least one microbe, any population of cells comprising said microbe, or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbe is characterized by the ability of growing on high salt concentration substrate/s.

In some specific embodiments, the present disclosure provides a method of producing at least one product of interest, the method comprising culturing at least one lactic acid microbe or any population of cells comprising said microbe on at least one substrate comprising high salt concentration, and recovering said at least one microbe, any population of cells comprising said microbe, or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbe is characterized by naturally residing in the digestive tract of at least one ruminant.

In some specific embodiments, the present disclosure provides a method of producing at least one product of interest, the method comprising culturing at least one lactic acid microbe or any population of cells comprising said microbe on at least one substrate comprising high salt concentration, and recovering said at least one microbe, any population of cells comprising said microbe, or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbe is characterized by being at least one bacterium of the Tetragenococcus genus. In some embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 1. In some further embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 2.

As used herein, a “product” or “product of interest” relevant to the present disclosure i.e. obtained by the methods of the present disclosure, or comprising at least one lactic acid microbial cell and/or population of cells comprising said at least one microbial cell, or any preparation or extract thereof as defined herein, may relate to any one of nutritional elements or supplements, cosmetics, pharmaceuticals, fertilizers, biofuels, enzymes, bioplastics, antibiotics, vitamins, biosurfactants, biopesticides, bioherbicides, bioinsecticides, and bioactive peptides.

In some embodiments, said product or product of interest is edible.

In some embodiments, said product is at least one food product, optionally said food product is an animal and/or human food or food supplement. In some further embodiments, the product, or derivative, or extract or preparation of said cell or population of cells may be employed for the production of a food product. In some further embodiments, the product, or derivative, or extract or preparation of said cell or population of cells may be employed for the production of a human food product or food supplement. In some further embodiments, the product, or derivative, or extract or preparation of said cell or population of cells may be employed for the production of an animal food product or food supplement.

In some embodiment, said product may be at least one human food product. As used herein, the term “food product” relates to any substance or item that is intended to be consumed for nutritional purposes. Specifically, food products may relate to human food products. Food products can vary greatly in their composition, preparation, and intended use. They can be raw ingredients, processed foods, or ready-to-eat meals, and they can come in various forms such as solid, liquid, semi-solid, or powder. Food products are typically categorized based on their nature, nutritional content, processing methods, and intended use.

In some embodiments, a food product may relate to processed foods. Processed foods that have undergone some form of processing, such as cooking, drying, freezing, canning, or packaging, to improve their shelf life, taste, texture, or convenience.

In some embodiments, a food product may relate to bakery products. This category includes bread, pastries, cakes, cookies, muffins, and other baked goods made from flour, sugar, eggs, and other ingredients.

In some embodiments, a food product may relate to dairy products. In some specific embodiments, dairy products may be products derived from milk such as milk itself, cheese, yogurt, butter, cream, and ice cream.

In some embodiments, a food product may relate to beverages. In some embodiments, beverages may encompass a wide range of liquid food products, such as juices, soft drinks, tea, coffee, energy drinks, sports drinks, and alcoholic beverages like beer, wine, and spirits. In some embodiments, a food product may relate to canned and preserved foods, these are foods that have been preserved through canning, pickling, or other preservation methods to extend their shelf life. Non limiting examples include canned fruits and vegetables, pickles, jams, and preserves.

In some embodiments, a food product may relate to snack foods. Snack foods are typically consumed between meals and are often designed for convenience and quick consumption. In some embodiments, snack foods may be chips, pretzels, popcorn, nuts, granola bars, or dried fruit.

In some embodiment, said product may be at least one animal food product or feed. As used herein, “animal food product” or “animal food” or "feed" refers to any substance or mixture of substances that is intended to be consumed by animals. Animal feed can come in various forms such as grains, forages, supplements, and processed feeds, and it is formulated to meet the specific nutritional requirements of different species of animals, including livestock, pets, and aquatic animals. The composition of animal feed can vary based on factors such as the species being fed, their age, their physiological status, and the intended purpose (e.g., growth, milk production, egg laying).

Non limiting examples of animal food or feed include but are not limited grains such as corn, wheat, barley, oats, and sorghum; forages like alfalfa, clover, grass hay, and silage; protein supplements such as soybean meal, cottonseed meal, and fish meal; mineral supplements like salt, limestone, and dicalcium phosphate; vitamin supplements including vitamin premixes containing vitamins A, D, E, and various B vitamins; processed feeds like pelleted feeds, textured feeds, and mixed rations; and aquafeed formulated for aquatic animals such as fish, shrimp, and other aquaculture species.

In some embodiment, said product may be at least one food supplement. As used herein, a “food supplement”, also known as a “dietary supplement”, is a product intended to provide additional nutrients that may be lacking in a person's diet. These supplements are typically consumed in the form of pills, capsules, tablets, powders, or liquids and are meant to complement, not replace, a balanced diet. Non limiting examples of food supplements may be vitamins, minerals, amino acids, fatty acids, enzymes, herbs, or other dietary substances. In some embodiments, said product may be at least one of vitamins, minerals, amino acids, fatty acids, enzymes, herbs, and/or other dietary substances. In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be a vitamin. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe may be riboflavin (vitamin B2). In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be niacin (vitamin B3). In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be folate (vitamin B9). In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be menaquinone (vitamin K2).

As mentioned above, lactic acid microbes are widely used for the production of lactic acid in several types of applications. Thus, in some embodiments, the product, derivative, extract or preparation of said cell or population of cells in accordance with the present disclosure may be lactic acid.

Lactic acids may be used for food preservation and flavor development. In some embodiments, the product, or derivative, or extract or preparation derived from the lactic acid microbe/ of said cell or population of cells may be a product for food fermentation. In some further embodiments, the product, or derivative, or extract or preparation derived from the lactic acid microbe/ of said cell or population of cells may be employed for the production of a product for food fermentation.

In some embodiments, the product, or derivative, or extract or preparation derived from the lactic acid microbe/ of said cell or population of cells may be a food additive and/or preservative. In some further embodiments, the product, or derivative, or extract or preparation of said cell or population of cells may be employed for the production of a food additive and/or preservative.

In some embodiments, the product, or derivative, or extract or preparation derived from the lactic acid microbe/ of said cell or population of cells may be a biodegradable plastic (e.g. Polylactic acid (PLA)). In some further embodiments, the product, or derivative, or extract or preparation derived from the lactic acid microbe/ of said cell or population of cells may be employed for the production of a biodegradable plastic (e.g. Polylactic acid (PLA)).

In some specific embodiments, the product, or derivative, or extract or preparation derived from the lactic acid microbe/ of said cell or population of cells may be lactate salts. Lactic acid microbes and their derivatives have significant applications in pharmaceuticals due to their ability to produce bioactive compounds, antimicrobial agents, and metabolic byproducts with therapeutic potential. In some embodiments, the product, or derivative, or extract or preparation of said cell or population of cells may be a pharmaceutical product. In some embodiments, the product, or derivative, or extract or preparation of said cell or population of cells may be employed for the production of a pharmaceutical product.

In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe may be an antimicrobial agent. In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be employed for the production of an antimicrobial agent, optionally a bacteriocin, organic acid, or exopolysaccharide with antibacterial, antifungal, or antiviral properties.

In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be a vaccine or vaccine adjuvant. In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be employed for the production of a vaccine or vaccine adjuvant, optionally for mucosal immunization, antigen presentation, or immune system modulation.

In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be a drug delivery system. In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be employed for the production of a drug delivery system, optionally in the form of encapsulated bioactive compounds, controlled-release formulations, or nanoparticle-based therapeutics.

In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be a biocompatible polymer or biopolymer precursor. In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be employed for the production of a biocompatible polymer or biopolymer precursor, optionally including poly-y-glutamic acid (y-PGA), polylactic acid (PLA), or exopolysaccharides for biomedical and pharmaceutical applications. As shown in Example 8, the lactic acid microbe in accordance with the present disclosure may produce volatile fatty acid (VFA) or alcohol. In some embodiments, the product may be volatile fatty acid (VFA). In some further embodiments, said VFA may be at least one of lactate, formate, acetate and/or propionate. In some other embodiments, the product may be alcohol. In some specific embodiments, the alcohol may be ethanol.

In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be a cosmetic formulation. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be employed for the production of a cosmetic formulation, optionally for skin care, hair care, or personal hygiene applications.

Eactic acid microbes are also widely used for or production of bioremediation and waste valorization. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be a bioremediation agent. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be employed for the production of a bioremediation agent, optionally for the degradation, detoxification, or removal of environmental pollutants, heavy metals, or organic waste.

In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be a biofuel precursor. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be employed for the production of a biofuel precursor, optionally for the generation of ethanol (bioethanol), butanol, or other renewable fuels through microbial fermentation.

In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be a biogas-enhancing agent. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be employed for the production of a biogasenhancing agent, optionally for improving methane yield, optimizing anaerobic digestion, or increasing the efficiency of biogas production from organic waste. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be a biofertilizer. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be employed for the production of a biofertilizer, optionally for enhancing soil health, promoting plant growth, or increasing nutrient bioavailability in agricultural applications.

In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe/ of said cell or population of cells may be a waste valorization product. In some embodiments, the product, or derivative, or extract, or preparation of said cell or population of cells may be employed for the production of a waste valorization product, optionally for converting organic waste into high-value products such as protein-rich biomass, organic acids, or industrially relevant biochemicals.

In some embodiments, said high salt concentration may be about to 1-8% or about 1-10%, or as further detailed above. In some embodiments, said high nitrogen concentration may such as about 3-10%, 3-15%, or as further detailed above.

In some specific embodiments, said at least one lactic acid microbe comprises at least one bacterium.

In some embodiments, the microbe suitable for the method of the present disclosure may be genetically modified in order to produce a specific product of interest from a culture medium comprising high salt concentrations.

In some embodiments, the method of the present disclosure may comprise a step of genetically modifying said at least one lactic acid microbe prior or during the culturing step.

In some embodiments, genetic modification of the lactic acid microbes is achieved using recombinant DNA technology, wherein genes encoding enzymes or biosynthetic pathways responsible for the production of specific compounds, such as proteins, peptides, vitamins, or bioactive molecules, are introduced into the microbial genome.

In some embodiments, genome editing techniques, such as CRISPR-Cas9, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats-associated transposases (CRISPR-associated transposases), are used. In some embodiments, such genome editing techniques may introduce targeted modifications that enhance native biosynthetic pathways or confer novel functionalities, including the ability to produce nutraceuticals, pharmaceuticals, or industrial enzymes.

In some embodiments, synthetic biology approaches are employed to design and construct synthetic metabolic pathways, allowing for the production of non-native metabolites such as polyunsaturated fatty acids, biofuels, or biopolymers.

In some embodiments, metabolic engineering is used to optimize endogenous microbial pathways to improve flux toward the biosynthesis of high-protein biomass, amino acids, organic acids, or bioactive secondary metabolites.

In some embodiments, heterologous gene expression is utilized to introduce genes from other microbes or higher organisms into lactic acid bacteria, enabling the biosynthesis of therapeutic proteins, recombinant enzymes, or food-grade compounds.

In some embodiments, horizontal gene transfer methods, including natural transformation, electroporation, conjugation, and protoplast fusion, are used to facilitate genetic exchange between microbial strains and introduce desired genetic traits.

In some embodiments, transposon mutagenesis is applied to modify gene regulatory elements and optimize gene expression for the production of metabolites or proteins of interest.

In some embodiments, inducible or constitutive promoters are engineered to regulate gene expression dynamically, allowing for controlled production of target compounds in response to specific environmental or chemical stimuli.

In some embodiments, RNA interference (RNAi) or antisense RNA strategies are employed to downregulate competing metabolic pathways, thereby increasing the efficiency of target metabolite biosynthesis.

As a result of these genetic modifications, in some embodiments, the engineered microbes are capable of producing an expanded range of products of interest. In some embodiments, these products include nutritional and functional biomolecules such as high-protein biomass, essential and non-essential amino acids, vitamins, riboflavin, folic acid and/or omega-3 fatty acids.

In some embodiments, the modified microbes produce industrial and environmental products, including biofuels such as ethanol, butanol, or hydrogen, biodegradable plastics such as polylactic acid and polyhydroxy alkanoates, biosurfactants, and biopolymers.

In some embodiments, the engineered microbes generate pharmaceutical and biomedical products, including recombinant therapeutic proteins, antimicrobial peptides, immunomodulatory compounds, and enzyme inhibitors for disease treatment.

In some embodiments, the microbes are genetically modified to produce agricultural and biocontrol agents, such as biofertilizers, biopesticides, and plant growth-promoting substances, as well as feed additives for livestock. In some embodiments, the engineered microbes are utilized for the production of food and beverage ingredients, including flavor-enhancing compounds, food preservatives such as bacteriocins, texture modifiers such as exopolysaccharides, and fermentation-derived food products.

In some embodiments, these genetic engineering strategies allow for the tailored development of lactic acid microbes for use in various industries, including biopharmaceuticals, food technology, renewable energy, and waste valorization, thereby promoting sustainable and high-value production processes.

In some embodiments, said bacteria are non-genetically modified bacteria. In some alternative embodiments, said bacteria are genetically modified bacteria.

In some more specific embodiments, said at least one bacterium is a non-aerobic bacteria.

In some embodiments, said at least one lactic acid microbe is anaerobic. In some other embodiments, said at least one lactic acid microbe is facultative anaerobic.

In some embodiments, said at least one lactic acid microbe naturally resides in the digestive tract of at least one ruminant. In some embodiments, said at least one lactic acid microbe naturally resides in the upper digestive tract. In some more specific embodiments, said at least one lactic acid microbe naturally resides in the rumen.

In some further specific embodiments, said lactic acid microbe is at least one bacterium of the Tetragenococcus genus.

In some embodiments, said bacteria is closely related to Tetragenococcus halophilus and/or Tetragenococcus koreensis.

In some embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 1. In some further embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 2.

Tetragenococcus halophilus and Tetragenococcus koreensis are halophilic lactic acid bacteria commonly found in fermented foods, particularly those with high salt concentrations. They play an essential role in flavor development, biopreservation, and enzyme production in various traditional and industrial fermentations. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe may be a fermented food ingredient.

In some embodiments, the product, or derivative, or extract, or preparation may be a bacteriocin or antimicrobial compound.

In some embodiments, the product, or derivative, or extract, or preparation may be a halophilic enzyme.

In some embodiments, the product, or derivative, or extract, or preparation may be a flavorenhancing metabolite, e.g. for high salt preparation.

In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe may be a lactic acid fermentation byproduct. In some embodiments, the product, or derivative, or extract, or preparation may be a bioprocessed salt-tolerant culture for food fermentation.

In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe may be a high-salinity adapted biomass for aquaculture feed.

In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe may be a salt-resistant starter culture for soy-based fermentations. In some embodiments, the product, or derivative, or extract, or preparation derived from the lactic acid microbe may be a preservative -producing microbial preparation for seafood processing.

In some embodiments, the product, or derivative, or extract, or preparation may be employed for the production of kimchi.

In some embodiments, the product, or derivative, or extract, or preparation may be employed for the production of sauerkraut. In some embodiments, the product, or derivative, or extract, or preparation may be employed for the production of soy sauce. In some embodiments, the product, or derivative, or extract, or preparation may be employed for the production of miso. In some embodiments, the product, or derivative, or extract, or preparation may be employed for the production of fermented seafood products, optionally fish sauce or shrimp paste. In some embodiments, the product, or derivative, or extract, or preparation may be employed for the production of pickled vegetables (e.g. kimchi). In some embodiments, the product, or derivative, or extract, or preparation may be employed for the production of fermented grain-based foods, such as soybean-based grain pastes (e.g. Doenjang, a Korean fermented soybean paste and Japanese Natto) or fermented rice-based alcoholic beverage (e.g. Makgeolli, a Korean rice wine).

In some other embodiments, at least one of the following is relevant to the substrate of the present disclosure:

(i) said substrate comprises an organic substrate

(iii) said substrate is a liquid substrate.

In another embodiments, wherein the organic substrate comprises an organic waste and/or industrial byproduct/s.

In some other embodiments, the organic substrate originates from at least one of:

(i) the food industry;

(ii) the agriculture industry; and

(iii) municipal solid waste. In yet another embodiment, the organic substrate comprises waste derived from anaerobic fermenters facilities and/or biogas facilities.

In some embodiments, the method of the present disclosure further comprises a pretreatment step of the at least one substrate prior to microbial culturing (as further detailed above). In some further embodiments, the method further comprises a washing step.

The term “whole cell”, “whole cell product”, “whole cell ingredient”, “whole cell protein” or the like as used herein refers to a product made from the lactic acid microbe biomass without undergoing a step of separating soluble proteins from other components of the biomass, especially solid components such as cell debris and/or cell wall. In certain embodiments, the process of producing a whole cell product may include a heating step to activate native nucleases (e.g., native endonucleases) to degrade some of the nucleic acids (DNA and RNA) present in the biomass.

In certain embodiments, the cells recovered by the recovering step may comprise intact cells. As used herein, the term “intact cell” refers to a non-lysed cell. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the cells may be intact cells.

In some embodiments, parts of the cells may be whole cells and parts of the cells may be lysed. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the cells may be whole cells. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the cells may be lysed cells.

In some specific embodiments, said product is a proteinaceous product. In some embodiments, the product, or derivative, or extract, or preparation may comprise a single-cell protein (SCP) biomass. In some other embodiments, the method may further comprise purifying proteins from said lactic acid microbe or protein isolate and/or protein concentrate.

In some embodiments, the method may further comprise combining protein isolate and whole cells from said lactic acid microbe. In some embodiments, the method may further comprise combining protein isolate and intact cells from said lactic acid microbe. In some embodiments, the method may further comprise combining protein isolate and lysed cells from said lactic acid microbe.

In some embodiments, the method may further comprise combining with at least one other edible ingredient or food additive.

In some embodiments, the method may further comprise combining with at least one plantbased protein source, at least one animal-based protein source, and/or at least one bacteria-based protein source.

In a third aspect, the present disclosure provides a product of interest comprising at least one lactic acid microbial cell and/or population of cells comprising said at least one microbial cell, or any preparation, derivative or extract thereof, or any product produced by said cells, wherein said at least one lactic acid microbial cell is characterized at least one of: (a) the ability of growing on high salt concentration substrate(s); (b) naturally residing in the digestive tract of at least one ruminant and (c) being at least one bacterium of the Tetragenococcus genus.

In some specific embodiments, the present disclosure provides a product of interest comprising at least one lactic acid microbial cell and/or population of cells comprising said at least one microbial cell, or any preparation, derivative or extract thereof, or any product produced by said cells, wherein said at least one lactic acid microbial cell is characterized by the ability of growing on high salt concentration substrate/s and naturally residing in the digestive tract of at least one ruminant.

In some further specific embodiments, the present disclosure provides a product of interest comprising at least one lactic acid microbial cell and/or population of cells comprising said at least one microbial cell, or any preparation, derivative or extract thereof, or any product produced by said cells, wherein said at least one lactic acid microbial cell is characterized by the ability of growing on high salt concentration substrate/s, naturally residing in the digestive tract of at least one ruminant and being at least one bacterium of the Tetragenococcus genus.

In some specific embodiments, the present disclosure provides a product of interest comprising at least one lactic acid microbial cell and/or population of cells comprising said at least one microbial cell, or any preparation, derivative or extract thereof, or any product produced by said cells, wherein said at least one lactic acid microbial cell is characterized by the ability of growing on high salt concentration substrate/s.

In some specific embodiments, the present disclosure provides a product of interest comprising at least one lactic acid microbial cell and/or population of cells comprising said at least one microbial cell, or any preparation, derivative or extract thereof, or any product produced by said cells, wherein said at least one lactic acid microbial cell is characterized by naturally residing in the digestive tract of at least one ruminant.

In some specific embodiments, the present disclosure provides a product of interest comprising at least one lactic acid microbial cell and/or population of cells comprising said at least one microbial cell, or any preparation, derivative or extract thereof, or any product produced by said cells, wherein said at least one lactic acid microbial cell is characterized by being at least one bacterium of the Tetragenococcus genus.

In some embodiments, said product or product of interest is edible.

In some embodiments, said product is a food product, optionally, said food product is an animal and/or human food or food supplement.

In some further embodiments, said at least one organic substrate further comprises high nitrogen concentration.

In some further specific embodiments, said at least one bacterium is a non-aerobic bacterium. In certain embodiments, said at least one lactic acid microbe is naturally present in the digestive tract of at least one ruminant.

In some specific embodiments, said lactic acid microbe is at least one bacterium of the Tetragenococcus genus.

(i) said substrate comprises an organic substrate;

(ii) composed of at least about 4% to about 25% dry material; and

(iii) said substrate is a liquid substrate.

In some other embodiments, the substrate comprises an organic waste and/or industrial byproduct/s.

In some further embodiments, the substrate originates from at least one of:

(i) the food industry;

(ii) the agriculture industry; and

(iii) municipal solid waste.

In some specific embodiments, the substrate comprises waste derived from anaerobic fermenters facilities and/or biogas facilities.

In certain embodiments, said cell comprises whole cells.

In some embodiments, the product may be a whole cell product. In some further embodiments, the product or food product according to the present disclosure may comprise less than 50% whole cells. In some embodiments, the product or food product according to the present disclosure may comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% whole cells.

In yet another embodiment, said cell comprises lysed cells or any preparation thereof. In some embodiments, the product or food product according to the present disclosure may comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of lysed cells.

In some embodiments, the product or food product according to the present disclosure may comprise less than 50% intact cells. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the cells may be intact cells.

In some specific embodiments, said product is a proteinaceous product.

As used herein a “proteinaceous product” refers to any substance or material that is primarily composed of protein or contains a significant amount of protein. In some embodiments, a proteinaceous product may refer to foods, food supplements, pharmaceutical products (such as vaccines, antibodies, or recombinant proteins), biotechnological products (such as such as enzymes for industrial applications or bio-based materials) or cosmetic products.

In some embodiments, said at least one proteinaceous food product comprises at least 10% protein content or concentration. In some embodiments, a proteinaceous product may comprise at least 10% protein content or concentration (e.g. dry weight), or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or 100% protein content (e.g. dry weight).

In some embodiment, the at least one product may be a proteinaceous food product. As used herein, the term “proteinaceous food product” relates to any food item that is primarily composed of protein or contains a significant amount of protein as one of its main components. Proteins are essential macronutrients that play crucial roles in the body, including muscle building, tissue repair, enzyme production, and immune function. Proteinaceous food products come naturally from both animal and plant sources. In some embodiments, the proteinaceous food product may relate to food analogue also known as a food substitute or food replacement or food alternative. A food analogue is a product designed to mimic the taste, texture, appearance, or functionality of another food item, typically a traditional or familiar food.

In some embodiments, the food analogue may relate to “meat substitute” or “dairy substitute”.

As used herein, the term “meat substitute”, also known as “meat analog”, “imitation meat”, “meat alternative”, and the like refers to a food composition having proteins partially derived from animal-based proteins but supplemented with non-animal protein source(s) or having proteins wholly derived from non-animal source(s). In certain embodiments, the meat substitute has proteins wholly derived from non-animal source(s). Meat substitutes may be in various forms, including a patty, meatball, crumble, sausage, jerky, loaf, filet, bacon, hot dog, or nugget.

As used herein, the term “dairy substitute”, “dairy alternative” is a product designed to mimic the taste, texture, and functionality of traditional dairy products such as milk, cheese, yogurt, and butter, but made without any animal-derived ingredients. Dairy substitutes include and are not limited to non-dairy milk, non-dairy creamer, non-dairy cream, non dairy yogurt, non-dairy whipped topping, and non-dairy ice cream.

In some embodiments, the food product of the present disclosure may further comprise at least one other edible ingredient or food additive.

In some embodiments, the product comprises a mix of proteins and oligosaccharides. In some embodiments, the product may comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% oligosaccharide content or concentration (e.g. dry weight).

In some embodiments, the product in accordance with the present disclosure may comprise protein isolate from said lactic acid microbe.

In some embodiments, the product in accordance with the aspects of the present disclosure may comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or 100% protein isolate and/or protein concentrate. In some other embodiments, the product in accordance with the present disclosure may comprise a combination of protein isolate and/or whole cells from said lactic acid microbe.

In a fourth aspect, the present disclosure provides an isolated bacterium of the Tetragenococcus genus originating from the digestive tract of at least one ruminant, wherein said bacterium is characterized by the ability of growing on high salinity conditions (or in high salt concentrations).

In some embodiments, said isolated bacterium originates from the upper digestive tract of at least one ruminant. In some specific embodiments, said isolated bacterium originates from the rumen of at least one ruminant.

In some embodiments, the isolated bacterium is capable to produce lactic acid.

In some embodiments, the isolated bacterium is edible.

In some embodiments, the isolated bacterium may be useful or used for food preservation. In some embodiments, the isolated bacterium may be useful or used for flavor development. In some embodiments, the isolated bacterium may be useful or used for generating lactic acid. In some embodiments, the isolated bacterium may be useful or used as a food additive, in biodegradable plastics, pharmaceuticals, and cosmetics.

In some embodiments, said bacterium is further capable of growing in high nitrogen concentrations.

In some specific embodiments, said bacterium is closely related to Tetragenococcus halophilus and/or Tetragenococcus koreensis.

In some further specific embodiments, the 16s rRNA of said bacterium comprises a nucleic acid sequence as denoted by at least one of: SEQ ID NO: 1 and/or SEQ ID NO: 2.

In a fifth aspect, the present disclosure provides a method of waste treatment, by converting at least one substrate originating from said waste and comprising high salt concentration into microbial biomass, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product or preparation thereof from the culture, wherein said at least one microbial cell is characterized by at least one of: (a) the ability of growing on high salt concentration substrate(s); (b) naturally residing in the digestive tract of at least one ruminant and (c) being at least one bacterium of the Tetragenococcus genus.

In some specific embodiments, the present disclosure provides a method of waste treatment, by converting at least one substrate originating from said waste and comprising high salt concentration into microbial biomass, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product or preparation thereof from the culture, wherein said at least one microbial cell is characterized by the ability of growing on high salt concentration substrate(s) and naturally residing in the digestive tract of at least one ruminant.

In some specific embodiments, the present disclosure provides a method of waste treatment, by converting at least one substrate originating from said waste and comprising high salt concentration into microbial biomass, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product or preparation thereof from the culture, wherein said at least one microbial cell is characterized by the ability of growing on high salt concentration substrate(s), naturally residing in the digestive tract of at least one ruminant and being at least one bacterium of the Tetragenococcus genus.

In some specific embodiments, the present disclosure provides a method of waste treatment, by converting at least one substrate originating from said waste and comprising high salt concentration into microbial biomass, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product or preparation thereof from the culture, wherein said at least one microbial cell is characterized by the ability of growing on high salt concentration substrate(s).

In some specific embodiments, the present disclosure provides a method of waste treatment, by converting at least one substrate originating from said waste and comprising high salt concentration into microbial biomass, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product or preparation thereof from the culture, wherein said at least one microbial cell is characterized by naturally residing in the digestive tract of at least one ruminant.

In some specific embodiments, the present disclosure provides a method of waste treatment, by converting at least one substrate originating from said waste and comprising high salt concentration into microbial biomass, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbe on said at least one substrate, and/or recovering said cells and/or any product or preparation thereof from the culture, wherein said at least one microbial cell is characterized by being at least one bacterium of the Tetragenococcus genus. In some embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 1. In some further embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 2.

In some embodiments, said at least one microbe is capable of consuming said at least one substrate.

In some embodiments, said microbial biomass comprises single cell protein biomass.

In some specific embodiments, said lactic acid microbe comprises the isolated bacterium of the Tetragenococcus genus as defined above.

All the definitions and embodiments mentioned above are applicable for the presently defined aspect of the present disclosure.

(a) culturing at least one lactic acid microbe or a population of cells comprising said microbe on at least one organic substrate comprising high salt concentration;

(b) converting at least a portion of the carbon content in said substrate into microbial biomass (in some embodiments, rather than releasing it as CO₂); and

(c) quantifying the reduction in CO₂ emissions resulting from said conversion to obtain carbon credits, wherein said at least one lactic acid microbe is characterized by at least one of: (a) the ability of growing on high salt concentration substrate(s); (b) naturally residing in the digestive tract of at least one ruminant and (c) being at least one bacterium of the Tetragenococcus genus.

In a specific embodiment, the present disclosure provides a method for reducing carbon emissions and/or generating carbon credits, the method comprising:

(c) quantifying the reduction in CO₂ emissions resulting from said conversion to obtain carbon credits, wherein said at least one lactic acid microbe is characterized by the ability of growing on high salt concentration substrate(s) and naturally resides in the digestive tract of at least one ruminant.

In a further specific embodiment, the present disclosure provides a method for reducing carbon emissions and/or generating carbon credits, the method comprising:

(c) quantifying the reduction in CO₂ emissions resulting from said conversion to obtain carbon credits, wherein said at least one lactic acid microbe is characterized by the ability of growing on high salt concentration substrate(s), naturally resides in the digestive tract of at least one ruminant and is at least one bacterium of the Tetragenococcus genus.

In some other embodiments, the present disclosure provides a method for reducing carbon emissions and/or generating carbon credits, the method comprising:

(b) converting at least a portion of the carbon content in said substrate into microbial biomass (in some embodiments, rather than releasing it as CO₂); and (c) quantifying the reduction in CO₂ emissions resulting from said conversion to obtain carbon credits, wherein said at least one lactic acid microbe is characterized by the ability of growing on high salt concentration substrate.

(c) quantifying the reduction in CO₂ emissions resulting from said conversion to obtain carbon credits, wherein said at least one lactic acid microbe is characterized by naturally residing in the digestive tract of at least one ruminant.

(c) quantifying the reduction in CO₂ emissions resulting from said conversion to obtain carbon credits, wherein said at least one lactic acid microbe is characterized by being at least one bacterium of the Tetragenococcus genus. In some embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 1. In some further embodiments, the 16s nucleic acid sequence of said bacteria may comprise a nucleic acid sequence as denoted by SEQ ID NO: 2.

In some embodiments, the method further comprises recovering said microbial biomass or any derivative, preparation, or product thereof. In some embodiments, the method of the present disclosure enables the reduction of carbon emissions and the generation of carbon credits by converting organic waste into microbial biomass, thereby preventing CO₂ release that would otherwise result from natural decomposition or alternative treatment methods.

In some embodiments, the microbial conversion process assimilates up to 50% of the available carbon from organic waste into microbial biomass rather than being emitted as CO₂. In some embodiments, the efficiency of carbon assimilation varies depending on the type of waste, microbial strain used, and environmental conditions, ranging from about 30% to about 60% under optimized conditions.

In some embodiments, the carbon savings achieved by this method depend on the composition of the organic waste. For instance, in some embodiments, waste streams with higher carbohydrate and protein content (e.g. , food processing residues, dairy byproducts) exhibit a higher carbon assimilation efficiency (-45-60%), while waste streams with a higher lignocellulosic fraction (e.g., agricultural waste, brewer’s spent grain) demonstrate a lower but still significant efficiency (-30-45%). In some embodiments, the method further comprises a pre-treatment step to improve carbon bioavailability and increase microbial assimilation rates.

In some embodiments, for every ton of organic waste processed, the method prevents between about 0.2 and about 0.5 tons of CO₂ emissions, depending on the specific carbon content of the substrate. In some embodiments, the estimated carbon credit value generated per ton of organic waste treated ranges from $10 to $25, based on prevailing carbon credit market prices. In some embodiments, the carbon credit generation potential scales proportionally with the amount of waste processed, such that an industrial-scale facility processing 100,000 tons of waste per year may generate carbon savings of up to 50,000 tons of CO₂ equivalent, corresponding to a potential revenue of $500,000 to $1.25 million per year from carbon credit trading.

In some embodiments, the microbial biomass produced in the process serves as a valuable byproduct for further commercialization, such as single-cell protein for feed applications or biodegradable polymers, providing additional economic and environmental benefits. In some embodiments, the carbon credit certification process involves life cycle assessment (LCA) methodologies and adherence to recognized standards, such as the Verified Carbon Standard (VCS) or the Gold Standard for Global Goals. In some embodiments, the method is compared to conventional waste management strategies such as composting or anaerobic digestion, which result in higher CO₂ or methane emissions due to incomplete carbon retention. In some embodiments, microbial biomass conversion is shown to have a 30-50% lower carbon footprint compared to these traditional waste treatment methods.

All the definitions and embodiments mentioned in the present disclosure are applicable for each and every suitable aspects of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. In some embodiments, the term "about" refers to ± 10 %.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of’ “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Throughout this specification and the Examples and claims which follow, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Specifically, it should understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures. More specifically, the terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of means “including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

EXAMPLES

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way.

Experimental procedures

MH+ growth medium

Mueller Hinton broth (Merck 10192) 7.35 g, Peptone (Merck 91249) 3.5g, yeast extract (Y 1625) 1.05G, NaCl 1.75g (for 0.5% NaCl in the medium) or 17.5g (for 5% NaCl in the medium) and few drops of resazurin were added to DW to a final volume of 350ml. The mix was boiled for few minutes, followed by cooling under N2 stream to remain anaerobic conditions. After the medium was cooled down to room temperature, NaHCO3 1.75g and cysteine-HCl 0.175g were added and the medium was autoclaved. Sugar mix (glucose + lactose mix or glucose or lactose at final concentration of 0.5% in the medium) was anaerobically added to the medium, following autoclave and cooling.

Gas Chromatography analysis

In each experiment, headspace gas was analyzed by sampling and injecting aliquots of 2 ml into a GC system (HP-5890 series II) equipped with Thermal Conductivity Detector (TCD) for carbon dioxide (CO2) measurements. Prior for the beginning of the experiment, calibration for the CO2 measurements was performed using pure 100% CO2 gas. Calibration samples were manually injected into the GC using a gas-tight syringe and standard curves were obtained from six different concentrations (0.1%, 0.5%, 2.5%, 5%, 10%, 20%) which were measured in triplicate. Each point on the standard curve was calculated as the average of those triplicates. The aliquots were injected into a ShinCarbon ST 100/120 packed column (RESTEK cat. C31405-03), with nitrogen as a carrier gas set to a flow rate of 30 ml/min and initial oven temperature of 180°C. The temperatures at the inlet and the detector were set at 120°C and 200°C, respectively.

Dry matter. Crude protein and amino acids tests

Dry matter tests were done according to the AOAC981.10 method (AO AC International). Crude protein tests (Kjeldahl method) were done as previously described in Latimer G.W et al. (Official Methods of Analysis of AOAC International. AOAC International; Gaithersburg, MD, USA: 2016). Hydrolysate tests were done according to AOAC 994.12 method of amino acid analyzer method (AOAC International).

16s and WGS analyses

16S rRNA sequencing of the X29 aNnbdiX X30 bacterNiab wiXas done by Hylabs LTD using Sanger. NbXiX29 and XN3b0iX Whole Genome Sequencing (WGS) (Hylabs) was performed according to the Illumina ‘TruSeq DNA PCR-FREE Sample Preparation Guide’ low sample (LS) protocol. Assembly of sequenced reads was performed by Kbase platform using IDBA assembly tool. Bioinformatics analysis was done by Rapid Annotation Using Subsystem Technology’ (RAST) platform (Aziz et al., 2008).

EXAMPLE 1

Isolation of two bacterial strains from the Tetragenococcus genus Two bacterial strains from the Tetragenococcus genus were isolated from their host (ovine) on MH+ agar growth medium. They were sequenced and the 16s rRNA analysis suggested that both of them are novel strains, named:

- NbiXX 29 - Tetragenococcus halophilus - R (Rumen)

- NbiXX 30 - Tetragenococcus koreensis - R (Rumen)

16s rRNA sequencing was carried out using Sanger and Whole Genome Sequencing (WGS) and showed the following results:

- NbiXX 29 - Tetragenococcus halophilus - R:

99.03% identity (1508/1523) with Tetragenococcus halophilus subsp. halophilus strain IAM 1676 16S ribosomal RNA, partial sequence NR_122102.1 or 98.84% identity (1535/1553) with Tetragenococcus halophilus subsp. halophilus strain DSM20339 NR_114788.2.

- NbiXX 30 - Tetragenococcus koreensis — R

99.8% identity (1271/1274 bp) to Tetragenococcus koreensis strain KCTC 3924 GenBank: CP027786.1

16S rRNA, small subunit ribosomal RNA of Tetragenococcus koreensis _R as denoted by SEQ ID NO: 2

EXAMPLE 2

Growth of bacterial isolates under different NaCl concentrations NbiX X29 growth was evaluated under two NaCl concentrations (0.5% and 5% NaCl) supplemented to the growth medium. Figure 1 demonstrates that addition of 5% NaCl stimulates NbiX29 growth up to around OD of 1.78, while 0.5% NaCl allows the growth of up to OD of 1.13, after 96h. NbiX29 had also another interesting feature regarding pigmentation under different salt concentration in the growth medium. Figure 2 shows NbiX29 concentrated samples (final concentration X25) following growth in MH+ medium supplemented with 0.5% NaCl and 5% NaCl.

While concentrated NbiX 29 that was grown under 0.5% NaCl demonstrated a light-yellow pigmentation, under 5% NaCl it was changed to brown- grey. Furthermore, the 5% pellets were weaker that the 0.5%' but much more sticker.

EXAMPLE 3

Growth of bacterial isolates in sludge originating from a mesophilic anaerobic digester, fed with agriculture waste NbiX 29 and NbiX30 were tested for their ability to grow in the challenging environment of sludge originating from a mesophilic AD (Anaerobic Digester), fed with agriculture waste (cow manure and dairy waste). Following sampling, the sludge samples were anaerobically processed - centrifuged and the liquid phase was separated, autoclaved and used as a growth medium for NbiX29 and NbiXv30. The AD based growth medium was chemically tested for NaCl and total nitrogen concentrations in order to evaluate its properties and potential use as novel source for animal feed production. The results demonstrated that the AD based growth medium contained relatively high values of salts (2-3%) and total nitrogen (5-8%) concentrations which can support the growth of specific tnicrobial agents. Figure 3 demonstrates ATLY29 and MnX30 growth rate as a function of the CO?. production, during 10 days growth experiment. Both strains showed a good growth ability, as A’biX 29 seemed to be more suitable to AD sludge with a shorter lag phase and a higher CO2 production that suggests a better growth.

EXAMPLE 4

Growth of bacterial isolates in dairy waste streams

EXAMPLE 5

Growth of bacterial isolates in BSG stream NbiX X29 was tested for its ability to grow in BSG (Brewer’s Spent Grain) based medium. This medium comprised of MH+ medium supplemented with 5% NaCl and 10% autoclaved BSG (w/v). BSG constitutes environmental burden as it can’t be disposed to the sewage or applied to the field and its handling and transportation to treatment facilities are expensive and often uneconomical. However, its chemical values are very high and include, inter alia, complex carbohydrates and proteins which can be used as carbon and nitrogen sources for microbial growth. Figure 5A-5B demonstrates X29 gNrboiwXth as a function of the CO₂ production, following a 72h days growth experiment. X29Nb shiXowed high capability to grow in BSG based medium as compared to growth in the control - minimal medium (> 40% of total CO₂ production compared to the cumulative CO₂ production of BSG alone and X29 alone). NbiX

EXAMPLE 6

Evaluation of protein content of the bacterial isolates under different salt concentrations

EXAMPLE 7

Evaluation of the amino acid profile of the bacterial isolates under different salt concentrations

EXAMPLE §

Characterization of growth of the bacterial isolates under different growth conditions

EXAMPLE 9

Whole Genomic Analysis

A genomic comparison was performed between M)iX29 and die four most close related T. halophilus strains (most similar to ZVbiX29 according to their 16s rRNA sequence), which their genomes are deposited in the NCBI database. Table 4 shows the 4 T. halophilus strains and their genomic properties.

The coding sequences were further analyzed and a function was attributed to most of the coding sequences (based on homology of known sequences). However, a function could not be attributed to about 19% of the coding regions, simply based on homology to known sequences and require further analysis.

Furthermore, the sequences of the coding regions of AbiX29 were compared to the most closely related sequences in the 4 T. halophilus strains mentioned above and a similarity percentage was determined for each one of these coding: sequences. The overall average similarity

In addition, a genomic comparison was performed between NbiX 30 and the three most close related T. koreensis strains (most similar to <VbiA'3O according to their 16s rRNA sequence), which their genomes are deposited in the NCB1 database. Table 6 shows the three T. koreensis strains and their genomic properties.

The coding sequences were further analyzed and a function was attributed to most of the coding sequences (based on homology of known sequences). However, a function could not be attributed to about 20% of the coding regions, simply based on homology to known sequences and require further analysis.

Furthermore, the sequences of the coding regions of AbiX30 were compared to the most closely related sequences in the three T. koreensis strains mentioned above and a similarity percentage was determined for each one of these coding sequences. The overall average similarity percentages of the coding sequences of AbiX30 in comparison with the three T. koreensis strains are as defined in Table 7 below.

EXAMPLE 10

Further isolation ami characterization of additional candidates of the Tetragenococcas genus from different rumen environments

More candidates are isolated from the Tetragenococcus genus from different rumen environments. The growth and yield production of the selected strains are then optimized.

These additional candidates are compared to the above mentioned X29 and NbiX 30 NbiX strains from the Tetragenococcus genus and to other non-rumen Tetragenococcus genus (in vitro) in terms of growth phenotype, crude protein content and amino acid profile (under different saline conditions) in order to analyze their potential to become SCP or other metabolites producers.

Fermentation assays are run in large scale to produce a few types of preliminary products.

EXAMPLE 11

In vivo safety evaluation

Objective

The objective of this trial was to evaluate the safety of live Ahn¥ 29 for animal use as a feed.

Study design

A total of 22 Balb/c male mice were utilized and divided into five groups of 2 (internal control) or 5 animals per group (vehicle and each dose). The number of groups and the total number of animals were based on previous studies demonstrating that this is the minimum number of animals required to obtain indicative/significant information. Three doses of live ATL¥ 29 suspended in anaerobic PBS (phosphate buffer saline) solution: 10⁷, 10⁸. 10⁵ cfu and a control sample (vehicle - PBS only), were administered orally (100μl) to each animal.

Trial lasted eight (8) consecutive days, during which the following tests examined: a. Mortality & morbidity - dally. b. Body weight monitoring - upon arrival, before Bacteria dosing, and on Days 2, 4, 6. and 8. c. Clinical signs - before Bacteria dosing, and on Days 2, 4, 6, and 8. cl. Stool collection - stool samples were collected on Days 2. 4, and 8. The stool samples were collected into a separate tube per animal, snap frozen, and labeled to reflect the mouse number and study day (4 feces samples were collected per animal per each collection point). All collected stools were kept frozen at ~80°C until further analysis. Termination - before termination (Day 8), animals were weighed, and clinical signs were recorded. The animals bled terminally, and blood was collected for serum preparation or collected as whole blood and sent for chemistry and CBC analysis, respectively. The GI tract was extracted. The large intestine (from rectum to cecum) was cut longitudinally, and each piece was snap frozen. In addition, the small intestine (the part between the stomach and the cecum) was cut and snap frozen as well.

Results a. Morbidity and Mortality Observation

No animal was found in morbid condition or died before termination day. b. Body Weight

No statistical differences were seen between Control group (Vehicle) and any of the bacteria administrated animals -- 10⁷, 10⁸’ 10⁹ cfu/animal (Figure 8). c. Clinical Observations

No animal showed adverse clinical signs or organ abnormalities during the study. d. Clinical Hematology

As seen in Table 8 and Table 9 no major differences were seen between Control animals (vehicle) and any of the bacteria administrated animals - 10⁷, 10⁸’ 10⁹ cfu/animal in their chemistry or hematology parameters.

Table 8: Complete Blood Count (CBC) parameters comparison between the different groups and the control. Statistical analysis (t -test ) of the different blood variables between the different bacteria groups at Day 8.

Table 9: Hematology parameters comparison between the different groups and the control.

Statistical analysis (t-test) of the different hematology parameters between the different bacteria groups at Day 8.

AbiX 29 was tested for its safety in Balb/c male mice, in a single oral administration with 3 different concentrations. Observations and tests included body weight and clinical signs follow up, gross pathology and clinical chemistry and hematology at termination (7 days post administration). Based on the study conducted, no visible changes were observed in any of the treated mice when compared to Control mice, thus it can be concluded that /VbiX 29 is safe for the mice under these conditions.

EXAMPLE 12

Carbon Credits

A carbon credit is a tradable permit that represents one ton of CO₂ (or equivalent greenhouse gases) reduced, avoided, or removed from the atmosphere. Companies and governments buy these credits to offset their emissions and meet sustainability goals. As for 2025, the price of carbon credits range between $20-$ 100 per ton of CO₂ eq. (Wood Mackenzie report - carbon market 2025 outlook - https://www.woodmac.com/news/opinion/carbon-markets-2025- outlook/).

/VacrobiX novel microbial strains enable the conversion of the carbon fraction in the waste streams into valuable biomass, saving and reducing CO₂ emissions that would otherwise be generated if organic wastes were disposed of in landfills or not optimally treated. > CO₂ savings example:

Assumptions:

• 1 ton of organic waste contains ~10%-40% dry matter with -50% carbon (C), on average (Schnurer et al. 2009. U2009:03 Swedish Gas Centre Report 207; Glowacki et al. 2022. Materials 15(10): 3703; Ahmad et al. 2019. Trends in Food Science & Technology 88: 361-72).

• Assuming carbon assimilation of -50% into microbial biomass instead of releasing it as CO₂ to the air (Shiloach and Fass. 2005. Biotechnology Advances 23(5): 345-57).

• If naturally decomposed, -1.83 tons of CO₂ would be released per ton of organic waste. Explanations: assuming that the carbon in the waste is fully oxidized to CO₂, and considering that carbon has an atomic mass of 12 atomic mass units (amu) and oxygen has an atomic mass of 16 amu, the molecular mass of CO₂ (CO₂ = 12 + 16x2) is 44 amu. Therefore, each ton of carbon can produce 44/12 (approximately 3.67) tons of CO₂.

Fresh waste example:

• 1 ton of fresh waste contains 25% of dry matter with 50% carbon.

Therefore, this waste can produce 0.25 x 0.5 x 3.67 = 0.46 tons of CO₂ upon complete decomposition.

• CO₂ prevented = 0.46 tons x 50% (carbon assimilation in biomass) = 0.23 tons CO₂ saved per ton of waste.

• Carbon credit value (-$50 per ton CO₂ in compliance markets): o Savings per ton of waste = 0.23 x $50 = $11.5

Thus, for 1,000 tons of waste treated, the CO₂ savings would be 230 tons, generating a potential $11,500 in carbon credits.

Claims

CLAIMS:

1. A method of producing microbial biomass from at least one substrate comprising high salt concentration, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbial cell on said at least one substrate, and/or recovering said cells and/or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by the ability of growing on high salt concentration substrate/s.

2. The method of claim 1, wherein said at least one substrate further comprises high nitrogen concentration.

3. The method of claim 1 or 2, wherein said at least one lactic acid microbial cell comprises at least one bacterium.

4. The method of claim 3, wherein said at least one bacterium is a non-aerobic bacteria.

5. The method of any one of claims 1 to 4, wherein said at least one lactic acid microbial cell naturally resides in the digestive tract of at least one ruminant.

6. The method of any one of claims 1 to 5, wherein said lactic acid microbial cell is at least one bacterium of the Tetragenococcus genus.

7. The method of any one of claims 1 to 6, wherein at least one of:

(i) said substrate comprises an organic substrate;

(iii) said substrate is a liquid substrate.

8. The method of any one of claims 1 to 7, wherein the substrate comprises an organic substrate, said organic substrate comprises an organic waste and/or industrial byproduct/s.

9. The method of any one of claims 1 to 8, wherein the substrate comprises an organic substrate, said organic substrate originates from at least one of: (i) the food industry;

(ii) the agriculture industry;

(iii) municipal solid waste.

10. The method of any one of claims 1 to 9, wherein the substrate comprises an organic substrate, said organic substrate comprises waste derived from anaerobic fermenters facilities and/or biogas facilities.

11. The method of any one of claims 1 to 10, wherein said microbial biomass comprises single cell protein biomass.

12. The method of claim 11, wherein the single cell protein biomass comprises at least 40% protein.

13. A method of producing at least one product of interest, the method comprising culturing at least one lactic acid microbial cell or any population of cells comprising said microbial cell on at least one substrate comprising high salt concentration, and recovering said at least one microbial cell, any population of cells comprising said microbial cell, or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by at least one of: (a) the ability of growing on high salt concentration substrate/s; (b) by naturally residing in the digestive tract of at least one ruminant; and (c) being at least one bacterium of the Tetragenococcus genus.

14. The method of claim 13, wherein said at least one bacterium is a non-aerobic bacteria.

15. The method of claim 13 or 14, wherein said product, derivative, extract or preparation of said cell or population of cells is at least one food product, optionally said food product is an animal and/or human food or food supplement.

16. The method of any one of claims 13 to 15, wherein said product, derivative, extract or preparation of said cell or population of cells is a proteinaceous product.

17. The method of any one of claims 13 to 16, wherein said product, derivative, extract or preparation of said cell or population of cells comprises single cell protein.

18. The method of any one of claims 13 to 17, wherein said at least one substrate further comprises high nitrogen concentration.

19. The method of any one of claims 13 to 18, wherein at least one of:

(i) said substrate comprises an organic substrate;

(iii) said substrate is a liquid substrate.

20. The method of claim 19, wherein the substrate comprises an organic substrate, said organic substrate comprises an organic waste and/or industrial byproduct/s.

21. The method of any one of claims 19 to 20, wherein the substrate comprises an organic substrate, said organic substrate originates from at least one of:

(i) the food industry;

(ii) the agriculture industry; and

(iii) municipal solid waste.

22. The method of any one of claims 19 to 21, wherein the substrate comprises an organic substrate, said organic substrate comprises waste derived from anaerobic fermenters facilities and/or biogas facilities.

23. The method of any one of claims 13 to 22, wherein the cells recovered comprise whole cells.

24. The method of any one of claims 13 to 22, wherein the cells recovered comprise lysed cells or any preparation thereof.

25. The method of any one of claims 13 to 24, further comprising purifying proteins from said lactic acid microbial cell thereby obtaining protein isolate and/or protein concentrate.

26. The method of claim 25, further comprising combining said protein isolate and whole cells from said lactic acid microbial cell .

27. A product of interest comprising at least one lactic acid microbial cell and/or population of cells comprising said at least one microbial cell, or any preparation, derivative or extract thereof, or any product produced by said cells, wherein said at least one lactic acid microbial cell is characterized by at least one of: (a) the ability of growing on high salt concentration substrate/s; (b) naturally residing in the digestive tract of at least one ruminant; and (c) being at least one bacterium of the Tetragenococcus genus.

28. The product of claim 27, wherein said at least one bacterium is a non-aerobic bacterium.

29. The product of any one of claims 27 and 28, wherein said product is a food product, optionally, said food product is an animal and/or human food or food supplement.

30. The product of any one of claims 27 to 29, wherein said product is a proteinaceous product.

31. The product of any one of claims 27 to 30, wherein said product, derivative, extract or preparation of said cell or population of cells comprises single cell protein.

32. The product of claim 27 or 31, wherein said at least one substrate further comprises high nitrogen concentration.

33. The product of any one of claims 27 to 32, wherein at least one of:

(i) said substrate comprises an organic substrate;

(ii) composed of at least about 4% to about 25% dry material; and

(iii) said substrate is a liquid substrate.

34. The product of claim 33, wherein the substrate comprises an organic substrate, said substrate comprises an organic waste and/or industrial byproduct/s.

35. The product of any one of claims 27 to 34, wherein the substrate originates from at least one of: (i) the food industry;

(ii) the agriculture industry; and

(iii) municipal solid waste.

36. The product of any one of claims 27 to 35, wherein the substrate comprises waste derived from anaerobic fermenters facilities and/or biogas facilities.

37. The product of any one of claims 27 to 36, wherein said cell comprises whole cells.

38. The product of any one of claims 27 to 36, wherein said cell comprises lysed cells or any preparation thereof.

39. The product of any one of claims 27 to 38, comprising protein isolate from said lactic acid microbial cell.

40. The product of any one of claims 27 to 99, comprising a combination of protein isolate and whole cells from said lactic acid microbial cell.

41. An isolated bacterium of the Tetragenococcus genus originating from the digestive tract of at least one ruminant, wherein said bacterium is characterized by the ability of growing on high salinity conditions.

42. The isolated bacterium of claim 41, wherein said bacterium is further capable of growing in high nitrogen concentrations.

43. The isolated bacterium of claim 41 or 42, said bacterium is closely related to Tetragenococcus halophilus and/or Tetragenococcus koreensis.

44. The isolated bacterium of any one of claims 41 to 43, wherein the 16s rRNA of said bacterium comprises a nucleic acid sequence as denoted by at least one of: SEQ ID NO: 1 and/or SEQ ID NO: 2.

45. A method of waste treatment, by converting at least one substrate originating from said waste and comprising high salt concentration into microbial biomass, the method comprising culturing at least one lactic acid microbial cell or a population of cells comprising said microbial cell on said at least one substrate, and/or recovering said cells and/or any product, derivative, extract or preparation thereof from the culture, wherein said at least one microbial cell is characterized by at least one of: (a) the ability of growing on high salt concentration substrate(s); (b) naturally residing in the digestive tract of at least one ruminant and (c) being at least one bacterium of the Tetragenococcus genus.

46. The method of claim 45, wherein said microbial biomass comprises single cell protein biomass.

47. The method according to claim 45 or 46, wherein said lactic acid microbial cell comprises the isolated bacterium of the Tetragenococcus genus as defined in in any one of claims 41-44.

48. A method for reducing carbon emissions and/or generating carbon credits, the method comprising:

49. The method of claim 48, further comprising recovering said microbial biomass or any derivative, preparation, or product thereof.