Lactobacillus salivarius with high antibacterial activity and application thereof

Abstract

The present application relates to a lactobacillus salivarius with high antibacterial activity and application thereof. Specifically, the present application provides the lactobacillus salivarius, preparation and application thereof. The lactobacillus salivarius of the present application has a preservation number of CGMCC No. 33044. The lactobacillus salivarius of the present application is resistant to high temperature and has antibacterial activity, and thus can be used for fermented feed, and can significantly improve the quality of the fermented feed.

Description

Technical Field

[0001] This invention belongs to the field of biological feed, specifically relating to a strain of Lactobacillus salivarius with high antibacterial activity and its application. Background Technology

[0002] Food safety issues and pathogenic bacterial infections occur frequently. Campylobacter jejuni is a common zoonotic pathogen. Due to widespread contamination of water sources and agricultural products, the likelihood of infection in humans and animals has greatly increased, leading to chronic digestive symptoms such as irritable bowel syndrome and inflammatory bowel disease. Antibiotics are currently the main means of prevention and treatment; however, overuse can cause secondary contamination, induce antibiotic resistance in pathogens, and disrupt the host's intestinal microecological balance. Therefore, exploring a safe, effective, and non-toxic probiotic that utilizes antagonistic effects against pathogens to prevent and treat pathogenic infections is of great significance.

[0003] Lactic acid bacteria are an important source of probiotics and constitute the natural gut microbiota of humans and most animals. During their metabolism, they can produce active substances such as organic acids, bacteriocins, extracellular polysaccharides, and hydrogen peroxide, which can inhibit the growth of pathogenic and putrefactive microorganisms. Due to their functions of antagonizing pathogenic bacteria, enhancing immune function, strengthening the intestinal barrier, and balancing gut microbiota, they have good application prospects and are expected to become green, safe, stable, and economical biological antibacterial agents to reduce or even replace the use of antibiotics.

[0004] This study screened a strain of Lactobacillus salivarius from the intestines of antibiotic-free wild boars. The antimicrobial peptides produced by fermentation can effectively inhibit the growth of Campylobacter jejuni. It is safe and non-toxic, has strong antibacterial properties and broad-spectrum antimicrobial activity, and is suitable for the fermentation of various meal products. Summary of the Invention

[0005] A strain of *Lactobacillus salivarius* screened from the intestines of antibiotic-free wild boar exhibits strong inhibitory effects against pathogenic bacteria such as *Campylobacter jejuni*. Furthermore, it can be used to prepare fermented feed samples, which also show strong inhibitory effects against *Campylobacter jejuni*. The resulting fermented rice bran meal, when fed to medium and large-sized pigs, improves feed conversion ratio, significantly reduces fecal volume, and also significantly reduces nitrogen and phosphorus content and ammonia emissions in the feces, thus reducing environmental pollution.

[0006] The first aspect of this invention provides a Lactobacillus salivarius with accession number CGMCC No. 33044.

[0007] The second aspect of the present invention provides a culture of Lactobacillus salivarius with accession number CGMCC No. 33044.

[0008] In one or more embodiments, the culture further comprises a culture medium.

[0009] In one or more embodiments, the culture medium is MRS culture medium.

[0010] In one or more embodiments, the culture medium contains peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, triammonium citrate, sodium acetate, glucose, Tween 80, magnesium sulfate, and manganese sulfate.

[0011] In one or more embodiments, the culture medium contains 5-15 g / L peptone, 2-10 g / L beef extract, 3-8 g / L yeast extract, 1-5 g / L dipotassium hydrogen phosphate, 1-5 g / L triammonium citrate, 3-8 g / L sodium acetate, 1-8 g / L glucose, 0.5-2 mL / L Tween 80, 0.1-0.5 g / L magnesium sulfate, and 0.01-0.2 g / L manganese sulfate.

[0012] A third aspect of the present invention provides a formulation containing Lactobacillus salivarius with accession number CGMCC No. 33044.

[0013] In one or more embodiments, the formulation is a microbial formulation.

[0014] The fourth aspect of the present invention provides a fermentation raw material containing Lactobacillus salivarius with accession number CGMCC No. 33044.

[0015] In one or more embodiments, the fermentation feedstock further comprises meal, enzymes, and optionally water.

[0016] In one or more embodiments, the meal is selected from one or more of soybean meal, rapeseed meal, cottonseed meal, peanut meal, sunflower meal, sesame meal, flaxseed meal, rice bran meal, tea meal, flaxseed meal, safflower meal, palm meal, palm kernel meal, coconut meal, chili meal, olive meal, corn distillers grains with solubles (DDGS), corn gluten meal, and beet meal. In one or more embodiments, the meal is one or more of soybean meal, rice bran meal, wheat bran meal, and palm meal.

[0017] In one or more embodiments, the enzyme is selected from one or more of proteases, pectinases, xylanases, α-galactosidases, and cellulases.

[0018] In one or more embodiments, the total amount of enzyme added is more than 100U per gram of meal, such as 100-1000U, 500-2000U or 100-500U.

[0019] In one or more embodiments, the water content is 20-60% based on the total mass of the fermentation feedstock.

[0020] In one or more embodiments, the water content is 20-40%, 30-40%, 35-50%, or 35-40% based on the total mass of the fermentation feedstock.

[0021] In one or more embodiments, 0.1-10 mL of *Lactobacillus salivarius* bacterial suspension is added per 100 g of meal. In one or more embodiments, the OD600 nm absorbance of the *Lactobacillus salivarius* bacterial suspension is 0-10 or 7-10. In one or more embodiments, the pH of the *Lactobacillus salivarius* bacterial suspension is 4-6 or 4-5. In one or more embodiments, the bacterial activity of the *Lactobacillus salivarius* bacterial suspension at 40-45°C is 0-1.0.

[0022] A fifth aspect of the present invention provides a fermentation composition, characterized in that the fermentation composition is obtained by fermentation of the fermentation raw materials described in any embodiment of the fourth aspect of the present invention.

[0023] In one or more embodiments, the fermentation temperature is 37-50°C, for example 37-45°C or 40-50°C.

[0024] In one or more embodiments, the fermentation time is 24-96 hours, for example 24-72 hours or 48-72 hours.

[0025] In one or more embodiments, after fermentation, a drying and pulverizing step is also included.

[0026] In one or more embodiments, the drying temperature is 60-70°C or 65-80°C, and preferably, the drying time is 5-10 hours or 6-12 hours.

[0027] In one or more embodiments, the fermentation composition is pulverized to a fineness of 40 mesh or higher.

[0028] In one or more embodiments, the fermentation composition is pulverized to 60 mesh or 80 mesh.

[0029] The sixth aspect of the present invention provides an alcohol extract of a fermentation composition, characterized in that the alcohol extract is prepared by the following method: thoroughly mixing the fermentation composition with an alcohol solution and extracting to obtain an alcohol extract of the fermentation composition; wherein the fermentation composition is as described in any embodiment of the fifth aspect of the present invention.

[0030] In one or more embodiments, the alcohol solution is an ethanol solution, preferably with a concentration of 70-90% or 80%-90%.

[0031] In one or more embodiments, the extraction temperature is 40-45°C.

[0032] In one or more embodiments, oscillation mixing is employed. In one or more embodiments, the oscillation rate is 100-150 rpm or 150-200 rpm, and / or the oscillation time is 3-5 hours or 2-3 hours.

[0033] In one or more embodiments, after extraction, optionally, a step of concentration is included to obtain an alcoholic extract of the fermentation composition.

[0034] The seventh aspect of the present invention provides a feed containing an alcohol extract of the fermentation composition described in any embodiment of the fifth aspect of the present invention or the fermentation composition described in any embodiment of the sixth aspect of the present invention.

[0035] In one or more embodiments, the fermentation composition is present in the feed at a concentration of 5-10% or 5-8%.

[0036] The eighth aspect of the present invention provides a method for increasing the reducing sugar content, metabolizable energy, digestible energy and / or organic acid content of a fermentation composition, the method comprising the step of obtaining the fermentation composition by fermentation using Lactobacillus salivarius as described in any embodiment of the first aspect of the present invention, the preparation as described in any embodiment of the third aspect of the present invention and / or the fermentation raw material as described in any embodiment of the fourth aspect of the present invention.

[0037] The ninth aspect of the present invention provides the use of Lactobacillus salivarius or microbial preparations containing Lactobacillus salivarius as described in any embodiment of the first aspect of the present invention, fermentation raw materials as described in any embodiment of the fourth aspect of the present invention, and / or fermentation compositions as described in any embodiment of the fifth aspect of the present invention in the preparation of feed.

[0038] In one or more embodiments, the application includes one or more of the following aspects: increasing the reducing sugar content of feed, increasing the metabolizable energy of feed, increasing the digestible energy of feed, increasing the organic acid content of feed, promoting the digestion and absorption of livestock, improving the feed conversion rate of livestock, improving the daily weight gain of livestock, reducing the feed conversion ratio of livestock, reducing the amount of livestock manure excreted, reducing the nitrogen content in livestock manure, reducing the phosphorus content in livestock manure, and reducing the ammonia emissions from livestock manure. Attached Figure Description

[0039] Figure 1 The growth curves and thermostable activity of *Lactobacillus salivarius* are shown, including OD value, pH value, and bacterial activity. Detailed Implementation

[0040] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0041] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0042] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0043] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0044] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0045] In this article, the sum of the percentages of all components in the composition is 100%.

[0046] In this article, "medium-sized and large pigs" refers to pigs that are in a growth stage between piglets and adult pigs during the pig farming process. Generally, medium-sized and large pigs refer to pigs that are 105-120 days old and weigh 30-60 kg.

[0047] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0048] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0049] The inventors have discovered a highly antibacterial strain of *Lactobacillus salivarius*, which exhibits a strong inhibitory effect on pathogenic bacteria such as *Campylobacter jejuni*. This strain can be used to prepare fermented feed samples, and the resulting fermented feed samples also show a strong inhibitory effect on *Campylobacter jejuni*. The fermented rice bran meal obtained from this strain, when fed to medium and large-sized pigs, can improve feed conversion ratio, significantly reduce fecal volume, nitrogen and phosphorus content in feces, and ammonia emissions, thereby reducing environmental pollution. This invention thus completes the present invention.

[0050] Heat-resistant and antibacterial Lactobacillus salivarius

[0051] This article first provides a *Lactobacillus salivarius*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33044. In an exemplary embodiment, the 16S RNA sequence of the *Lactobacillus salivarius* described herein is shown in SEQ ID NO: 1.

[0052] Microbial strains are generally preserved in the form of inoculum (such as powder or liquid) and usually require activation before use. Depending on the strain type and preservation method, a suitable activation method can be selected. Conventional methods can be used to activate *Lactobacillus salivarius*, such as using MRS medium. A typical MRS medium includes peptone, yeast, sodium acetate, Tween 80, magnesium sulfate heptahydrate, beef extract, glucose, diammonium citrate, potassium dihydrogen phosphate, manganese sulfate heptahydrate, and water. For solid MRS medium, an appropriate amount of agar powder can be added to this formula. The proportions of these ingredients can be adjusted according to actual needs. Activation conditions can be conventional, such as incubation at 25-40℃ or 35-40℃ for 10-24 hours or 15-36 hours. Single colonies of the bacterial strain can be directly transferred to a suitable fresh solid culture medium (such as MRS medium) and allowed to grow and multiply for a period of time (e.g., 2-5 days). Then, they can be inoculated into a liquid culture medium (such as MRS medium). The inoculation amount can be selected according to actual needs; for example, two to three loops can be inoculated into 10-50 mL or 15-30 mL of liquid culture medium. The culture is then activated at 30-40°C or 35-40°C for 10-20 hours or 15-20 hours to obtain an activated bacterial solution. In some embodiments, the OD600nm absorbance of the activated Lactobacillus salivarius solution is 0-10 or 7-10. In some embodiments, the pH of the activated Lactobacillus salivarius solution is 4-6 or 4-5. In some implementations, the activated bacterial culture of *Lactobacillus salivarius* has a bacterial activity of 0-1.0 at 40-45°C, or 0.5-1.1 at 25-30°C, or 0.9-1.1 at 30-40°C, and a bacterial activity greater than 0 and less than 0.1 at 45-50°C.

[0053] In some embodiments, this document also provides a culture of *Lactobacillus salivarius* with accession number CGMCC No. 33044. This culture contains *Lactobacillus salivarius* as described herein and a culture medium. The culture medium can be any commonly used culture medium for culturing *Lactobacillus salivarius* known in the art, including media used for preparing seed culture of *Lactobacillus salivarius*, and media used for fermentation using *Lactobacillus salivarius*.

[0054] In some embodiments, the culture medium is MRS medium. More preferably, the medium contains peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, triammonium citrate, sodium acetate, glucose, Tween 80, magnesium sulfate, and manganese sulfate. In some embodiments, the medium contains 5-15 g / L peptone, 2-10 g / L beef extract, 3-8 g / L yeast extract, 1-5 g / L dipotassium hydrogen phosphate, 1-5 g / L triammonium citrate, 3-8 g / L sodium acetate, 1-8 g / L glucose, 0.5-2 mL / L Tween 80, 0.1-0.5 g / L magnesium sulfate, and 0.01-0.2 g / L manganese sulfate. When a solid culture medium is required, an appropriate amount (e.g., 10-20 g / L) of agar powder can be added to the above medium.

[0055] In some embodiments, the culture comprises the supernatant obtained by centrifuging an activated bacterial solution.

[0056] The *Lactobacillus salivarius* strain described in this study exhibits high-temperature resistance (40-55℃ or 40-45℃) and broad-spectrum antibacterial activity, demonstrating good inhibitory effects against pathogenic bacteria, including but not limited to *Escherichia coli* CICC10413, *Salmonella typhimurium* TA100, *Staphylococcus aureus* CGMCC1.1861, and *Campylobacter jejuni* NFM100, especially *Campylobacter jejuni* NFM100. The antibacterial activity of this *Lactobacillus salivarius* strain can be detected using common methods, such as the Oxford cup method. Typically, the supernatant obtained after centrifuging the activated bacterial culture (e.g., at 8000-1000 rpm for 5-10 minutes) is added to an Oxford cup to assess its antibacterial activity.

[0057] Therefore, in some embodiments, this document also provides a formulation containing *Lactobacillus salivarius* with accession number CGMCC No. 33044. In some embodiments, the formulation is a microbial preparation. In some embodiments, the formulation is an antibacterial agent. In some embodiments, the microbial preparation is a compound microbial preparation, containing, in addition to *Lactobacillus salivarius*, other bacteria commonly used in feed fermentation, including but not limited to one or more of *Lactobacillus plantarum* (LP), *Lactobacillus casei* (LC), and *Pediococcus lactis* (PC). There are no particular limitations on the content of each bacterium in the compound microbial preparation and their ratio; generally, each bacterium may be present in the microbial compound preparation of this invention at a level higher than its conventional dosage in feed fermentation. For example, in the compound microbial preparation of this invention, the content of each bacterium is generally not less than 1 × 10⁻⁶. 8 Cells / mL, preferably not less than 1×10 9 per mL.

[0058] Fermentation raw materials, fermentation composition, and alcohol extract of the fermentation composition.

[0059] The *Lactobacillus salivarius* described in this article belongs to the lactic acid bacteria family and can be used to prepare fermentation feedstocks. Therefore, this article also provides a fermentation feedstock containing *Lactobacillus salivarius* with accession number CGMCC No. 33044.

[0060] In this document, fermentation raw materials may include commonly used plants or animals, byproducts of plant processing, and byproducts of animal processing, including various crop straw powders, leaf and weed powders, melon vine powders, fruit pomace, dried sugarcane bagasse, rice husk powder, rice bran, distiller's grains, brewer's grains, sugar residue, vinegar residue, starch residue, cassava residue, citric acid residue, soy sauce residue, monosodium glutamate residue, edible fungus residue, powder residue, tofu residue, medicinal residue, oil residue, oilseed cake, bran, cottonseed meal, moldy feed, slaughterhouse waste, swill, leftover food, chicken manure, and other waste. In an exemplary embodiment, the fermentation raw materials in this document include plants and their byproducts, such as meal. The oilseed meal referred to in this article can be any type of oilseed meal commonly used in feed in this field, usually a byproduct of oilseed or grain processing or after processing, such as oilseed cake or meal after defatting or partially defatting, including but not limited to soybean meal, rapeseed meal, cottonseed meal, peanut meal, sunflower meal, sesame meal, flaxseed meal, rice bran meal, tea meal, flaxseed meal, safflower meal, palm meal, palm kernel meal, coconut meal, chili meal, olive meal, corn distillers grains with solubles, beet meal, etc., preferably one or more of soybean meal, rice bran meal, wheat bran meal and palm meal.

[0061] Fermentation raw materials may also contain enzymes and water.

[0062] The enzymes suitable for use in this invention can be various enzymes conventionally used in the art for fermentation and enzymatic hydrolysis (such as of meal), including but not limited to one or more of proteases, pectinases, xylanases, α-galactosidases, and cellulases. In some embodiments, the invention uses proteases, such as one or more of alkaline proteases, neutral proteases, and acidic proteases. In some embodiments, the invention uses xylanases and / or proteases. Typically, the amount of enzyme used can be the conventional amount used in the fermentation of raw materials in the art, for example, more than 100 U per gram of meal (such as soybean meal or rice bran meal), such as 100-1000 U, 500-2000 U, or 100-500 U of enzyme.

[0063] The amount of water used is typically 25-85% of the raw material mass. In some embodiments, the weight ratio of raw material (such as meal) to water is not higher than 10, for example, not higher than 5, such as 1-3 or 2.5-3.0. In some embodiments, the water content is 20-60% based on the total mass of the fermentation raw material, for example, 20-40%, 30-50%, 35-50%, or 35-40%.

[0064] In this article, the fermentation feedstock contains *Lactobacillus salivarius* in the form of an inoculum, such as a liquid or powder. As mentioned earlier, the *Lactobacillus salivarius* inoculum can be activated and then added to the fermentation feedstock. The amount of activated inoculum added can be conventional, such as 0.2-10 mL per 100 g of meal, 1-10 mL per 100 g of fermentation feedstock, or 1-3 mL per 100 g of fermentation feedstock. In some embodiments, the *Lactobacillus salivarius* is in solution form.

[0065] This document also provides a method for fermenting meal, which includes the step of fermenting meal using *Lactobacillus salivarius* with accession number CGMCC No. 33044. Therefore, the method herein includes the step of fermenting a fermentation feed containing *Lactobacillus salivarius*, meal, enzymes, and water. In some embodiments, the method herein includes the step of mixing the *Lactobacillus salivarius*, meal, enzymes, and water described herein. Typically, during fermentation, the meal and enzymes are first mixed, and then the *Lactobacillus salivarius* and water described herein are added to form the fermentation composition (e.g., fermented meal).

[0066] Typically, the fermentation temperature is 37-50℃, for example, 37-45℃ or 40-50℃. The fermentation time can be 24-96 hours, for example, 24-72 hours or 48-72 hours. The fermentation environment can be a closed environment. After fermentation, a drying and pulverizing step may be included, in which the mixture is dried at 60-70℃ or 65-80℃ for 5-10 hours or 6-12 hours. The fermented composition is usually pulverized to a fineness of 40 mesh or higher, for example, 60 mesh or 80 mesh.

[0067] In some embodiments, this document also provides a fermentation composition containing meal, *Lactobacillus salivarius*, protease, and water, or obtained by fermentation of the fermentation feedstock described in any embodiment of this document. The type and amount of meal, the type and amount of protease, and the amount of water, etc., can be as described above. The fermentation composition of the present invention can be a mixture prepared for fermentation (i.e., not yet fermented) (i.e., the fermentation feedstock described herein), a mixture during fermentation, or a mixture after fermentation. Preferably, the fermentation composition described herein is prepared by the method described in any embodiment of this document.

[0068] In some embodiments, this document also provides an alcoholic extract of a fermentation composition, prepared by thoroughly mixing the fermentation composition with an alcohol solution and extracting to obtain the alcoholic extract of the fermentation composition. In some embodiments, after extraction, the solid components are further filtered off, and the resulting supernatant is the alcoholic extract of the fermentation composition. In this document, the alcoholic extract of the fermentation composition includes the solid components and the supernatant obtained after filtering off the solid components, and also includes components obtained by concentrating the supernatant.

[0069] Alcohol extraction can be performed using conventional methods, typically employing an ethanol solution with a concentration of approximately 80%. To ensure thorough and homogeneous mixing, the mixture of alcohol solution and fermentation composition can be shaken together, for example, at 40-45°C and 100-150 rpm or 150-200 rpm for 3-5 hours or 2-3 hours. Filtration to remove solid components can be done using common methods, such as centrifugation at 8000-10000 rpm or 8000-12000 rpm for 5-10 minutes or 5-8 minutes. Concentration methods are not particularly limited and can include evaporation (e.g., rotary evaporation, metal bath), drying, etc.

[0070] Feed containing fermented compositions

[0071] Feed typically includes energy sources, protein sources, mineral sources, vitamin sources, and optional additives, wherein the protein sources include fermentation compositions. Therefore, in some embodiments, this document also provides a feed containing the fermentation composition described in any embodiment herein.

[0072] In some embodiments, the fermented composition in the feed described herein comprises 5-10% or 5-8%. In some embodiments, the fermented composition in the feed described herein is one or more of fermented soybean meal, fermented rice bran meal, fermented wheat bran meal, and fermented palm meal, preferably fermented soybean meal and / or fermented rice bran meal.

[0073] The protein raw materials can be commonly used raw materials in the field that contain high levels of protein (e.g., 40-50% or 40-60%), such as one or more of animal protein, plant protein, and / or microbial protein. Animal protein can be by-products of livestock or poultry processing, including inedible carcasses, bones, blood, and plasma from livestock or poultry obtained through high-temperature cooking, defatting, drying, or pulverization, such as fish meal, meat and bone meal, and plasma protein powder. Plant protein can be plant protein extracts, by-products of oilseed crop extraction, and fermentation products of by-products of oilseed crop extraction. Preferred by-products of oilseed crop extraction include one or more of soybean meal, cottonseed meal, peanut meal, rapeseed meal, sesame meal (sesame residue), sunflower seed cake, flaxseed cake, coconut meal, walnut meal, and palm kernel meal, and more preferably one or more of soybean meal, rice bran meal, wheat bran meal, and palm kernel meal. The byproducts of oilseed crops after oil extraction may be the same as or different from those of oilseed meals. Similarly, the fermentation products of the byproducts of oilseed crop oil extraction may be the same as or different from those byproducts. Plant protein extracts include legume protein extracts, cereal protein extracts, hemp protein extracts, and algae protein extracts, preferably selected from soy protein concentrate, soy protein isolate, pea protein concentrate, pea protein isolate, broad bean protein concentrate, broad bean protein isolate, wheat gluten protein (gluten), wheat protein isolate, zein, zein, flaxseed protein, spirulina protein, etc.

[0074] The specific protein sources and amounts in the protein feed can be adjusted according to the actual feeding species. For example, if the feed is fish feed, the protein feed may include byproducts of fish processing or processing, plant protein extracts, byproducts of oilseed crop extraction, and fermented products of oilseed crop extraction byproducts. In these embodiments, the protein feed in this article includes fishmeal, soybean meal, fermented soybean meal, soybean protein concentrate, and wheat gluten. For example, if the feed is poultry feed, the protein feed may include byproducts of oilseed crop extraction and fermented products of oilseed crop extraction byproducts. In some embodiments, the content of byproducts of oilseed crop extraction in the feed, by weight of total feed, is 1-2% or 1-5%, and the content of fermented products of oilseed crop extraction byproducts is 5-8% or 3-8%. In some embodiments, the protein feed in this article includes soybean meal, fermented soybean meal, rice bran, and fermented rice bran. The content of the protein feed, by weight of total feed, can be 20-30%, 25-30%, or 23-25%.

[0075] Energy feed ingredients can be commonly used energy-providing raw materials, such as grains, grain processing by-products, and oils. Grains can be those with high carbohydrate content or containing a large amount of easily digestible and absorbable starch, including but not limited to corn, wheat, barley, sorghum, rice, and oats. Grain processing by-products include but are not limited to corn gluten meal, corn germ meal, corn bran, wheat bran, barley bran, barley germ meal, sorghum bran, sorghum germ meal, rice bran, rice husk powder, oat bran, and oat germ meal. The amount of grains added can be 50% or more, such as 60-65% or 60-70%. The amount of grain processing by-products added can be 1-5%, such as 2-5% or 3-4%. Adding an appropriate amount of oil (such as 1-3% or 1.0-1.5%) to the feed can increase the energy content of the feed and also improve its palatability. The oils and fats can be vegetable oils and / or animal oils. Vegetable oils include, but are not limited to, palm oil, soybean oil, sunflower seed oil, peanut oil, rapeseed oil, corn oil, safflower seed oil, sesame oil, rice bran oil, rice bran oil, flaxseed oil, olive oil, hazelnut oil, pumpkin seed oil, grapeseed oil, evening primrose oil, Sichuan pepper seed oil, almond oil, wheat germ oil, perilla seed oil, pecan oil, almond oil, cashew oil, macadamia nut oil, pistachio oil, palm kernel oil, and coconut oil, preferably one or more of soybean oil, corn oil, rapeseed oil, palm oil, and coconut oil. Animal oils include, but are not limited to, lard, beef tallow, mutton tallow, duck tallow, chicken tallow, fish oil, and shrimp oil, preferably one or more of fish oil, lard, mutton tallow, and beef tallow.

[0076] Mineral feed ingredients provide calcium, phosphorus, sodium, and trace elements for the animals being fed. Calcium sources are typically limestone powder (mainly calcium carbonate) and shell powder; phosphorus sources are typically dicalcium phosphate and calcium dihydrogen phosphate; sodium sources can be table salt; and trace elements mainly include iron, copper, zinc, manganese, iodine, and selenium. By total feed weight, calcium content can be 3-4%, phosphorus content can be 2-6% or 2-3%, sodium content can be 3-4%, and trace element additions are minimal. By total feed weight, calcium dihydrogen phosphate is the primary mineral feed ingredient, with a content of 2-6% or 2-3%.

[0077] Vitamin raw materials can be commonly used, such as fat-soluble vitamins A, D, E, and K, and B vitamins (such as vitamins B1, B2, B6, B12, B23, B24, B25, B26, B28, B29, B20 ... 12 (etc.) and water-soluble vitamins such as vitamin C.

[0078] The most commonly used additives are amino acid additives, such as methionine and / or lysine. They can also be enzyme preparations such as amylase, protease, and cellulase, probiotics (such as lactic acid bacteria and bifidobacteria) and prebiotics, antioxidants (such as ethoxyquinoline and butylated hydroxyanisole) or antifungal agents (such as calcium propionate and sodium diacetate), and nutritional additives (such as phospholipids, especially soybean phospholipids).

[0079] The feed described in this article can be in conventional form, but can take various forms depending on the species being fed. For example, fish feed is usually in powder or pellet form with good buoyancy or sinking properties, and is stable in water. Poultry feed is usually in larger pellets or powder form, making it easier for poultry such as chickens, ducks, and geese to peck at. Poultry feed is usually in blocks, pellets, or powder form with a mesh size of 4-10 or larger. Livestock feed is usually in larger pellets or powder form.

[0080] The feed described in this article can be prepared using conventional feeding methods. You can refer to the industry standards for animal husbandry, such as feeding two to three times a day, with the amount of feed being 3-4% of the animal's body weight.

[0081] Applications and methods

[0082] This document also provides a method for increasing the reducing sugar content, metabolizable energy, digestible energy, and / or organic acid content of a fermentation composition. The method includes the step of fermenting a feed using *Lactobacillus salivarius* and / or fermentation raw materials as described in any embodiment of this document, and then applying the feed to the animal being fed. Fermentation treatment improves the nutritional quality of the meal, making it more easily digestible and absorbable by other animals. Preferably, the animal being fed is poultry, livestock, or aquaculture animals, more preferably livestock such as pigs (including piglets, medium and large pigs, and adult pigs), cattle, and sheep.

[0083] In this article, metabolic energy refers to the energy remaining after subtracting fecal energy, urinary energy, and gas energy from the digestive tract from total energy, which is the energy in nutrients that can be utilized by a passive object.

[0084] In this article, digestible energy refers to the total energy of feed consumed by an animal minus fecal energy. Digestible energy (DE) = Total energy (GE) - Fecal energy (FE). Commonly used experimental methods include the total fecal and urine collection method, the indicator method, and the in vitro digestion test method.

[0085] In this article, organic acids refer to a class of organic compounds containing hydroxyl groups. Common organic acids include citric acid, malic acid, succinic acid, and lactic acid. Applicable testing methods include ion chromatography, acid-base titration, and HPLC. In an exemplary implementation, acid-base titration is used to detect organic acids.

[0086] This document also provides the use of Lactobacillus salivarius, fermentation feedstock, fermentation composition and / or alcohol extract of fermentation composition as described in any embodiment herein in the preparation of feed.

[0087] In some implementations, the application includes one or more of the following aspects: increasing the reducing sugar content of feed, increasing the metabolizable energy of feed, increasing the digestible energy of feed, increasing the organic acid content of feed, promoting the digestion and absorption of livestock, improving the feed conversion rate of livestock, increasing the daily weight gain of livestock, reducing the feed conversion ratio of livestock, reducing the amount of livestock manure excreted, reducing the nitrogen content in livestock manure, reducing the phosphorus content in livestock manure, and reducing the ammonia emissions from livestock manure.

[0088] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0089] Example 1: Screening and Identification of Lactic Acid Bacteria

[0090] The contents of small intestine collected from antibiotic-free pigs in Guangxi were screened for bacterial strains, and a strain of Lactobacillus salivarius W was isolated. It was smooth, pillow-shaped, milky white, opaque, smooth, and round, and Gram-positive.

[0091] The sequencing results of the 16S RNA gene are as follows (SEQ ID NO: 1):

[0092]

[0093] The obtained 16S rDNA sequence of the strain was compared with the NCBI website using BLAST. Combined with morphological observation and physiological and biochemical characteristics, the strain was identified as *Lactobacillus salivarius*. This strain was deposited on December 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC No. 33044.

[0094] Example 2: Determination of growth characteristics of lactic acid bacteria

[0095] 1) Seed activation

[0096] Lactic acid bacteria strains from glycerol tubes were streaked onto MRS solid plates and incubated at 37°C for 2 days. Single colonies were then picked and inoculated into MRS test tubes and incubated for 16 hours before use.

[0097] 2) Growth and acid production of lactic acid bacteria

[0098] The seeds obtained from the in vitro activation were inoculated into test tubes containing 10 ml of MRS medium at a 1% inoculation rate and incubated at 37°C. Every 2 hours, one test tube was taken out to measure pH and OD. The absorbance at OD600 nm and pH were measured every 2 hours using the turbidimetric method.

[0099] 3) Effect of culture temperature on strain growth

[0100] The seeds obtained from in vitro activation were inoculated at a 1% inoculation rate into test tubes containing 10 ml of MRS medium. These tubes were then incubated at 25℃, 30℃, 37℃, 40℃, 45℃, 50℃, and 55℃ for 14 hours. After incubation, the tubes were spread and counted. The ratio of the number of viable bacteria at different temperatures to the number of viable bacteria at 37℃ reflects the viability at that temperature.

[0101] 4) Results of growth characteristics of Lactobacillus salivarius

[0102] The results of the growth and temperature tolerance characteristics of *Lactobacillus salivarius* are shown in [the table below]. Figure 1 The activated bacterial solution of the strain has an OD600nm absorbance of 0-10, a pH < 4, and a survival rate of 50.5% under culture conditions of 45℃, indicating that the strain is heat-resistant.

[0103] Example 3: Detection of antibacterial activity of Lactobacillus salivarius using the Oxford cup method

[0104] 1) Activation culture of indicator bacteria

[0105] Indicator bacteria: enterotoxigenic *Escherichia coli* CICC10413, *Salmonella typhimurium* TA100, *Staphylococcus aureus* CGMCC1.1861, *Campylobacter jejuni* NFM100.

[0106] Pathogenic bacteria such as *Escherichia coli*, *Salmonella* and *Staphylococcus aureus* preserved in glycerol tubes are inoculated into a culture medium. For *Escherichia coli*, *Salmonella* and *Staphylococcus aureus*, the culture medium used is broth medium: 10.0 g of peptone, 3.0 g of beef extract powder, 5.0 g of sodium chloride, 1000 mL of distilled water. The culture is carried out on a shaker at 37°C and 200 r / min for 16-18 h, and the obtained product is set aside for later use.

[0107] The specific culture method for *Campylobacter jejuni* is as follows: put 10 mL of commercially available Brucella broth solid medium into a cell flask, add 10 mL of sterilized BHI liquid medium before use, inoculate *Campylobacter jejuni* preserved in a glycerol tube into the liquid medium, culture at 37°C in a tri-gas incubator for 40 hours, until a layer of red bacterial biofilm forms on the surface of the liquid medium.

[0108] Cool the sterilized Columbia blood agar base medium to 45-50°C, slowly add sheep blood along the wall of the flask, shake gently to avoid foaming as much as possible until the mixture is uniform. Take plates with a diameter of about 90 mm, pour 18-20 mL of the agar mixture into each plate respectively, spread it evenly inside the plate, and place it on a horizontal table to solidify. Absorb an appropriate amount of indicator bacterial liquid, spread it evenly, then place Oxford cups uniformly at equal intervals in each plate and set aside for later use.

[0109] 2) Preparation of lactic acid bacteria fermentation supernatant

[0110] Pick single colonies of *Lactobacillus salivarius* from Example 2 and inoculate them respectively into sterilized glass test tubes containing 10 mL of MRS liquid medium, culture at 37°C for 16 h to obtain a strain fermentation liquid, centrifuge at 4°C and 8000 r / min for 10 minutes, and retain the supernatant.

[0111] 3) Bacteriostatic effect of fermentation supernatant on indicator bacteria

[0112] Take 100 μL of indicator bacterial liquid and spread it on a solid medium, fix Oxford cups on the solid medium, add 200 μL of *Lactobacillus salivarius* fermentation supernatant dropwise into the Oxford cups, culture at 37°C for 18 h, then measure the diameter d of each bacteriostatic zone for evaluation. Photograph representative plates. Evaluation criteria for bacteriostatic performance: when d<10 mm, it means no bacteriostatic effect; when 10 mm<d<15 mm, it means moderate bacteriostatic activity; when d>15 mm, it means high bacteriostatic activity.

[0113] Additional notes: 1) A larger bacteriostatic zone indicates a better bacteriostatic effect; 2) The bacteriostatic zone refers to the full diameter of the circular transparent zone; 3) The outer diameter of the Oxford cup is 8 mm.

[0114] The antibacterial results are shown in Table 1 below.

[0115] Table 1: Results of the antibacterial activity of fermentation supernatant against different indicator bacteria

[0116]

[0117] It is evident that *Lactobacillus salivarius* has a broad-spectrum antibacterial effect, especially against *Campylobacter jejuni*, which is significantly more effective than existing strains.

[0118] Example 4: Preparation of Fermented Feed Raw Materials and Determination of Antibacterial Effect of Samples

[0119] Take 100g of commercial meal and place it in a self-sealing bag. Add protease at a ratio of 100-500U / g of meal and mix well. Add water and lactic acid bacteria mixture in a certain proportion. The specific formula is shown in Table 2 below. Mix well again and place the sample in a 40℃ constant temperature incubator for 72h. After drying the sample in a 65℃ oven for 6h, pulverize it and pass it through a 60-mesh sieve.

[0120] Table 2: Fermentation Formula

[0121]

[0122] 1) Preparation of fermented meal extract and control group

[0123] Weigh the sample and mix it with 80% ethanol solution. Shake well and incubate at 45℃ and 150 rpm for 3 hours. Centrifuge at 8000 rpm for 5 minutes. Collect the supernatant and evaporate it to half its original volume in a 65℃ metal bath.

[0124] Separately, 40% and 80% ethanol solutions were prepared without any sample and subjected to the same operation as control groups.

[0125] 2) Determination of antibacterial activity of fermented meal

[0126] The antibacterial activity of *Lactobacillus salivarius* was detected using the Oxford cup method, with ethanol as a blank control and meal as a control. The evaluation of antibacterial performance was the same as in Example 3.

[0127] 3) Antibacterial effect of fermented meal extract on indicator bacteria

[0128] The distribution of bacteria in the wet sample after fermentation is shown in Table 3 below, and the antibacterial effect of the fermented meal extract is shown in Table 4 below.

[0129] Table 3: Distribution of bacteria in wet samples after fermentation

[0130] viable bacteria count / CFU / g Lactic acid bacteria bacteria yeast mold soybean meal ND <![CDATA[2*10 2 ]]> ND ND Fermented soybean meal (without lactic acid bacteria) ND <![CDATA[3*10 7 ]]> ND <![CDATA[4.5*10 5 ]]> Fermented soybean meal (Lactobacillus salivarius) <![CDATA[5*10 8 ]]> <![CDATA[3.4*10 2 ]]> ND ND Rice bran meal ND <![CDATA[5.2*10 3 ]]> ND <![CDATA[3.9*10 2 ]]> Fermented rice bran meal (without added lactic acid bacteria) ND <![CDATA[4.7*10 7 ]]> ND <![CDATA[4.5*10 5 ]]> Fermented rice bran meal (Lactobacillus salivarius) <![CDATA[6.7*10 8 ]]> <![CDATA[6.9*10 2 ]]> ND 30

[0131] Table 4: Results of the antibacterial activity of fermented meal extracts against different indicator bacteria

[0132]

[0133]

[0134] As shown in the table above, fermented soybean meal and fermented rice bran meal prepared from Lactobacillus salivarius both have good antibacterial effects.

[0135] Example 5: Fermented rice bran meal (FRBM) prepared from Lactobacillus salivarius

[0136] The parameter changes of fermented rice bran meal obtained by fermentation with Lactobacillus salivarius in this paper were detected, and the results are shown in Table 5 below.

[0137] The detection indicators and corresponding detection methods are as follows: Moisture, GB / T10358; Crude protein, GB / T6432; Ash, GB / T6438; Small peptides, QB / T2653-2008; Organic acids, GB12456; Crude fat, GB / T14772-2008; Crude fiber, GB / T6434-2022; Energy, Detection method: Approximate nutrient analysis method; Reducing sugar, GB 5009.7-2016.

[0138] Table 5: Changes in Indicators of Fermented Rice Bran Meal

[0139]

[0140]

[0141] As shown in Table 5 above, fermentation significantly increases reducing sugars, metabolizable energy, digestible energy, and organic acids.

[0142] Example 6: Effects of fermented rice bran meal prepared with Lactobacillus salivarius on medium and large pigs

[0143] Fermented rice bran meal was added to the feed formulation at 5% and 8% respectively, and the pigs were fed with these two types of fermented rice bran meal. The specific feed formulations are shown in Table 6 below, and the feeding results are shown in Table 7 below.

[0144] Table 6: Feed Formulation (by weight)

[0145] variety Comparison 5% FRBM 8% FRBM corn 65％ 61.0％ 61.5％ soybean meal 25％ 24.0％ 23.0％ bran 4％ 4.0％ 1.5％ premix 6％ 6.0％ 6.0％ Fermented rice bran 5.0％ 8.0％ total 100％ 100％ 100％ crude protein 16.46％ 16.49％ 16.17％

[0146] Note: The premix provides 8000 IU of vitamin A, 32100 IU of vitamin D, 15 IU of vitamin E, 3 mg of vitamin K, 83 mg of vitamin B, 35 mg of niacin, 20 mg of pantothenic acid, 12 mg of copper, 80 mg of iron, 46 mg of manganese, 50 mg of zinc, 0.35 mg of iodine, and 0.40 mg of selenium per kilogram of feed.

[0147] Table 7: Experimental Results

[0148]

[0149]

[0150] It can be seen that the basal weight of pigs in the experimental groups was similar, while that in the control group was slightly higher. Under the same feeding amount, the daily weight gain of fermented rice bran meal was 0.05-0.1 kg higher than that of the control group, and the feed conversion ratio was 0.2-0.35 lower. This indicates that the application of fermented rice bran meal in fattening pigs promoted digestion and absorption and improved feed conversion rate.

[0151] Example 7: Effects of fermented rice bran meal prepared from Lactobacillus salivarius on pig manure of medium and large pigs

[0152] The fecal excretion, nitrogen emission, phosphorus emission, and ammonia emission of the pigs fed in Example 6 were measured, and the results are shown in Table 8 below.

[0153] Table 8: Nitrogen and phosphorus excretion in pig manure

[0154]

[0155] Note: Different letters in the upper right corner of the same column indicate a significant difference between the two, while the same letter indicates no significant difference.

[0156] It is evident that as the amount of fermented rice bran meal added increases, the amount of feces decreases significantly, as do the nitrogen and phosphorus content in the feces and the ammonia emissions.

Claims

1. Lactobacillus salivarius with accession number CGMCC No.33044.

2. A culture of Lactobacillus salivarius with accession number CGMCC No. 33044.

3. The culture as described in claim 2, characterized in that, The culture also contains a culture medium; preferably, the culture medium is MRS medium; more preferably, the culture medium contains peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, triammonium citrate, sodium acetate, glucose, Tween 80, magnesium sulfate, and manganese sulfate; preferably, the culture medium contains 5-15 g / L peptone, 2-10 g / L beef extract, 3-8 g / L yeast extract, 1-5 g / L dipotassium hydrogen phosphate, 1-5 g / L triammonium citrate, 3-8 g / L sodium acetate, 1-8 g / L glucose, 0.5-2 mL / L Tween 80, 0.1-0.5 g / L magnesium sulfate, and 0.01-0.2 g / L manganese sulfate.

4. A formulation containing Lactobacillus salivarius with accession number CGMCC No. 33044; preferably, the formulation is a microbial preparation.

5. A fermentation feedstock containing *Lactobacillus salivarius* with accession number CGMCC No. 33044, preferably, the fermentation feedstock further contains meal and enzymes. Preferably, the meal is selected from one or more of soybean meal, rapeseed meal, cottonseed meal, peanut meal, sunflower meal, sesame meal, flaxseed meal, rice bran meal, tea meal, flaxseed meal, safflower meal, palm meal, palm kernel meal, coconut meal, chili meal, olive meal, corn distillers' grains meal, and beet meal, and more preferably from one or more of soybean meal, rice bran meal, wheat bran meal, and palm meal. Preferably, the enzyme is selected from one or more of protease, pectinase, xylanase, α-galactosidase, and cellulase. Preferably, the total amount of enzyme added is more than 100U per gram of meal, such as 100-1000U, 500-2000U or 100-500U.

6. A fermentation composition, characterized in that, The fermentation composition is obtained by fermenting the fermentation raw material according to claim 5, and the fermentation composition preferably includes fermented meal; Preferably, the fermentation temperature is 37-50℃, for example 37-45℃ or 40-50℃. Preferably, the fermentation time is 24-96 hours, for example, 24-72 hours or 48-72 hours. Preferably, after fermentation, the process further includes a drying and pulverizing step. Preferably, the drying temperature is 60-70℃ or 65-80℃. Preferably, the drying time is 5-10 hours or 6-12 hours. Preferably, the fermentation composition is pulverized to a mesh size of 40 or higher, for example, 60 mesh or 80 mesh.

7. An alcoholic extract of a fermentation composition, characterized in that, The alcohol extract is prepared by the following method: thoroughly mixing the fermentation composition with an alcohol solution and extracting to obtain an alcohol extract of the fermentation composition; wherein the fermentation composition is as described in claim 6. Preferably, the alcohol solution is an ethanol solution, and preferably, the concentration of the ethanol solution is 70-90% or 80%-90%. Preferably, oscillation mixing is employed; more preferably, the oscillation rate is 100-150 rpm or 150-200 rpm, and / or the oscillation time is 3-5 hours or 2-3 hours. Preferably, after extraction, optionally, the step further includes concentration to obtain an alcoholic extract of the fermentation composition.

8. Feed containing the fermentation composition of claim 6 or the alcohol extract of claim 7.

9. A method for increasing the reducing sugar content, metabolizable energy, digestible energy, and / or organic acid content of a fermentation composition, the method comprising the step of obtaining the fermentation composition by fermentation using Lactobacillus salivarius as claimed in claim 1, the formulation as claimed in claim 4, and / or the fermentation feedstock as claimed in claim 5.

10. The use of Lactobacillus salivarius or a microbial preparation containing Lactobacillus salivarius as described in claim 1, the fermentation raw material as described in claim 5, and / or the fermentation composition as described in claim 6 in the preparation of feed; Preferably, the application includes one or more of the following aspects: increasing the reducing sugar content of feed, increasing the metabolizable energy of feed, increasing the digestible energy of feed, increasing the organic acid content of feed, promoting the digestion and absorption of livestock, increasing the feed conversion rate of livestock, increasing the daily weight gain of livestock, reducing the feed conversion ratio of livestock, reducing the amount of livestock manure excretion, reducing the nitrogen content in livestock manure, reducing the phosphorus content in livestock manure, and reducing the ammonia emissions from livestock manure.

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