Isolation and application of lactobacillus pentosus

Abstract

The present application relates to a lactobacillus pentosus strain, and its application. Specifically, the present application provides a lactobacillus pentosus strain, its preparation and application. The lactobacillus pentosus strain of the present application has a preservation number of CGMCC No. 33043. The lactobacillus pentosus strain of the present application can be applied to poultry feed to improve the quality of poultry eggs.

Description

Technical Field

[0001] This invention belongs to the field of feed application, specifically relating to the isolation and application of a strain of Lactobacillus pentosus. Background Technology

[0002] The consolidation process in the egg-laying hen industry is accelerating, gradually moving towards standardization and large-scale production. With increasing consumer focus on food safety and nutrition, the market prospects for branded eggs are broad. Branded eggs offer advantages such as traceability and guaranteed quality. Egg quality evaluation involves multiple aspects, including shell quality, egg weight, shell strength, shell thickness, egg shape index, shell color, specific gravity, and surface cleanliness. Various factors influence egg quality, including genetics, management practices, and nutritional levels. Regarding nutritional levels, the intake of nutrients such as protein, carbohydrates, and fats all affect egg quality. The scaling up and standardization of the egg-laying hen industry is conducive to improving egg quality, while good egg quality can promote the high-quality development of the industry.

[0003] Scientific feed formulation and feed additives can improve the production performance of laying hens and the quality of eggs. Soybean meal, a commonly used feed ingredient, is rich in protein and amino acids, but it also contains various anti-nutritional factors, such as glycinin and β-conglycinin, which can hinder the digestion and absorption of nutrients. Fermented soybean meal, after fermentation, can significantly reduce the content of anti-nutritional factors and improve the digestibility and absorption of nutrients.

[0004] Therefore, there is an urgent need in this field to use fermented soybean meal in laying hen diets to improve egg quality and enhance the standardization and scale of the laying hen industry. Summary of the Invention

[0005] This study investigates the use of fermented soybean meal fermented with Lactobacillus fermentum in laying hen diets, explores the impact of fermented soybean meal on egg quality, and develops new strains for application.

[0006] The first aspect of this invention provides a Lactobacillus pentosus, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33043.

[0007] The second aspect of the present invention provides a culture of Lactobacillus pentosus with accession number CGMCC No. 33043.

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

[0009] In one or more embodiments, 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.

[0010] 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.

[0011] A third aspect of the present invention provides a formulation containing Lactobacillus pentosus with accession number CGMCC No. 33043.

[0012] In one or more embodiments, the formulation is a microbial formulation.

[0013] The fourth aspect of the present invention provides a fermentation raw material containing Lactobacillus pentosus with accession number CGMCC No. 33043.

[0014] In one or more embodiments, the fermentation feedstock further includes meal, enzymes, and optionally water.

[0015] 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 and beet meal.

[0016] In one or more embodiments, the meal is selected from 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 enzyme is a protease, such as one or more of alkaline protease, neutral protease, and acidic protease.

[0019] 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.

[0020] In one or more embodiments, the content of meal is 70-80% or 72-75% based on the total weight of the fermentation feedstock.

[0021] In one or more embodiments, the water content is 20-60% by total mass of the fermentation feedstock, for example 20-40%, 35-50%, or 35-40%.

[0022] The fifth aspect of the present invention provides a fermentation composition obtained by fermenting 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.

[0027] In one or more embodiments, the drying time is 5-10 hours or 6-12 hours.

[0028] In one or more embodiments, the fermentation composition is pulverized to a fineness of 40 mesh or higher, for example, 60 mesh or 80 mesh.

[0029] A sixth aspect of the present invention provides a feed containing a fermented composition as described in any embodiment of the fifth aspect herein. In one or more embodiments, the fermented composition is present in the feed at a concentration of 5-10% or 5-8%.

[0030] In one or more embodiments, the feed is poultry feed.

[0031] The seventh aspect of the present invention provides a method for improving the quality of poultry eggs, the method comprising the step of preparing poultry feed using Lactobacillus pentosus, such as accession number CGMCC No. 33043, fermentation raw materials as described in any embodiment of the fourth aspect of the present invention, or fermentation compositions as described in any embodiment of the fifth aspect of the present invention, wherein the poultry eggs are derived from the poultry.

[0032] The eighth aspect of the present invention provides the use of Lactobacillus pentosus with accession number CGMCC No. 33043, fermentation raw materials as described in any embodiment of the fourth aspect of the present invention, or fermentation compositions as described in any embodiment of the fifth aspect of the present invention, or feed as described in any embodiment of the sixth aspect of the present invention in the preparation of feed, or in the improvement of poultry egg quality.

[0033] In one or more embodiments, the improvement of poultry egg quality includes one or more of the following characteristics: (1) increasing the hardness, strength and / or thickness of the eggshell, (2) increasing the weight and / or freshness of the egg, (3) improving the likability of the egg, (4) increasing the color of the yolk, (5) increasing the aroma of the egg, (6) reducing the fishy smell of the egg, (7) increasing the texture, elasticity and / or delicacy of the egg, and (8) reducing the rate of defective eggs, dirty eggs, bloody eggs, broken eggs, soft-shelled eggs and / or deformed eggs.

[0034] In one or more embodiments, the poultry eggs include chicken eggs, duck eggs, goose eggs, quail eggs, pigeon eggs, and turkey eggs, preferably chicken eggs. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0040] In this article, the sum of the percentages of all components in the composition is 100%.

[0041] In this article, "watermarked egg" refers to an eggshell with watermark-like marks, usually semi-transparent spots or patterns.

[0042] 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.

[0043] 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.

[0044] The inventors screened a strain of *Lactobacillus pentosus* from fermented sauerkraut and tested its fermentation performance. Tests showed that it can be used for fermenting oilseed meals, increasing the content of small peptides and organic acids. More unexpectedly, applying the fermented samples to laying hen feed effectively improved egg quality and reduced the formation of watermarked eggs. This completes the invention.

[0045] Lactobacillus pentosaccharide used for fermentation

[0046] This article provides a strain of Lactobacillus pentosus, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33043.

[0047] 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. *Lactobacillus pentosaceus* can be cultured using conventional methods, such as 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. The proportions of these ingredients can be adjusted according to actual needs. Culture conditions can be conventional, such as incubating at 25-40℃ or 35-40℃ for 10-20 hours or 15-20 hours. Single colonies of the bacterial strain can be directly transferred to a suitable fresh solid culture medium (such as MRS medium), allowed to grow and multiply for a period of time (e.g., 2-5 days), and then inoculated into a liquid culture 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℃ or 35-40℃ for 10-20 hours or 15-20 hours to obtain an activated bacterial solution (seed culture). In an exemplary embodiment, the OD value of the activated bacterial solution of *Lactobacillus pentosus* described herein is 5-12, 5-10, or 8-12.

[0048] In some embodiments, this document also provides a culture of *Lactobacillus pentosus* with accession number CGMCC No. 33043. This culture contains *Lactobacillus pentosus* as described herein and a culture medium. The culture medium can be any commonly used culture medium for culturing *Lactobacillus pentosus* known in the art, including media used for preparing *Lactobacillus pentosus* seed culture or activated culture, and media used for fermentation using *Lactobacillus pentosus*.

[0049] 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.

[0050] The *Lactobacillus pentosus* described herein can be used as a probiotic additive. Therefore, this article also provides a formulation containing *Lactobacillus pentosus* with accession number CGMCC No. 33043. In some embodiments, the formulation is a microbial preparation. In some embodiments, the microbial preparation is a compound microbial preparation, which, in addition to the *Lactobacillus salivarius*, contains other bacteria commonly used in feed fermentation, including but not limited to *Lactobacillus plantarum* (LP), *Lactobacillus casei* (LC), and *Pediococcus lactis* (PC). There are no special limitations on the content of each bacterium in the compound microbial preparation and the ratio between them. Generally, each bacterium can be present in the microbial compound preparation of this invention at an amount greater than its conventional dosage in feed fermentation.

[0051] Fermentation raw materials, fermentation composition

[0052] The *Lactobacillus pentosus* described in this article belongs to the lactic acid bacteria family and can be used as a fermentation feedstock. Therefore, this article also provides a fermentation feedstock containing *Lactobacillus pentosus* with accession number CGMCC No. 33043.

[0053] In this document, fermentation raw materials may include various agricultural crop straw powder, leaf and weed powder, melon vine powder, 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, oil cake meal, bran, cottonseed meal, moldy feed, slaughterhouse by-products, swill, leftover food, chicken manure, and other waste. In an exemplary embodiment, the fermentation raw materials in this document include meal. The term "oil meal" in this article refers to commonly used feed meals in this field, typically byproducts of oilseed or grain processing. The raw materials for these meals are byproducts of oil extraction, usually defatted or partially defatted oilseed cakes or meals. Oilseeds include, but are not limited to, palm kernels, soybeans, sunflower seeds, peanuts, rapeseed, corn, safflower seeds, sesame seeds, rice, rice bran, flaxseeds, olives, hazelnuts, pumpkin seeds, grape seeds, evening primrose, Sichuan pepper seeds, almonds, wheat germ, perilla seeds, pecans, cashews, macadamia nuts, pistachios, and coconuts. Preferably, the raw materials for these meals are 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. More preferably, they are one or more of soybean meal, rice bran meal, wheat bran meal, and palm meal. Preferably, the meal content, based on the total weight of the fermented raw materials, can be 70-80% or 72-75%.

[0054] Typically, the raw materials used for fermentation also include enzymes and water.

[0055] The enzymes suitable for use in this invention can be various enzymes conventionally used in the art for fermenting and enzymatically hydrolyzing soybean 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. Typically, the amount of enzyme used can be the conventional amount used in soybean meal fermentation in the art, for example, more than 100U per gram of soybean meal, such as 100-2000U, 500-2000U, or 100-500U of enzyme.

[0056] The amount of water used is typically 25-85% of the mass of the meal raw material, for example, 35-50% or 35-40%. In some embodiments, the weight ratio of meal raw material 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 weight of the fermented meal of the present invention, for example, 20-40%.

[0057] In this article, *Lactobacillus pentosus* is used as a fermentation agent, such as in bacterial solution or powder. As mentioned earlier, activated bacterial solution or seed culture of this *Lactobacillus pentosus* can be added to the fermentation raw materials. The amount of activated bacterial solution added can be conventional, with a volume of 0.1-10 mL per 100 g of meal, for example, 0.5-8 mL, 1.0-5.0 mL, or 2.0-8.0 mL per 100 g of meal. The activated bacterial solution of *Lactobacillus pentosus* can be diluted before being added to the meal raw materials, for example, diluted 3-80 times, 5-40 times, or 8-20 times.

[0058] This document also provides a method for fermenting meal, which includes the step of fermenting meal using *Lactobacillus pentosus* with accession number CGMCC No. 33043. Therefore, the method of this document includes the step of fermenting a fermented meal feed containing *Lactobacillus pentosus*, meal, enzymes, and water. In some embodiments, the method of this document includes the step of mixing the *Lactobacillus pentosus*, meal, enzymes, and water described herein. Typically, during fermentation, the meal and enzymes are first mixed, and then the *Lactobacillus pentosus* and water are added to form the fermented meal.

[0059] Typically, the fermentation temperature is 37-50℃, for example, 37-45℃. 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, which can be done at 60-70℃ or 65-80℃ for 5-10 hours or 6-12 hours. Usually, the fermented meal is pulverized to a fineness of 40 mesh or higher, for example, 60 mesh or 80 mesh.

[0060] In some embodiments, this document also provides a fermentation composition of *Lactobacillus pentosus* with accession number CGMCC No. 33043, or obtained by fermentation of the fermentation feedstock described in any embodiment of this document. In this fermentation composition, 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 this invention can be a mixture prepared for fermentation (i.e., before fermentation) (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.

[0061] The fermentation composition described herein contains 50.6-50.7% protein, has a pH of 4.6-4.7, contains more than 17% acid-soluble protein (as a percentage of dry matter), for example 17.8-17.9%, and has a viable lactic acid bacteria count of at least 10. 3 cfu / g, for example 3.52*10 3 -3.55*10 3The concentration of CFU / g and the moisture content is 8-12%. Typically, the fermentation composition is dried and pulverized before its protein content, pH, acidity, acid-soluble proteins, and viable lactic acid bacteria count are determined.

[0062] Feed containing fermented composition

[0063] This document provides a feed containing the fermented composition described herein. Feeds typically include energy sources, protein sources, mineral sources, vitamin sources, and optional additives. In the feed described herein, the protein source comprises the fermented composition as described in any embodiment herein.

[0064] 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 is usually high, above 50%, 60%, or 70%, for example, 60-65%. The amount of grain processing by-products added can be 1-5%, for example, 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, especially helpful in winter or during the peak growth and egg production period of chickens, and can also improve the palatability of the feed. 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, tallow, mutton tallow, duck tallow, chicken tallow, fish oil, and shrimp oil, preferably one or more of fish oil, lard, mutton tallow, and tallow. In some embodiments, the energy ingredient content is 60-65% or 60-62% by total feed weight. In some embodiments, the corn content is 60-61% and the bran content is 2-5% by total feed weight.

[0065] Protein feed ingredients can be conventional feed ingredients, such as those containing high protein content (e.g., 40-60% or 40-50%), including but not limited to byproducts of oilseed pressing and fermentation products of these byproducts. Oilseed pressing byproducts, as mentioned above, include 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, corn gluten meal, and beet meal. Preferably, they are 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. The oilseed pressing byproducts in the protein feed ingredients can be the same as or different from the meal ingredients. The amount of protein feed ingredients added, by total feed weight, can be 15-30% or 15-25%. In some embodiments, the content of the fermentation composition may be 3-15%, 3-8%, or 5-10% based on the total weight of the feed.

[0066] Mineral feed ingredients provide calcium, phosphorus, sodium, and trace elements for the feed organisms. Calcium sources are typically limestone powder (mainly calcium carbonate) and shell powder; phosphorus sources are typically dicalcium 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 0.4-0.6%, sodium content 0.3-0.4%, and trace element additions are minimal. By total feed weight, the mineral feed ingredient is limestone, with a content of 8-10% or 9-12%.

[0067] 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.

[0068] 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).

[0069] 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.

[0070] In some implementations, the feed described herein is poultry feed, such as chicken feed, duck feed, goose feed, quail feed and turkey feed, preferably chicken feed, especially laying hen feed.

[0071] Applications and methods

[0072] This document also provides the use of *Lactobacillus pentosus* with accession number CGMCC No. 33043, fermentation feed as described in any embodiment of this document, feed containing the fermentation composition described in any embodiment of this document in feed preparation, or in improving the quality of poultry eggs. Preferably, the improvement of poultry egg quality includes one or more of the following characteristics: (1) increasing the hardness, strength, and / or thickness of the eggshell; (2) increasing the weight and / or freshness of the egg; (3) improving the egg's appeal; (4) increasing the yolk color; (5) increasing the egg's aroma; (6) reducing the egg's fishy smell; (7) increasing the egg's texture, elasticity, and / or delicacy; and (8) reducing the rate of defective eggs, dirty eggs, bloody eggs, broken eggs, soft-shelled eggs, and / or deformed eggs. In an exemplary embodiment, the fermentation composition includes fermented soybean meal.

[0073] In some embodiments, this document also provides the use of *Lactobacillus pentosus* with accession number CGMCC No. 33043 in the preparation of feed, or in the improvement of poultry egg quality. Preferably, the feed is poultry feed. Preferably, the improvement of poultry egg quality is as described above.

[0074] In this article, poultry eggs include, but are not limited to, chicken eggs, duck eggs, goose eggs, quail eggs, pigeon eggs, and turkey eggs, with chicken eggs being preferred.

[0075] The present invention has the following beneficial effects:

[0076] This invention provides a strain of Lactobacillus pentosus and prepares fermented soybean meal samples for feeding laying hens, which improves egg quality, reduces the frequency of watermarked eggs, and improves egg flavor.

[0077] 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.

[0078] Lactobacillus plantarum M, with accession number CGMCC No. 1.557, was purchased from the Institute of Microbiology, Chinese Academy of Sciences.

[0079] MRS liquid culture medium formula: 10g peptone, 5g Angel yeast extract, 5g sodium acetate, 1g Tween 80, 0.2g magnesium sulfate heptahydrate, 10g beef extract, 20g glucose, 2g diammonium citrate, 2g potassium dihydrogen phosphate, 0.05g manganese sulfate heptahydrate, 1L water.

[0080] MRS solid medium formulation: Based on MRS liquid medium, add 20 g / L of agar.

[0081] Moisture content was determined using the method specified in GB / T6435-2006.

[0082] The detection of acid-soluble proteins was performed according to the GB9005.5-2010 method.

[0083] The viable count was determined using the plate count method.

[0084] Acidity was determined by titration.

[0085] Example 1: Screening and identification of Lactobacillus pentosus

[0086] The strain of this invention was isolated from pickled cabbage in Guangxi Zhuang Autonomous Region. The specific steps are as follows:

[0087] Take 1g of pickled cabbage sample and resuspend it in 9mL of sterile physiological saline, then shake well. Take at least 1mL of the liquid and add it to 9mL of sterile physiological saline, shaking well to obtain a dilution with a concentration of 10⁻¹. Repeat the above steps to perform serial dilutions, obtaining dilutions of 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, and 10⁻⁷. Select one dilution of 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, or 10⁻⁷, and spread 200μL onto MRS solid medium, performing two replicates for each dilution. All operations are performed under aseptic conditions. After spreading, incubate at 37°C. After 48 hours, the plates were removed. Under aseptic conditions, colonies with a diameter of 1-3 mm, exhibiting characteristics of lactic acid bacteria (white, grayish-white, or milky yellow, smooth or slightly rough surface), were picked from the single colonies on the plates and streaked onto new MRS solid plates for purification. The plates were then incubated at 37°C for 24 hours. Microscopic observation revealed a short rod-shaped bacterial strain. PCR amplification of the bacterial 16S rDNA was performed using the bacterial culture. The primers used were those known in the art.

[0088] Upstream primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO:2)

[0089] Downstream primer 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO:3)

[0090] The PCR system (50 μL) is as follows:

[0091] Premix taq (2×) 25μL Upstream primer 27F (20 μM) 1μL Downstream primer 1492R (20 μM) 1μL DNA template 1μL <![CDATA[ddH2O]]> 22μL

[0092] The PCR program was as follows: ① 94℃ for 5 min; ② 94℃ for 30 s, 50℃ for 30 s, 72℃ for 30 s, for 30 cycles; ③ 72℃ for 5 min.

[0093] After the PCR reaction was completed, the PCR products were confirmed by nucleic acid electrophoresis analysis and then sent to Sangon Biotech (Shanghai Co., Ltd.) for sequencing. The 16S rDNA sequence of the sampled bacteria was obtained and compared with the 16S rDNA sequences indexed in the NCBI website. The isolated bacteria were confirmed to be the non-engineered wild-type Lactobacillus pentosaccharide.

[0094] The sequencing results of the 16S RNA gene are as follows (SEQ ID NO: 1):

[0095]

[0096] The strain is Lactobacillus pentosus, which was deposited on December 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC No. 33043.

[0097] Example 2:

[0098] 2.1 Preparation of Fermented Soybean Meal Samples

[0099] Lactic acid bacteria culture: A single colony of *Lactobacillus pentosus* was picked and inoculated into a sterile glass tube containing 15 ml of MRS liquid medium. The culture was incubated at 37°C for 16 hours. The cultured solution was then stored at 4°C for later use. The OD value of the culture was 8. The MRS medium formula was: 10 g peptone, 5 g Angel yeast extract, 5 g sodium acetate, 1 g Tween 80, 0.2 g magnesium sulfate heptahydrate, 10 g beef extract, 20 g glucose, 2 g diammonium citrate, 2 g potassium dihydrogen phosphate, 0.05 g manganese sulfate heptahydrate, and 1 L water.

[0100] Solid sample preparation: Take 50g of commercial soybean meal (from Dahai Grain and Oil Industry (Fangchenggang) Co., Ltd.) and place it in a self-sealing bag. Add protease (200,000 U / g alkaline protease) at a ratio of 500 U / g soybean meal and mix well to form sample a. Mix 1mL of lactic acid bacteria solution and 18.8g of water evenly and pour it into sample a. Mix well again, remove all air from the bag, seal the bag, and incubate the sample in a 40℃ constant temperature incubator for 72h. After 72h of incubation, dry the sample in a 65℃ oven for 6h, then pulverize it through a 60-mesh sieve. Determine the protein content, acid-soluble protein, pH, and acidity. Acidity is determined by titration.

[0101] The results are shown in Table 1 below. Sample 1 is without strain; Sample 2 is a fermentation sample with Lactobacillus pentosus added; and Sample 3 is a fermentation sample with Lactobacillus plantarum M added.

[0102] Table 1: Detection indicators of various fermentation samples

[0103]

[0104] As shown in Table 1 above, the samples prepared from *Lactobacillus pentosus* showed superior performance compared to other strains, increasing the content of small peptides and organic acids in the meal. After drying, the viable count of lactic acid bacteria in the samples was consistently above 10. 3 CFU / g, compared to commercially available lactic acid bacteria inoculants (live count 10). 9 The cfu / g ratio varies considerably.

[0105] 2.2 Effects of different amounts of lactic acid bacteria added on fermented soybean meal samples

[0106] Lactic acid bacteria culture: A single colony of *Lactobacillus pentosus* was picked and inoculated into a sterile glass tube containing 15 ml of MRS liquid medium. The culture was incubated at 37°C for 16 hours. The cultured broth was then stored at 4°C for later use. At this point, the OD value of the fermentation broth was approximately 8. The MRS medium formula was as follows: 10 g peptone, 5 g Angel yeast extract, 5 g sodium acetate, 1 g Tween 80, 0.2 g magnesium sulfate heptahydrate, 10 g beef extract, 20 g glucose, 2 g diammonium citrate, 2 g potassium dihydrogen phosphate, 0.05 g manganese sulfate heptahydrate, and 1 L water.

[0107] Solid sample preparation: 100g of commercial soybean meal (from Dahai Grain & Oil Industry (Fangchenggang) Co., Ltd.) was placed in a self-sealing bag. Protease (200,000 U / g alkaline protease) was added at a concentration of 500 U / g of soybean meal and mixed thoroughly to form sample a. The fermentation broth of the lactic acid bacteria strain and water (specific amounts shown in Table 2 below) were poured into sample a and mixed thoroughly again. The air in the bag was removed, and the bag was sealed. The sample was then incubated at 40℃ for 72 hours. After 72 hours of incubation, the sample was dried in a 65℃ oven for 6 hours, then pulverized and passed through a 60-mesh sieve. Protein content, acid-soluble protein, pH, and acidity were measured. Acidity was determined by titration. The results are shown in Table 3.

[0108] Table 2: Experimental Formula

[0109]

[0110]

[0111] Table 3: Detection of various indicators in fermentation samples

[0112]

[0113] As shown in Table 3 above, when 2% of different levels of lactic acid bacteria were added, the fermented soybean meal sample had better quality. This condition was used to prepare the fermented soybean meal for the feeding experiment in the subsequent examples.

[0114] Example 3: Design and Trial Management of Feeding Formula

[0115] 1. Experimental formulation and grouping: The diet was formulated with reference to NRC (1994) and chicken feeding standards (NYT 332004), as detailed in Table 4 below.

[0116] 2. Experimental species: Agricultural University No. 3 laying hen.

[0117] 3. Experimental Management: The experiment adopted cage rearing, with each replicate housed in a single cage. The chicken house equipment and water system were disinfected before the experiment. The laying hens had free access to feed and water. During the experiment, lighting, temperature, ventilation, and immunization were all conducted according to standard procedures. The chicken house was cleaned and disinfected regularly.

[0118] Table 4: Feed Formulation Composition (%)

[0119]

[0120]

[0121] The premix contains: VA 3000 IU, VD3 1500 IU, VE 40 mg, VK3 4.5 mg, VB 22.515 mg, VC 110 mg; biotin 0.15 mg, inositol 40 mg, folic acid 1.3 mg; iodine 1.2 mg, manganese 8.5 mg, molybdenum 1 mg, copper 6.5 mg, zinc 53 mg; selenium 0.35 mg, iron 45 mg.

[0122] Example 4: Experimental Results – Egg Defect Rate

[0123] Egg defect rate refers to the proportion of substandard eggs caused by various reasons during the production process, including dirty egg rate, blood egg rate, broken shell egg rate, soft shell egg rate, and deformed egg rate. Layering hens were fed a diet supplemented with Lactobacillus pentosus, and the results were observed through a periodic trial. The results of the trials are shown in Tables 5 and 6.

[0124] Table 5: Changes in Egg Defect Rate Over Two Weeks

[0125]

[0126] Table 6: Changes in Egg Defect Rate Over Four Weeks

[0127]

[0128] Note: In the same column, different lowercase letters indicate significant differences (P<0.05), while the same letter or no letter indicates no significant difference (P>0.05).

[0129] Table 5 shows that after two weeks of feeding, the rates of dirty eggs, bloody eggs, broken eggs, and deformed eggs in the *Lactobacillus pentosus* group were significantly lower than those in the control group. Table 6 shows that after four weeks of observation, the defective egg rate in the *Lactobacillus pentosus* group further decreased. Therefore, compared with the control group, the defective egg rate in the *Lactobacillus pentosus* group was lower, and the effect became more pronounced with increasing feeding time.

[0130] Example 5: Experimental Results – Egg Quality

[0131] Three different brands of eggs were purchased from the market (Hema Supermarket) and tested along with eggs from a breeding experiment. Egg quality indicators generally include: egg shape index (length to width ratio, normal range: 1.28-1.43); eggshell strength and thickness (reflecting shell quality, generally positively correlated); yolk color (scored using the Roche colorimetric method); and freshness indicators (generally evaluated using Haugh value and yolk index, with a Haugh value between 72-82 considered edible). The results are shown in Table 7 below.

[0132] Table 7: Egg Quality

[0133]

[0134]

[0135] As can be seen, the egg shape index of each group of eggs was similar, all within the normal range. The eggshell thickness and strength of the group with added Lactobacillus pentosus were the highest, slightly higher than those of Huangtian'e and Zhengda antibiotic-free eggs; the grain-fed fresh eggs were the worst. The egg weight of the Lactobacillus pentosus group was the largest, along with Huangtian'e and Zhengda eggs, while the weight of other groups was relatively smaller. Huangtian'e eggs were the reddest, and compared with the control group, the yolk color of the Lactobacillus pentosus group showed a trend of increasing. It is evident that, compared with the control group, the Lactobacillus pentosus group had the highest eggshell quality and egg freshness, and thus the highest grade evaluation.

[0136] Example 6 Experimental Results – Eggshell Quality Scoring

[0137] The Novus eggshell scoring system primarily evaluates and scores eggshells using light, effectively reflecting the presence of watermarks in eggs. The scoring criteria are as follows: 0 points: Opaque, very few tiny bright spots, no black spots. 1 point: Slightly translucent, a few tiny bright spots, no black spots. 2 points: Slightly translucent, many tiny bright spots, no black spots. 3 points: Moderately translucent, many tiny bright spots, a few black spots. 4 points: Highly translucent, tiny and large bright spots, many black spots. 5 points: Extremely high translucent, various bright spots, many black spots. Lower scores indicate fewer watermarks or better quality eggs; higher scores indicate more obvious watermarks, poorer shell membrane airtightness, and easier liquid leakage, which shortens the egg's shelf life. The scoring results are shown in Table 8 below.

[0138] Table 8: Eggshell Quality Scoring

[0139]

[0140]

[0141] Table 8 shows that the frequency of eggs with watermarks in the Lactobacillus pentosus group was lower, and the scores were also lower, indicating that the eggs were of better quality. It is evident that Lactobacillus pentosus watermark eggs had the lowest frequency and overall scores were better than those of Huangtian'e and Zhengda antibiotic-free brand eggs.

[0142] Example 7 Experimental Results – Tasting Test

[0143] Five eggs were randomly selected from each group for steaming. After steaming, each egg was cut into four pieces and tasted by eight evaluators. The evaluators scored the eggs based on the above indicators, with scores of 1, 2, and 3, where 1 is the weakest and 3 is the strongest.

[0144] Table 9: Egg Tasting Test

[0145]

[0146]

[0147] It is evident that Lactobacillus pentosus histones have a higher preference for color, while Grain Fresh has the lowest. Yellow Swan egg yolks have a higher preference for color, which is related to their highest color intensity. Eggs have the strongest egg flavor and the least fishy smell. In terms of texture, they have the best elasticity and delicacy.

Claims

1. Lactobacillus pentosus with accession number CGMCC No.33043.

2. A culture of Lactobacillus pentosus with accession number CGMCC No. 33043.

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 pentosus with accession number CGMCC No. 33043; preferably, the formulation is a microbial preparation.

5. A fermentation feedstock containing *Lactobacillus pentosus* with accession number CGMCC No. 33043, preferably, the fermentation feedstock further includes 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 proteases, pectinases, xylanases, α-galactosidases, and cellulases; more preferably, the enzyme is a protease, such as one or more of alkaline proteases, neutral proteases, and acidic proteases. Preferably, the total amount of enzyme added is more than 100U per gram of meal, such as 100-1000U, 500-2000U, or 100-500U. Preferably, the content of meal is 70-80% or 72-75% based on the total weight of fermentation raw materials.

6. A fermentation composition, characterized in that, The fermentation composition is obtained by fermenting the fermentation raw material according to claim 5, preferably, the fermentation composition is 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 fermented meal is pulverized to a fineness of 40 mesh or higher, for example, 60 mesh or 80 mesh.

7. A feed containing the fermentation composition of claim 6; preferably, the fermentation composition is fermented meal; preferably, the content of the fermentation composition in the feed is 5-10% or 5-8%; preferably, the feed is poultry feed.

8. A method for improving the quality of poultry eggs, characterized in that, The method includes the step of preparing feed for the poultry using Lactobacillus pentosus, such as accession number CGMCC No. 33043, the fermentation feedstock as described in claim 5, or the fermentation composition as described in claim 6.

9. The use of Lactobacillus pentosus with accession number CGMCC No. 33043, the fermentation raw material as described in claim 5, the fermentation composition as described in claim 6, or the feed as described in claim 7 in the preparation of poultry feed, or in the improvement of poultry egg quality.

10. The application as described in claim 9, characterized in that, The improvement of poultry egg quality includes one or more of the following characteristics: (1) increasing the hardness, strength and / or thickness of the eggshell, (2) increasing the weight and / or freshness of the egg, (3) improving the likability of the egg, (4) increasing the color of the yolk, (5) increasing the aroma of the egg, (6) reducing the fishy smell of the egg, (7) increasing the texture, elasticity and / or delicacy of the egg, and (8) reducing the rate of defective eggs, dirty eggs, bloody eggs, broken eggs, soft-shelled eggs and / or deformed eggs. Preferably, the poultry eggs include chicken eggs, duck eggs, goose eggs, quail eggs, pigeon eggs, and turkey eggs, with chicken eggs being the most preferred.

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