Tibetan pig source enterobacter lewis TP1 and application of Tibetan pig source enterobacter lewis TP1 in fermentation of highland barley vinasse feed

Abstract

The invention discloses a strain of Tibetan pig source enterobacter ludwigii TP1 and application thereof in fermentation of highland barley vinasse feed, belongs to the field of microbial fermentation, and provides the strain of enterobacter ludwigii TP1 which is preserved in Guangdong Microbial Culture Collection Center on June 19, 2025, and the preservation number is GDMCC No: 66554. The strain has good degradation capacity on highland barley vinasse fibers, and simulation experiments prove that the strain can completely adapt to the intestinal environment of Tibetan pigs. When the feed additive is applied to Tibetan pig breeding, the digestion and utilization rate of highland barley vinasse feed can be improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microbial fermentation, and particularly relates to a strain of Enterobacter ludwigii TP1 from Tibetan pigs and application of the strain in fermentation of highland barley dreg feed. BACKGROUND

[0002] In the field of livestock breeding, efficient utilization of feed resources and development of high-quality feed have always been key issues. Tibetan pigs, as a characteristic pig breed in the plateau region of China, are known for their tolerance to roughage and delicious meat. Studies have shown that the apparent digestibility of forage fiber and semi-fiber by grazing Tibetan pigs is significantly higher than that of Tibetan pigs in confinement and ordinary commercial pigs (such as Duroc Landrace Yorkshire, DLY pigs). This strong fiber digestion ability is closely related to the rich fiber-degrading bacteria in the intestinal tract of Tibetan pigs (Chinese Agricultural Science and Technology, 2022, 53(9):3063-3078). Isolating efficient fiber-degrading bacteria from the intestinal tract of Tibetan pigs is of great significance for improving the utilization rate of fiber in feed and reducing breeding costs.

[0003] Highland barley, as the main crop in the plateau region, its dreg is the by-product after highland barley brewing. Highland barley dreg is rich in a large amount of nutrients, such as dietary fiber, crude protein, crude fat, amino acids, and various vitamins, trace elements, enzymes, β-glucan, γ-aminobutyric acid and other bioactive substances (CN patent, 202310158903.0, 2023-03-05). Through research and comparison, the content of protein in highland barley dreg is more than 20%, which has obvious advantages compared with the content of crude protein in sorghum dreg which is generally not more than 10% (CN patent, 202310158903.0, 2023-03-05). However, due to the high content of fiber and other indigestible components in highland barley dreg, the utilization rate of animals is low when it is used directly as feed (CN patent, 202310158903.0, 2023-03-05). If efficient fiber-degrading bacteria can be used to ferment highland barley dreg, it will significantly improve its nutritional value and utilization rate as feed.

[0004] At present, the research on the fermentation of highland barley wine lees mostly uses common strains such as Aspergillus oryzae (China Cattle Industry Science, 2021, 47(6):51-55). Although this strain can degrade fiber to some extent, it is not related to the intestinal environment of Tibetan pigs, and its adaptability to the intestinal microecosystem of Tibetan pigs is poor. When applied in Tibetan pig feed, its fiber degradation efficiency is restricted by the intestinal environment, making it difficult to fully play its role. According to the research data, the neutral detergent fiber (NDF) degradation rate of Aspergillus oryzae fermented highland barley wine lees for 7 days was only 13.4%, and the acid detergent fiber (ADF) degradation rate was only 7.5%. Moreover, its carboxymethyl cellulase activity decreased significantly on the 7th day of fermentation (from 2066.70 U·g⁻¹ on the 6th day to 1497.24 U·g⁻¹), which cannot sustain efficient fiber degradation. In addition, its optimal fermentation condition is static culture at 30°C, which is not optimized for the 39°C body temperature and anaerobic physiological environment of Tibetan pig intestines.

[0005] Enterobacter ludwigii is a member of the Enterobacter genus, which is a gram-negative, facultative anaerobic bacterium widely found in various environments (Invert Neurosci. 2019 Oct 22;19(4):13). A patent has shown that Enterobacter ludwigii (CCTCC M 2015182) can degrade β-mannan components in hemicellulose by secreting β-mannanase, and has the ability to degrade hemicellulose. Hemicellulose, as a natural polysaccharide, is the second most abundant renewable component in lignocellulosic biomass, second only to cellulose; its global annual output is about 60 billion tons (Rao J. Prog Polym Sci. 2023 Mar 14;140:101675). It is worth noting that current research on the isolation and study of Enterobacter ludwigii mostly focuses on soil and plant planting sites in natural environments, and there is no related report on collecting this strain from Tibetan pigs, which leaves room for further exploration of the habitat distribution and potential function of this strain.

[0006] Currently, the research on Tibetan pig-derived fiber-degrading bacteria mainly focuses on common fiber-degrading bacteria such as Bacillus (Animal Nutrition, 2023, 35 (8): 5067-5076). There is less research on Enterobacter ludwigii, which is relatively rare but distributed in the intestinal tract of Tibetan pigs. In the development of highland barley distiller's grains, although various microbial fermentation has been tried to improve its quality, there is a lack of targeted application research on specific strains from Tibetan pigs. Developing highland barley distiller's grains based on Tibetan pig-derived Enterobacter ludwigii has great potential to improve the efficiency of Tibetan pig breeding and promote the development of the livestock industry in highland areas, and needs further research. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a strain suitable for the intestinal environment of Tibetan pigs and capable of efficiently degrading highland barley distiller's grains.

[0008] The technical solution of the present application is a strain of Enterobacter ludwigii TP1, which was deposited at the Guangdong Microbial Culture Collection Center on June 19, 2025, with the accession number GDMCC No: 66554.

[0009] The application of the above-mentioned Enterobacter ludwigii TP1 in highland barley distiller's grains fermentation.

[0010] Further, the application is to inoculate Enterobacter ludwigii TP1 in highland barley distiller's grains, thereby obtaining highland barley distiller's grains with reduced fiber content.

[0011] Compared with the prior art, the present application has the following beneficial effects:

[0012] 1. The Tibetan pig-derived Enterobacter ludwigii TP1 strain of the present application has better adaptability to the digestive physiological environment of Tibetan pigs. Compared with other strains, it is more beneficial to improve the digestion and utilization rate of highland barley distiller's grains when applied in Tibetan pig breeding.

[0013] 2、The Enterobacter ludwigii TP1 strain of the Tibetan pig source of the application has significant advantages compared to the Enterobacter ludwigii in the application (CCTCC M 2015182), first, in the degradation substrate range, MY271 is mainly aimed at beta-mannan in hemicellulose (such as glucomannan in konjac powder), and the degradation is realized by producing beta-mannanase, and the advantage is that the enzyme activity can reach 62.76 U / mL, which is higher than that of some strains such as Aspergillus niger 27.4 U / mL, but is limited to the degradation of a single type of hemicellulose.

[0014] 3、In terms of application value, MY271 is mainly used for industrial enzyme production and mannose oligosaccharide production, serving the fields of food additives, papermaking and the like, while the TP1 strain of the application is directly applied to the improvement of highland barley wine dregs feed, and the nutritional value of the feed is improved by degrading complex fiber, which is more in line with the actual demand of low utilization rate of roughage in agricultural production, and solves the problem that MY271 cannot directly act on composite feed fiber; in addition, from the effect pertinence, the enzyme activity data of MY271 is based on pure substrate fermentation (such as konjac powder 14 g / L, 31 ℃ fermentation for 48 h, etc.), while the degradation rate of the application is based on the actual feed in vitro enzyme experiment, which can better reflect the effect in the actual application scene, compared with the above, the fiber degradation capacity of TP1 strain in complex actual system has more practical significance.

[0015] Preservation information:

[0016] Enterobacter ludwigii TP1 was preserved in Guangdong Microbial Culture Collection Center, No. 59 Building, Guangzhou Martyrs' Road 100 Courtyard, on June 19, 2025, and the preservation number is: GDMCC No: 66554. DETAILED DESCRIPTION

[0017] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0018] In the application, the determination of crude fiber (CF) and neutral detergent fiber (NDF) is carried out according to the guidelines of the American Official Chemists Association (AOAC, 2007 edition), and the determination of acid detergent fiber (ADF) is carried out according to the guidelines of the American Official Chemists Association (AOAC, 2007 edition).

[0019] Example 1: Isolation, purification and preservation of Enterobacter ludwigii TP1 strain of Tibetan pig source

[0020] Purpose of the test: Isolate and screen strains with high fiber degradation capacity from Tibetan pig manure.

[0021] Test plan:

[0022] Sample collection and processing: Take 5 g of fresh feces from healthy Tibetan pigs, cut the feces in half on a clean bench, and take the center sample into 45 mL of sterile water containing steel balls, shake for 30 min to prepare a bacterial suspension.

[0023] Enrichment culture: Take 1 mL of bacterial suspension and inoculate into 20 mL of enrichment medium (formula: CMC-Na 1 g, yeast powder 1 g, KH2PO40.1 g, NaCl 1 g, MgSO4·7H2O 0.03 g, water 100 mL, 121℃ sterilization for 20 min), shake culture at 37℃, 200 r / min for 48 h.

[0024] Primary screening and purification: Take 50 μL of enriched bacterial solution and add it to the center of Congo red fiber medium, incubate at 37℃ for 3 days, stain with 1 mg / mL Congo red staining solution for 10 min, then decolorize with 1 mol / L NaCl solution for 15 min, select colonies with transparent hydrolysis rings. Take single colonies for 4 times of streaking and purification, confirm negative bacilli by Gram staining, name it TP1, measure the cellulase activity (filter paper enzyme activity is 3.2 U / mL), and finally store it in -80℃ refrigerator by glycerol preservation method (15 % glycerol).

[0025] Identification: The TP1 strain is observed under a microscope and is rod-shaped, with negative Gram staining. After incubation on LB solid medium for 24 h, the colonies are ivory white, with neat edges, no bulge, dry surface, and no wrinkles. 16S rRNA sequencing confirms that it has 99.8 % homology with Enterobacter ludwigii, so the strain is named Enterobacter ludwigii-TP1.

[0026] Preservation: Preserved in the Guangdong Microbial Culture Collection Center on June 19, 2025, with the preservation number GDMCC No: 66554.

[0027] Example 2: In vitro enzymatic hydrolysis test of Enterobacter ludwigii TP1 strain

[0028] Test purpose: To verify the degradation ability of the strain on the fiber in highland barley wine lees.

[0029] Test plan:

[0030] Strain activation: Inoculate the frozen bacterial solution into liquid LB medium at a 10 % inoculation amount, incubate at 37℃ for 24 h, then transfer 5 % of the inoculation amount to fresh LB medium for continuous culture for 24 h to prepare a bacterial suspension (OD 600= 1.2, the number of colonies 1.5 x 10 8 CFU / mL).

[0031] Control group and experimental group grouping: take 50 g of highland barley vinasse, crush through 40 mesh screen, divide into 2 groups:

[0032] Control group: highland barley vinasse + 200 mL sterilized LB medium

[0033] Experimental group: highland barley vinasse + 200 mL bacterial suspension

[0034] Culture conditions: load the sample into a fermentation bag, and place in a 37 ℃ incubator for static fermentation for 14 days

[0035] Detection: after fermentation, dry at 105 ℃ to constant weight, and determine the contents of crude fiber (CF), acid detergent fiber (ADF), and neutral detergent fiber (NDF)

[0036] Test results:

[0037] Control group: CF 15.61 %, NDF 35.12 %, ADF 24.47 %

[0038] Experimental group: CF 11.43 %, NDF 26.69 %, ADF 19.16 %

[0039] Degradation rate: CF 26.96 %, NDF 22.74 %, ADF 22.02%

[0040] Conclusion: the strain has a significant degradation effect on the fiber components in highland barley vinasse, and is superior to the non-inoculated control group.

[0041] Example 3: In vitro fermentation test of Enterobacter ludwigii TP1 strain

[0042] Test purpose: simulate the intestinal environment of Tibetan pigs, and verify the deep fermentation effect of the strain on highland barley vinasse.

[0043] Test scheme:

[0044] (I) Preparation of reagents:

[0045] A. Trace element solution: In a 50 mL beaker, add 12.5 mg of manganese chloride, 10 mg of ferrous sulfate, 12.5 mg of zinc chloride, 25 mg of copper chloride, 25 mg of cobalt chloride, 25 mg of selenium dioxide, 125 mg of nickel chloride, 125 mg of sodium molybdate, 15.65 mg of sodium vanadate, and 125 mg of boric acid, respectively. Dissolve with 10 mL of 0.02 mol / L hydrochloric acid, transfer to a 500 mL volumetric flask, and then dilute to volume with water. Then, divide and store in 500 mL color reagent bottles.

[0046] B. Vitamin phosphate solution: In a 500 mL beaker, add 27.35 g of potassium dihydrogen phosphate, 10.2 mg of biotin, 10.25 mg of folic acid, 82 mg of calcium pantothenate, 82 mg of nicotinamide, 82 mg of riboflavin, 82 mg of thiamine hydrochloride, 82 mg of pyridoxine hydrochloride, 10.2 mg of p-aminobenzoic acid, and 10.25 mg of cobalamin, respectively. Dissolve with 100 mL of distilled water, transfer to a 500 mL volumetric flask, and then dilute to volume with water. Then, divide and store in 500 mL brown reagent bottles after filtration, sterilization, and sealing.

[0047] C. Bicarbonate solution: Weigh 82 g of anhydrous Na2CO3 and add it to a 1 L beaker. Dissolve with 1 L of boiling water, continue to aerate, and use immediately. Aerate again for 20 minutes before use.

[0048] D. Resazurin solution: (Red in the presence of free oxygen, colorless in anaerobic conditions, as the anaerobic indicator for this experiment). 0.1 % (W / V): 100 mg of resazurin is dissolved in 100 ml of water in a 100 mL beaker. (Add 1 ml of resazurin per liter of medium).

[0049] E. Hematin chloride solution: Boil clean water in a water boiler, weigh 0.1 g of green hematin and add it to a 10 mL beaker, add 5 mL of 0.56 mol / L NaOH solution, dissolve, transfer to a 500 mL volumetric flask, and dilute to volume with boiling water while continuously aerating.

[0050] F. Fatty acid solution: Weigh 4 g of sodium hydroxide into a 50 mL beaker, dissolve with 100 mL of distilled water, transfer to a 500 mL volumetric flask, add 200 mL of distilled water, shake well, then add 3.43 mL of acetic acid, 1.50 mL of propionic acid, 0.92 mL of butyric acid, and 0.28 mL of valeric acid, respectively, and then dilute to 500 mL. Divide and store in 500 mL color reagent bottles for future use.

[0051] G. Preparation of reducing agent solution: 20.5 g of Na2S·9H2O was weighed into a 50 mL beaker, dissolved with a small amount of water, transferred to a 1 L volumetric flask, and then made up to volume with boiling water while continuously bubbling CO2. After filtration and sterilization, it was stored in a serum bottle at 4 ℃.

[0052] (II) Test process:

[0053] Preparation of simulated Tibetan pig intestinal environment culture solution: microbial culture solution was prepared according to the following order and proportion: 10 mL of (A) trace element acid solution→1 mL of indigo solution→50 mL of (C) bicarbonate solution→placed in a microwave oven and heated to boiling, and CO2 was bubbled to room temperature→1 g of cysteine hydrochloride was added before dispensing, and the pH value was adjusted to about 6-8 (adjusted with 0.2 mol / L HCl and 0.2 mol / L NaOH solution)→10 mL of fermentation substrate solution obtained by mixing fresh pig manure with PBS was added→dispensed into fermentation bottles→1 mL of (G) reducing agent Na2S and 1 mL of (B) vitamin phosphate solution were added to each fermentation bottle→obtained culture solution; then CO2 was bubbled to quickly seal, and sealed with an aluminum cap. Placed in a 39 ℃, 50 r / min shaker for 7 d fermentation. After fermentation, the fermentation was terminated with an ice bag, the fermentation liquid was collected and placed in a cryotube, and the residue after fermentation was collected, washed, dried, weighed, and then stored at 4 ℃ for standby use;

[0054] (III) Determination index and method

[0055] Determination of crude fiber, acid detergent fiber, and neutral detergent fiber in raw materials and residues.

[0056] Test results:

[0057] Each group was set with 3 replicates, each with 10 parallels, then CO2 was bubbled to quickly seal, and sealed with an aluminum cap. Placed in a 39 ℃, 50 r / min shaker for 7 d fermentation. After fermentation, the fermentation was terminated with an ice bag, the fermentation liquid was collected and placed in a cryotube, and the residue after fermentation was collected, washed, dried, weighed, and then stored at 4 ℃ for standby use.

[0058] (III) Determination index and method

[0059] Determination of crude fiber (CF), acid detergent fiber (ADF), and neutral detergent fiber (NDF) in residues.

[0060] Test results:

[0061] Control group: CF 13.46 %, NDF 25.16 %, ADF 18.39 %

[0062] Test group: CF 9.18%, NDF 18.41%, ADF 15.22%

[0063] Degradation rate: CF 31.95%, NDF 26.72%, ADF 17.51%

[0064] Conclusion: The strain has a significant degradation effect on the fiber components in highland barley wine lees in the simulated Tibetan pig intestinal environment.

Claims

1. A strain of Enterobacter ludwigii TP1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 19, 2025, with accession number GDMCC No: 66554.

2. The application of Enterobacter ludwigii TP1 as described in claim 1 in the fermentation of highland barley lees.

3. The application according to claim 2, characterized in that, The application involves inoculating highland barley distillers' grains with Enterobacter ludwigii TP1 to obtain highland barley distillers' grains feed with reduced fiber content.