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CN-121975655-A - Preparation method and application of pig intestinal glutamic acid enriched flora

CN121975655ACN 121975655 ACN121975655 ACN 121975655ACN-121975655-A

Abstract

The invention relates to the field of animal nutrition and intestinal microecology regulation and control, and discloses a preparation method and application of pig intestinal glutamic acid enriched flora. Comprises obtaining the small intestine content of healthy lactation piglet, preparing intestinal bacteria inoculum under anaerobic condition, inoculating in culture medium with glutamic acid, and continuously subculturing to enrich the directionally enriched intestinal bacteria and metabolites thereof capable of metabolizing glutamic acid and producing mixed active amino acid metabolites. According to the invention, by screening dominant flora with glutamic acid metabolism function in pig small intestine and establishing a mother-child integrated transplanting system, early intervention on piglet intestinal development and healthy intestinal bacteria establishment can be realized, piglet intestinal flora structure and metabolism function are regulated, intestinal development is promoted, and growth performance is improved.

Inventors

  • DAI ZHAOLAI
  • JI JING
  • Yang Aoyu
  • DUAN SHUAI
  • HAN DANDAN
  • ZHANG XIUWEN

Assignees

  • 中国农业大学

Dates

Publication Date
20260505
Application Date
20260107

Claims (10)

  1. 1. The preparation method of the pig intestinal glutamic acid enriched flora is characterized by comprising the following steps: (1) Obtaining the small intestine content of healthy lactation piglets, and preparing an intestinal fungus inoculum under anaerobic conditions; (2) Inoculating the intestinal fungus inoculum into a culture medium added with glutamic acid, and performing continuous subculture to enrich specific flora capable of metabolizing the glutamic acid and generating active amino acid metabolites, thus obtaining glutamic acid enriched flora; The glutamic acid-enriched bacterial population comprises Lactobacillus, veillonella, megasphaera genus microorganisms.
  2. 2. The method according to claim 1, wherein the step (1) comprises placing the small intestine content in a 50 mL sterilized centrifuge tube containing sterilized glass beads and continuously fed with CO 2 , adding sterilized anaerobic medium at a ratio of 10% (w/v), rapidly screwing the tube cap, shaking vigorously for 1 min, centrifuging at 4 ℃ under 250 Xg for 5min, filtering the centrifuged supernatant with sterilized 4-layer gauze, and collecting in a sterilized serum bottle continuously fed with CO 2 as an intestinal inoculum; The anaerobic culture medium comprises：0.1-0.5 g CaCl 2 2H 2 O、0.2-1.0 g MgSO 4 7H 2 O, 0.5-1.5 g K 2 HPO 4 , 2-4 g KH 2 PO 4 , 8-12 g NaHCO 3 , 1-2.5 g NaCl, 1-2.5 g lactose, 0.5-2 g fucose, 0.5-2.5 g maltose, 0.5-2.5 g cellobiose, 1.5-5 g tryptone, 1.5-5 g peptone, 3-7 g yeast extract, 1.5-5 g beef extract, 3-7 g glucose, 0.3-0.7 g cysteine hydrochloride, 0.8-1.5 mL tween-80 solution, 0.8-1.5 mL resazurin solution, 20-30 mL heme solution, 10-30 mL vitamin K 1 solution and 8-15 mL fatty acid solution; the preparation method of the resazurin solution comprises dissolving 1 g resazurin in 1L deionized water; the preparation method of heme solution comprises dissolving 1 g heme in a small amount of 50 mM NaOH solution, and fixing volume to 1L; The preparation method of vitamin K 1 solution comprises dissolving 10mg vitamin K 1 in 0.2L 100% ethanol, filtering, and sterilizing; the fatty acid solution was prepared by dissolving 140. Mu.L of acetic acid, 60. Mu.L of propionic acid, 36. Mu.L of butyric acid and 12. Mu.L of valeric acid in 20 mL 200 mM NaOH.
  3. 3. The method according to claim 2, wherein the culture medium used in the step (2) is prepared by adding 1 mL glutamic acid mother liquor, 100. Mu.L of fatty acid solution and 200. Mu.L of vitamin K1 solution to the anaerobically-packed and 115℃and autoclaved 7.7. 7.7 mL anaerobic culture medium of 15: 15 min to give a final concentration of glutamic acid of 2 to 7 mmol/L, preheating 30min in a 37℃water bath, adding 1 mL intestinal inoculum, and/or, The continuous subculture method comprises the steps of carrying out primary culture in a 37 ℃ incubator for 48 h%, inoculating fermentation broth of the primary culture for 48 h as an inoculum of secondary culture in a preheated sterilization culture medium according to an inoculum size of 5% -20%, culturing for 24h in the 37 ℃ incubator and as a1 st generation of fermentation, culturing for 24h as a1 st generation, preferably carrying out continuous transmission for 5 th generation, and carrying out glycerol preservation on the bacterial broth at a fermentation end point.
  4. 4. A method according to any one of claims 1-3, wherein the healthy lactating piglets of step (1) are piglets of the age of 7-21 days after birth, preferably 14 days.
  5. 5. A method according to any one of claims 1-3, wherein the small intestine content originates from the jejunum and/or ileum of a piglet.
  6. 6. A glutamic acid-enriched enterobacteria agent prepared by the method of any one of claims 1 to 5.
  7. 7. A method of improving intestinal health or promoting growth of a suckling piglet comprising applying the glutamic acid-enriched enterobacteria agent of claim 6 to the living environment of the piglet or to the body surface of a sow or orally; The methods are for non-disease diagnosis and treatment purposes.
  8. 8. The method of claim 7, wherein the administering comprises administering the glutamic acid-enriched enterobacteria agent to the udder and/or the piglet active area of the sow in a spray.
  9. 9. The method of claim 7 or 8, wherein the administration is initiated within one week of birth of the piglet and continues until weaning.
  10. 10. Use of any one of the following glutamic acid-enriched enterobacteria agents of claim 6: 1) The preparation method is used for preparing a product for improving intestinal health of the suckling piglets, reducing diarrhea rate, relieving weaning stress of the piglets or improving the growth performance of the piglets before and after weaning; 2) Is used for piglet cultivation.

Description

Preparation method and application of pig intestinal glutamic acid enriched flora Technical Field The invention relates to the field of animal nutrition and intestinal microecology regulation and control, in particular to a preparation method and application of pig intestinal glutamic acid enriched flora. Background Intestinal microorganisms are a complex population, which includes bacteria, fungi, archaea, viruses, etc., are important components for maintaining animal health, and are closely related to digestion, immunization, and metabolism of hosts. The problems of diarrhea, reduced feed intake, slow growth and the like often occur in the early stage of piglets due to imperfect intestinal development and strong weaning stress. The traditional solution means mainly depend on antibiotics or nutritional additives, but long-term use of antibiotics can cause drug resistance and microorganism disorder, which is unfavorable for sustainable development of the breeding industry. Intestinal bacterial transplantation (IMT) differs from fecal bacterial transplantation (FMT) in that as an important means of reconstructing the structure of the intestinal flora, the effect is expected to be superior to fecal bacterial transplantation because the initial flora is derived from the intestinal tract, and the intestinal bacteria are closer to targeted and regulated intestinal segments (e.g., small intestine) than to fecal-like flora. However, currently, in livestock and poultry production, no intestinal fungus transplantation technology aiming at specific metabolic functions (such as amino acid metabolism) is reported. Functional amino acids (e.g., tryptophan, arginine, glutamic acid, branched chain amino acids) play a key role in protein synthesis and cell growth, but also have important effects on immune function, intestinal health, and metabolic homeostasis. Glutamic acid is a main energy substrate of intestinal epithelial cells of piglets, and can promote mucous membrane repair and immunoregulation. However, not all amino acids are able to efficiently enter the blood circulation system through the intestinal barrier and are absorbed and utilized by the body. Intestinal flora plays a key role in amino acid synthesis and metabolism, and amino acid derivatives produced by intestinal flora may contribute to regulating intestinal immune function, maintaining the integrity of the intestinal barrier. Therefore, by screening and enriching intestinal flora with glutamic acid metabolic potential, the sow integrated transplantation is hopefully improved in early intestinal health of piglets. Disclosure of Invention The invention aims to provide a preparation method and application of pig intestinal glutamic acid enriched flora. According to the invention, by screening dominant flora with glutamic acid metabolism function in pig small intestine and establishing a mother-child integrated transplanting system, early intervention on intestinal development of piglets can be realized, intestinal flora structure and metabolism function of piglets can be regulated, intestinal development can be promoted, and growth performance can be improved. In order to achieve the object of the present invention, in a first aspect, the present invention provides a method for preparing a pig intestinal glutamic acid-enriched flora (enterobacteria), comprising the steps of: (1) Obtaining the small intestine content of healthy lactation piglets (such as Du X long X big three-way hybrid piglets), and preparing the intestinal fungus inoculum under anaerobic conditions; (2) Inoculating the flora inoculation liquid into a culture medium added with glutamic acid, and carrying out continuous subculture to enrich specific flora capable of metabolizing the glutamic acid and producing active amino acid metabolites, thus obtaining glutamic acid enriched flora. Further, the glutamic acid-enriched bacterial population mainly includes, but is not limited to, lactobacillus, veillonella, megasphaera bacteria genus microorganisms. Further, step (1) comprises placing the small intestine content in a 50 mL sterilizing centrifuge tube containing sterilized glass beads and continuously introducing CO 2, adding sterilized anaerobic culture medium at a ratio of 10% (w/v), rapidly tightening the tube cover, vigorously shaking for 1 min, centrifuging at 4 deg.C and 250×g for 5min, filtering the centrifuged supernatant with sterilized 4 layers of gauze, and collecting in a sterilized serum bottle continuously introducing CO 2 as an intestinal inoculum; The anaerobic culture medium comprises：0.1-0.5 g CaCl22H2O, 0.2-1.0 g MgSO47H2O, 0.5-1.5 g K2HPO4, 2-4 g KH2PO4, 8-12 g NaHCO3, 1-2.5 g NaCl, 1-2.5 g lactose, 0.5-2 g fucose, 0.5-2.5 g maltose, 0.5-2.5 g cellobiose, 1.5-5 g tryptone, 1.5-5 g peptone, 3-7 g yeast extract, 1.5-5 g beef extract, 3-7 g glucose, 0.3-0.7 g cysteine hydrochloride, 0.8-1.5 mL tween-80 solution, 0.8-1.5 mL resazurin solution, 20-30 mL heme solution, 10-30