CN115466697B - Lactobacillus reuteri HLRE05 and application thereof - Google Patents
Lactobacillus reuteri HLRE05 and application thereof Download PDFInfo
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- CN115466697B CN115466697B CN202211156178.6A CN202211156178A CN115466697B CN 115466697 B CN115466697 B CN 115466697B CN 202211156178 A CN202211156178 A CN 202211156178A CN 115466697 B CN115466697 B CN 115466697B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/173—Reuteri
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
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- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Animal Husbandry (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus reuteri HLRE05 and application thereof. The name of the lactobacillus reuteri HLRE05 is Lactobacillus reuteri HLRE, and the lactobacillus reuteri HLRE05 is preserved in China Center for Type Culture Collection (CCTCC) with the number of M2022092 in the year 2022, 01 and 18. The invention provides a lactobacillus reuteri HLRE05 for high yield of the reuteri, the concentration of the reuteri in the fermentation liquor is as high as 9.98g/L, which is far higher than the yield of 3.7g/L of the standard strain lactobacillus reuteri ATCC6475, and the invention has wide industrial application prospect, can be applied to the preparation of microbial feed additives, and can greatly promote the growth and development of piglets and improve the immunity of the piglets; the method can also be applied to the preparation of the fermented yoghourt, ensures the nutritional value and the health care function of the fermented yoghourt, reduces the risk of product pollution, and can obviously prolong the shelf life of the fermented yoghourt on the premise of not changing the physicochemical property of the yoghourt.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus reuteri HLRE05 and application thereof.
Background
During the processing, transportation and storage of food and animal feed, the food and animal feed is extremely easy to be polluted by food-borne pathogenic bacteria such as bacillus cereus, staphylococcus aureus, listeria monocytogenes and the like, and the food or animal feed after being eaten can have great influence on the health of people and animals and bring great economic loss.
For example, yogurt can be contaminated with microorganisms during processing, transportation, storage, sale, and consumption, and after contamination can cause acute short-term effects of food-borne diseases or long-term hazards with chronic long-term effects. At present, food industry mainly can prevent food deterioration and prolong the quality guarantee period of food by adding artificial synthetic chemical additives. However, a great deal of researches show that excessive intake of the synthetic chemical additive has different degrees of loss on human health, and along with continuous importance of related departments and continuous enhancement of health consciousness of consumers, a new pollution reduction method is very necessary.
Also, diarrhea, for example, is one of the diseases with very high incidence in the course of piglet cultivation, and is one of the important factors affecting the survival rate of piglets. Diarrhea can affect the normal growth and mental state of piglets, and seriously lead to dehydration death of piglets. The diarrhea of piglets is caused by incomplete development of intestinal tracts in vivo and poor immunity, and the period is the period when intestinal flora begins to form, and is extremely easily affected by external factors, especially infection of pathogenic bacteria and viruses such as escherichia coli, salmonella and the like, so that diarrhea symptoms of piglets are caused. In particular, most of the drugs currently on the market for treating bacterial diarrhea are antibiotic symptomatic treatments. However, the antibiotic treatment can affect the balance of intestinal flora of piglets, damage intestinal barriers, generate super bacteria and remain in the product to seriously threaten human health, and have great side effects. With the general ban of antibiotics in the feed industry, the development of a novel feed additive with no side effect and obvious diarrhea prevention effect is a hotspot in the feed industry.
The main component of the reuterin is 3-hydroxy propanal, the polymerization and hydration of which in aqueous solution are reversible, and a dynamic equilibrium mixed system of monomers, dimers and hydrates of the 3-hydroxy propanal is formed, namely the reuterin. The reuterin keeps activity in a wider pH value range (4.1-7.0), has resistance to protease and lipase, has good thermal stability, has inhibition effect on gram-negative bacteria, gram-positive bacteria and yeast, is considered as a potential additive suitable for food biological preservation, and has great industrial application potential. However, at present, few documents report that the metabolism of lactobacillus reuteri produces the reuterin, and the yield is low and is not higher than 50mmol/L (3.7 g/L).
Therefore, the development of the lactobacillus reuteri which can specifically metabolize and produce the reuteri and has high yield can enrich the resources of the probiotics strain, and simultaneously provides more potential value for the lactobacillus reuteri in industrial production application.
Disclosure of Invention
The first object of the present invention is to provide a lactobacillus reuteri strain with high yield of reuterin, which is isolated from fresh excreta of healthy poultry by the inventor, has a pdu gene cluster encoding the reuterin, and can produce a broad-spectrum antibacterial substance of the reuterin by metabolizing glycerol, wherein the name of the lactobacillus reuteri HLRE05 is Lactobacillus reuteri HLRE05, which has been preserved in the China center for type culture collection (China) at the month 18 of 2022, and the sequence of 16S rRNA is shown as SEQ ID No. 1:
AGGCCCCTGCGGTGTGCTTATACTTGCAAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATTACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTTACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCTTTATGGAGGGAGCCGCCCTAAGCCGAAACCAAAGGGG。
under the same fermentation conditions, compared with the standard strain Lactobacillus reuteri ATCC6475, the concentration of the lactobacillus reuteri HLRE05 in the fermentation liquid is as high as 9.98g/L, which is far higher than the yield (about 50mmol/L, namely 3.7 g/L) of the standard strain Lactobacillus reuteri ATCC6475, and the lactobacillus reuteri has the excellent characteristics of gastric acid resistance and bile salt resistance, and has wide industrial application prospect.
A second object of the invention is the use of the Lactobacillus reuteri HLRE05 according to the invention for the preparation of a microbial feed additive. The lactobacillus reuteri HLRE05 disclosed by the invention can be successfully adhered to an organism and planted with intestinal tract metabolism oligosaccharides to generate small molecular organic acid substances such as lactic acid, short chain fatty acid and the like and various hydrolytic enzymes, participate in the synthesis of in-vivo B-group vitamins and convert inorganic selenium into organic selenium which can be utilized by the organism, and can ferment glycerol in vivo to generate natural antibacterial substances of reuterin, so that the growth of various diarrhea-related pathogenic bacteria in an intestinal epithelial environment is inhibited, the dominant position of lactobacillus in the intestinal tract is maintained, the growth and development of piglets are greatly promoted, and the immunity of the piglets is improved.
Based on the above situation, the invention also provides a microbial feed additive and a microbial feed containing the microbial feed additive, and the preparation method of the microbial feed additive comprises the following steps: inoculating the lactobacillus reuteri HLRE05 into MRS liquid culture medium containing glycerol, and fermenting to obtain the lactobacillus reuteri. Preferably, the fermentation conditions are: the fermentation temperature is 37 ℃, the inoculation amount is 1 percent, and the fermentation time is 24 hours. Preferably, the fermentation is further dried (by spray drying). More preferably, the spray drying conditions are: the material concentration is 10%, the air inlet temperature is 150 ℃, and the air outlet temperature is 75 ℃.
A third object of the present invention is the use of the Lactobacillus reuteri HLRE05 of the invention in the preparation of fermented yoghurt. The method is obtained by fermenting fresh milk by lactobacillus reuteri HLRE05 and specifically comprises the following steps: adding 0.1kg of the lactobacillus reuteri HLRE05 into each ton of fresh milk, and fermenting for 4-5 hours at the temperature of 40 ℃; then adding glycerin according to the proportion of 4.6kg per ton of fresh milk, homogenizing, and then continuing fermentation under the same condition until the final acidity reaches 70 DEG T.
The lactobacillus reuteri HLRE05 disclosed by the invention can prevent food-borne pathogenic bacteria infection in the fermented yoghourt, so that the nutritional value and the health care function of the fermented yoghourt are ensured, the risk of product pollution is reduced, the risk of intestinal tract food-borne pathogenic bacteria infection is relieved, and the shelf life of the fermented yoghourt can be obviously prolonged on the premise of not changing the physicochemical property of the yoghourt.
The beneficial effects of the invention are as follows: the invention provides a lactobacillus reuteri HLRE05 for high yield of the reuteri, the concentration of the reuteri in the fermentation liquor is as high as 9.98g/L, which is far higher than the yield of 3.7g/L of the standard strain lactobacillus reuteri ATCC6475, and the invention has wide industrial application prospect, can be applied to the preparation of microbial feed additives, and can greatly promote the growth and development of piglets and improve the immunity of the piglets; the method can also be applied to the preparation of the fermented yoghourt, ensures the nutritional value and the health care function of the fermented yoghourt, reduces the risk of product pollution, and can obviously prolong the shelf life of the fermented yoghourt on the premise of not changing the physicochemical property of the yoghourt.
Drawings
FIG. 1 is a diagram showing the results of verifying whether Lactobacillus reuteri HLRE05 carries the pdu gene;
FIG. 2 is a graph showing the results of a standard curve for the quantification of reuterin;
FIG. 3 is a graph showing the results of the production and quantification of reuterin;
FIG. 4 is a graph showing the results of inhibiting food-borne pathogenic bacteria in a fermentation broth of Lactobacillus reuteri HLRE 05;
FIG. 5 is a graph showing the change in the number of food-borne pathogenic bacteria in Lactobacillus reuteri HLRE05 fermented yogurt and normal fermented milk;
fig. 6 is a graph showing the change of physicochemical properties of lactobacillus reuteri HLRE05 fermented yogurt and common fermented yogurt under storage conditions of 4 ℃.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects and effects of the present invention.
Example 1:
isolation and identification of lactobacillus reuteri HLRE 05:
(1) Configuration of the selective Medium: 15.0g raffinose, 15.0g sodium acetate, 10.0g peptone, 6.0g monobasic potassium phosphate, 5.0g yeast extract, 2.0g ammonium acetate, 1.32mL glacial acetic acid, 1.0g Tween 80, 0.57g magnesium sulfate, 0.12g manganese sulfate, 0.003g ferrous sulfate were weighed, distilled water was added to a constant volume of 1L, and autoclaved at 121℃for 15min.
(2) Isolation and identification of lactobacillus reuteri:
collecting fresh excrement of poultry, adding 1g of PBS buffer solution into 9mL of the fresh excrement, carrying out gradual gradient dilution, uniformly coating 100 mu L of the diluted solution on a selective culture medium plate, and culturing the mixture for 48 hours at 45 ℃ under anaerobic conditions. Single colonies with different colony morphologies are picked, streaked and separated on an MRS plate, and purified culture is carried out. The morphological characteristics of the cells were observed under a microscope, colonies in rod form were selected for PCR, and the PCR products were sent to Bio-company for sequencing analysis.
And (3) analyzing and comparing the base sequence of the separated strain 16S rRNA after sequencing with NCBI database, constructing a phylogenetic tree to finally determine the strain as lactobacillus reuteri, finding that the homology with a standard strain of lactobacillus reuteri reaches 99.79%, and naming the strain as lactobacillus reuteri HLRE05.
(3) Gene screening for encoding reuterin
The genome DNA of the Lactobacillus reuteri HLRE05 is used as a template, a specific primer pdu (5 '-CCTGAAGTAAAYCGCATCTT-3' at the upstream and 5'-GAAACYATTTCAGTTTATGG-3' at the downstream) is utilized for PCR amplification, and the product is identified by 1.2% agarose gel electrophoresis, and the result is shown in figure 1.
Example 2:
lactobacillus reuteri fermentation to produce reuterin and quantification thereof:
(1) Preparation of a LewItalin standard curve
The standard curve establishing method comprises the following steps: under acidic conditions, the reuterin is dehydrated to acrolein, soTaking 0-1.0mg/mL of acrolein solution as a standard substance, incubating with tryptophan at 37 ℃ for 30min to form a purple compound, measuring the absorbance at 560nm by using an enzyme-labeling instrument, taking the mass concentration as the abscissa, and OD 560 A standard curve was drawn for the ordinate and used to quantify the unknown concentration of reuterin, and the standard curve results are shown in figure 2.
(2) The method for producing the reuterin by lactobacillus reuteri fermentation comprises the following steps: inoculating Lactobacillus reuteri HLRE05 into MRS culture solution, anaerobically culturing for 24h, collecting thalli, re-suspending in 250mmol/L glycerol, incubating for 2h at 37deg.C in an anaerobic incubator, filtering the supernatant with 0.22 μm filter membrane, and collecting filtrate to obtain high concentration of reuterin.
(3) Quantification of reuterin: 0.1mL of the filtrate was taken, 0.075mL of a tryptophan solution (dissolved in 50mmol/L hydrochloric acid) and 0.3mL of concentrated hydrochloric acid were added, the OD value was measured at 560nm after reaction at 37℃for 30min, and the concentration of the produced reuterin by Lactobacillus reuteri HLRE05 was 9.98g/L, and the production process and the results are shown in FIG. 3.
The same method conditions determine the standard strain Lactobacillus reuteri ATCC6475, which produces a concentration of 3.7g/L of reuterin, well below the Lactobacillus reuteri HLRE05.
Example 3:
antibacterial properties of reuterin:
taking 2 μl of fermentation broth of Lactobacillus reuteri HLRE05 obtained in example 2 and culturing for 24 hr, dripping at the center of MRS plate surface, culturing under anaerobic condition until bacterial lawn grows, and culturing 10mL containing 10 6 CFU/mL indicator bacteria (common food-borne pathogenic bacteria) and 0.7% LB culture medium containing 250mmol/L glycerol are fully and uniformly mixed, covered on a flat plate, after solidification, anaerobic cultured for 2 hours at 37 ℃, then transferred to an aerobic environment for culturing for 24 hours, and the size of a bacteriostasis zone is measured, wherein the measurement result is shown in figure 4, and the reuterin has good inhibition effect on five common food-borne pathogenic bacteria.
Example 4:
tolerance of lactobacillus reuteri HLRE05 to acids, bile salts:
inoculating activated over-night cultured lactobacillus reuteri HLRE05 bacterial liquid into an autoclaved MRS liquid culture medium with hydrochloric acid adjusted to pH of 2 and 3 respectively according to an inoculum size of 1.0%, performing constant-temperature anaerobic culture at 37 ℃, respectively taking 0h bacterial liquid and 3h bacterial liquid for gradient dilution, performing colony count, and calculating the survival rate. The survival rate is the ratio of the number of viable bacteria in the culture fluid to the number of viable bacteria at 0h, expressed in%.
Taking activated overnight cultured lactobacillus reuteri HLRE05 bacterial liquid, dissolving bovine bile salt in MRS liquid culture medium, autoclaving to make final concentration of bovine bile salt reach 0.15% and 0.3%, anaerobic culturing at 37 ℃ at constant temperature, respectively taking bacterial liquid gradient dilutions of 0, 3 and 6h for colony counting, and calculating survival rate. The survival rate is the ratio of the number of viable bacteria in the culture fluid to the number of viable bacteria at 0h, expressed in%.
The experimental results are shown in tables 1, 2 and 3, and it can be seen that lactobacillus reuteri HLRE05 has better tolerance to both acidic environment and high bile salt environment.
TABLE 1 tolerance of Lactobacillus reuteri HLRE05 in acidic Environment
| Acidic environment (pH) | 2 | 3 |
| Treatment time (h) | 3 | 3 |
| Survival (%) | 88.09±0.16 | 96.36±1.04 |
TABLE 2 tolerance of Lactobacillus reuteri HLRE05 in 0.15% bovine bile salt environment
TABLE 3 tolerance of Lactobacillus reuteri HLRE05 in 0.30% bovine bile salt environment
Example 5:
(1) A microbial feed additive is prepared by the following steps:
lactobacillus reuteri HLRE05 was inoculated into glycerol-added MRS liquid medium and fermented according to the following conditions: the fermentation temperature is 37 ℃, the inoculation amount is 1 percent, and the fermentation time is 24 hours; after the fermentation is completed, spray drying is performed according to the following conditions: the material concentration is 10%, the air inlet temperature is 150 ℃, the air outlet temperature is 75 ℃, and the fermentation product containing the reuterin is prepared and used as a feed additive.
(2) Comparative test for preventing diarrhea of piglets
Selecting 60 piglets which are of the same age and have similar weight as feeding objects, and dividing the piglets into A, B groups according to the principle that the weight and the proportion of male and female are the same, wherein group A is 30 experimental groups, male and female are half and half, and feeding the piglets by using feed containing the microbial feed additive; group B is blank 30, male and female halves, and is fed with feed containing no microbial feed additive. The experimental results after three months of feeding are shown in table 4.
Table 4 effect of the feed additive containing Rauyimycin on preventing diarrhea
| Index (I) | Control group | Experimental group |
| Daily feed intake (g) before feeding | 95.16±2.18 | 94.55±1.84 |
| Daily intake (g) after feeding | 114.39±3.41 | 143.49±2.77 |
| Daily gain (g) before feeding | 19.15±0.56 | 19.56±0.81 |
| Daily gain (g) after feeding | 25.11±1.17 | 32.48±0.46 |
| Number of diarrhea after feeding (head) | 5 | 1 |
| Mortality (%) | 0 | 0 |
As can be seen from table 4: the microbial additive prepared by the embodiment of the invention can obviously increase the appetite of piglets, promote the food intake and digestion of the piglets, improve the daily gain of the piglets, and obviously reduce the diarrhea rate of the piglets, so that the microbial additive prepared by the invention has stronger weight gain and immunity improving effects on the piglets and has extremely strong diarrhea preventing capability.
Example 6:
a fermented yoghurt prepared by the steps of:
(1) Acceptance of raw milk: after fresh milk is collected, the quality detection is carried out on the fresh milk by utilizing an infrared spectrum cow milk analyzer, the fat content is more than or equal to 3.1 percent, the protein content is more than or equal to 2.95 percent, the color is milky white or yellowish, the detection of antibiotics is negative, no foreign matters visible to naked eyes exist, and the quality safety of raw milk is ensured;
(2) Purifying: removing impurities and precipitates in milk and reducing the number of microorganisms by using a high-speed centrifuge and a filter;
(3) And (3) cooling: the purified raw milk should be cooled rapidly by a plate heat exchanger, and the temperature is kept at 4-10deg.C to inhibit bacterial reproduction;
(4) Homogenizing: preheating the mixture to 55 ℃ (50-60 ℃ all right), and then pumping the milk into a high-pressure homogenizer for homogenization;
(5) Sterilizing: sterilizing for 300s in a high-temperature environment of 90-95 ℃ by a heat exchanger;
(6) And (3) cooling: cooling the sterilized material to 40-42 ℃ and pumping the material into a fermentation tank;
(7) Inoculating: adding 0.1kg of lactobacillus reuteri HLRE05 into each ton of milk, and keeping the first-stage fermentation at 40 ℃ for 4 hours to avoid the interference of the fermentation of the lactobacillus reuteri with the acidification of the lactobacillus; adding glycerol according to the standard of 4.6kg per ton of milk, homogenizing, performing second fermentation until final acidity reaches 70 deg.C, stirring, cooling to room temperature (20-25deg.C), and packaging;
(8) And (3) refrigerating: and (5) transferring the product to a refrigeration house and refrigerating.
(9) And (3) a finished product: and (5) after the inspection is qualified, obtaining a finished product, and storing at 2-7 ℃.
Example 7:
(1) The fermented yoghurt prepared in example 6 is used as an experimental group, the commercial lactobacillus reuteri DSM20056 prepared by the method of example 6 is used as a control group, 0.01kg of food-borne pathogenic bacteria including staphylococcus aureus, listeria monocytogenes, escherichia coli and salmonella (1:1:1:1) are added, and the pollution is subjected to homogenizing simulation treatment. LB medium (1% tryptone, 0.05% yeast extract and 1% sodium chloride) plates were used for counting and dynamic monitoring of total changes in food-borne pathogens over 24h. The experimental results are shown in fig. 5, and it can be seen that the addition of lactobacillus reuteri HLRE05 can significantly inhibit the contamination of food-borne pathogenic bacteria in fermented yogurt products.
(2) The comparison results of physicochemical properties of the experimental group and the control group in this example are shown in fig. 6, and it can be seen that the fluctuation of the pH value, acidity and viscosity of the fermented yogurt of the present invention is relatively small and the sensory evaluation is significantly higher in the 28-day shelf life.
The present invention is not limited to the above embodiments, but is merely preferred embodiments of the present invention, and the present invention should be construed as being limited to the above embodiments as long as the technical effects of the present invention are achieved by the same means. Various modifications and variations are possible in the technical solution and/or in the embodiments within the scope of the invention.
Claims (10)
1. Lactobacillus reuteri strainLactobacillus reuteri) The HLRE05 is characterized in that the lactobacillus reuteri HLRE05 is preserved in China Center for Type Culture Collection (CCTCC) with the number of CCTCC M2022092 in the year 2022, 01 and 18.
2. Lactobacillus reuteri HLRE05 according to claim 1, characterized in that the sequence of the 16S rRNA of lactobacillus reuteri HLRE05 is shown in SEQ ID No. 1.
3. Use of lactobacillus reuteri HLRE05 according to claim 1 or 2 for the preparation of a microbial feed additive.
4. The preparation method of the microbial feed additive is characterized by comprising the following steps of: inoculating lactobacillus reuteri HLRE05 according to claim 1 or 2 into MRS liquid medium containing glycerol, and fermenting.
5. The method according to claim 4, wherein the fermentation conditions are: the fermentation temperature is 37 ℃, the inoculation amount is 1 percent, and the fermentation time is 24 hours.
6. The method according to claim 4, wherein the fermentation is further followed by spray drying under the following conditions: the material concentration is 10%, the air inlet temperature is 150 ℃, and the air outlet temperature is 75 ℃.
7. A microbial feed additive produced by the production method according to any one of claims 4 to 6.
8. Use of lactobacillus reuteri HLRE05 according to claim 1 or 2 for the preparation of fermented yoghurt.
9. A fermented yoghurt obtained from the fermentation of fresh milk by probiotics comprising lactobacillus reuteri HLRE05 according to claim 1 or 2.
10. A process for the production of fermented yoghurt as claimed in claim 9, comprising the steps of: adding 0.1kg of the lactobacillus reuteri HLRE05 into each ton of fresh milk, and fermenting for 4-5 hours at the temperature of 40 ℃; then adding glycerin according to the proportion of 4.6kg per ton of fresh milk, homogenizing, and then continuing fermentation under the same condition until the final acidity reaches 70 DEG T.
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| CN101946850A (en) * | 2009-12-11 | 2011-01-19 | 中国农业科学院饲料研究所 | Lactobacillus reuteri fermented liquid feed, preparation method and application thereof |
| CN105062933A (en) * | 2015-09-11 | 2015-11-18 | 北京博锦元生物科技有限公司 | Lactobacillus reuteri and application thereof |
| CN110734879A (en) * | 2019-11-13 | 2020-01-31 | 东北农业大学 | Lactobacillus reuteri LR-CO21 and application thereof |
| CN114806929A (en) * | 2022-03-29 | 2022-07-29 | 山东凤凰生物科技股份有限公司 | Lactobacillus reuteri LR4009 capable of highly producing reuterin and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101946850A (en) * | 2009-12-11 | 2011-01-19 | 中国农业科学院饲料研究所 | Lactobacillus reuteri fermented liquid feed, preparation method and application thereof |
| CN105062933A (en) * | 2015-09-11 | 2015-11-18 | 北京博锦元生物科技有限公司 | Lactobacillus reuteri and application thereof |
| CN110734879A (en) * | 2019-11-13 | 2020-01-31 | 东北农业大学 | Lactobacillus reuteri LR-CO21 and application thereof |
| CN114806929A (en) * | 2022-03-29 | 2022-07-29 | 山东凤凰生物科技股份有限公司 | Lactobacillus reuteri LR4009 capable of highly producing reuterin and application thereof |
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