A kind of Lactobacillus amylovora ZC01 and its application
By screening Lactobacillus acetobili ZC01 from corn slurry and determining its optimal growth conditions, the existing Lactobacillus acetobili is solved, and its wide application and safety in the fields of livestock breeding, food and medicine has been achieved.
Patent Information
- Application Number
- CN202411820798.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-04-04
AI Technical Summary
The limited source and safety issues of existing Lactobacillus acetica limit their scope of application and value.
Lactobacillus acetica ZC01 was screened from corn slurry and its optimal growth conditions were determined, including a temperature of 34°C and an environment with pH=6 to ensure its safety and applicability.
Lactobacillus starchycete ZC01 has low fermentation costs, is suitable for commercialization, and shows antibacterial and virus properties. It is suitable for animal husbandry, food and pharmaceutical fields.
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Figure CN119752691A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a starch-eating lactobacillus ZC01 and an application thereof. Background Art
[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.
[0003] Lactic acid bacteria (LAB) are a group of Gram-positive bacteria that can ferment sugars and produce lactic acid as the main metabolite. They are commonly found in dairy products, pickled products, and the intestinal mucosa of humans or animals. Lactic acid bacteria have been found to have the effects of inhibiting the growth of gastrointestinal pathogens, alleviating lactose intolerance, immunoregulation, anti-cancer, and lowering blood pressure. There are many types of lactic acid bacteria, such as Lactobacillus, Lactococcus, Pediococcus, Streptococcus, and Enterococcus.
[0004] Lactobacillus amylovorus is a type of lactic acid bacteria that has antibacterial and antiviral properties. Among them, Lactobacillus amylovorus from pig intestines has always been valued, and scholars around the world have conducted extensive research on Lactobacillus amylovorus. However, due to the limited source of Lactobacillus amylovorus and safety issues, its application scope and application value are also greatly limited. Summary of the invention
[0005] In view of the shortcomings of the prior art, the first object of the present invention is to provide a Lactobacillus amylovora ZC01.
[0006] The second object of the present invention is to provide the application of the above-mentioned Lactobacillus amylovora ZC01.
[0007] To achieve the above object, the present invention adopts the following technical solution: A starch-eating lactobacillus ZC01, the storage date of the starch-eating lactobacillus ZC01 is July 1, 2024, the storage location is the General Microbiology Center of China Microorganism Culture Collection Administration, and the storage number is CGMCC No.31126.
[0008] Furthermore, the Lactobacillus amylovora ZC01 is activated in a solid MRS culture medium and then cultured in a liquid MRS culture medium.
[0009] Furthermore, the Lactobacillus amylovora ZC01 is cultured at 34°C.
[0010] Furthermore, the Lactobacillus amylovora ZC01 is cultured in an environment of pH=6.
[0011] Furthermore, the Lactobacillus amylovora ZC01 is cultured in an anaerobic environment.
[0012] The application of the above-mentioned Lactobacillus amylovora ZC01 is used in the fields of animal husbandry, medicine and food.
[0013] Beneficial effects: (1) The present invention screens out amylovora bacillus ZC01 from corn pulp for the first time and screens out the optimal growth conditions. The fermentation cost of the amylovora bacillus ZC01 is low, which is conducive to commercialization. The safety evaluation results show that the amylovora bacillus ZC01 is non-hemolytic and does not produce azoreductase and nitroreductase.
[0014] (2) Lactobacillus amylovora ZC01 has antibacterial and antiviral properties and can be used in animal husbandry, food and pharmaceutical fields. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein: Figure 1 This is a diagram of the genome sequencing results of Lactobacillus amylovora ZC01; Figure 2 is the phylogenetic tree of Lactobacillus amylovora ZC01; Figure 3 is the OD of Lactobacillus amylovora ZC01 cultured at different temperatures and pH values. 600 Statistical charts, Figure 3 a is the OD of Lactobacillus amylovora ZC01 at different temperatures 600 Statistical charts, Figure 3 b is the OD of Lactobacillus amylovora ZC01 cultured at different pH values 600 Statistical charts; Figure 4 This is the result of hemolytic test. Figure 4 a is Bacillus subtilis Hemolytic test results chart, Figure 4 b is E. coli DH5α hemolytic test results chart, Figure 4 c is a graph showing the hemolytic activity test results of Lactobacillus amylovora ZC01; Figure 5 Schematic diagram of the ability to produce azoreductase. Figure 5 a is a schematic diagram of the azoreductase production ability of Lactobacillus amylovora ZC01. Figure 5 b is Bacillus subtilis Schematic diagram of azoreductase production capacity; Figure 6 This is a schematic diagram of the ability of Lactobacillus amylovora ZC01 to produce bioamines. Figure 6 a is a schematic diagram of the tyrosine production ability of Lactobacillus amylovora ZC01. Figure 6 b is a schematic diagram of the histidine production ability of Lactobacillus amylovora ZC01; Figure 7 This is a schematic diagram of the nitroreductase production ability of Lactobacillus amylovora ZC01. Figure 7 a is a schematic diagram of the nitroreductase production capacity of LB liquid culture medium, Figure 7 b is a schematic diagram of the nitroreductase production ability of Lactobacillus amylovora ZC01. Figure 7 c is E. coli Schematic diagram of DH5α's ability to produce nitroreductase. DETAILED DESCRIPTION
[0016] The following description further describes the specific details of the present invention for a full understanding of the present invention. The terms used in the description of the present invention are only used to illustrate the advantages and features of the present invention and are not intended to limit the present invention.
[0017] Unless otherwise defined, all professional and scientific terms used in the present invention have the same meanings as those understood by technicians in the technical field of the present invention. Unless otherwise specified, the drugs or reagents used in the present invention are used in accordance with the product instructions or the conventional methods of use in the field. The technical solution of the present invention is further described according to the drawings and specific embodiments of the specification.
[0018] The culture medium and formulation used in the following examples are: Seed culture medium: glucose 40-150 g / L; ammonium sulfate 0-15 g / L; potassium dihydrogen phosphate 0-5 g / L; magnesium sulfate 0-5 g / L; inorganic salts 0-200 mg / L; vitamins 0-200 mg / L; corn steep liquor powder 1-40 g / L.
[0019] Fermentation medium: glucose 40-150 g / L; ammonium sulfate 0-15 g / L; potassium dihydrogen phosphate 0-5 g / L; magnesium sulfate 0-5 g / L; inorganic salts 0-200 mg / L; vitamins 0-200 mg / L; corn steep liquor powder 1-40 g / L; feed medium is supplemented during the fermentation process.
[0020] Feed medium: glucose 400-600 g / L. The feeding principle is to add feed when the residual sugar is lower than 2.0 g / L.
[0021] Culture conditions: culture temperature 34℃, pH=6.
[0022] Seed culture medium, fermentation medium and feed medium generally contain carbon source, nitrogen source, inorganic salt and vitamin. Glucose is used as carbon source; ammonium sulfate is used as inorganic nitrogen source; potassium dihydrogen phosphate is phosphorus source; inorganic salt is a cofactor of enzyme; vitamin generally acts as coenzyme component; corn steep liquor powder is organic nitrogen source.
[0023] Lactobacillus amylovora is fermented at high density, the cells are collected by centrifugation, and then freeze-dried in a freeze dryer.
[0024] Example 1 Screening of Lactobacillus amylovora: Corn processing enterprises obtain dilute corn slurry samples from corn soaking tanks or dilute corn slurry storage tanks. Use the dilution spreading method to isolate single clones, use MRS solid medium, anaerobic culture, 30°C. Pick the largest number of clones for expansion culture, and then perform 16S rDNA PCR sequencing identification.
[0025] Example 2 (1) Morphological identification ① Colony characteristics Color and transparency: Colonies of Lactobacillus amylovora are usually white, smooth, with clean edges and opaque.
[0026] Shape and size: The colony shape can be round or nearly round, soft in texture, and without pigment. On the culture medium, the colony size may be small (such as 2-3 mm).
[0027] Growth conditions: Lactobacillus amylovora grows well in suitable culture medium and conditions. Its optimal pH is 6, the culture medium is RMS medium, and the optimal temperature is 34°C.
[0028] ②Cell morphology Cell shape: Lactobacillus amylovora is a Gram-positive bacterium with diverse cell morphologies. It can be convexly round, arranged in chains, or exist singly.
[0029] Other characteristics: no spores, no production of metabolites such as indole and hydrogen sulfide.
[0030] ③Dyeing characteristics Gram stain: Lactobacillus amylovora is a Gram-positive bacterium and appears purple in Gram stain.
[0031] (2) Molecular biological identification The 16S rDNA sequence result of Sangon Biotechnology (Shanghai) Co., Ltd. is shown in SEQ ID No.1.
[0032] SEQ ID No.1: Figure 1 This is the result of genome sequencing of Lactobacillus amylovora ZC01. The results show that the whole genome of Lactobacillus amylovora ZC01 is 2.06 Mb in length, with a GC content of 37.98% and 2075 genes.
[0033] Figure 2 This is the phylogenetic tree of Lactobacillus amylovora ZC01.
[0034] Example 3 Optimization of temperature and pH for Lactobacillus amylovora.
[0035] Take the culture medium with the same composition and pH, culture it at different temperatures for the same time, and measure the OD 600 value to determine the optimum temperature.
[0036] Take the culture medium with the same composition and different pH values, culture it at the optimal temperature for the same time, and measure the OD 600 The optimum pH was determined by the value of
[0037] Figure 3 is the OD of Lactobacillus amylovora ZC01 cultured at different temperatures and pH values. 600 Statistical charts, Figure 3 a is the OD of Lactobacillus amylovora ZC01 at different temperatures 600 Statistical charts, Figure 3 b is the OD of Lactobacillus amylovora ZC01 cultured at different pH values 600 The statistical results show that the optimal culture temperature of Lactobacillus amylovora ZC01 is 34°C, and the optimal culture pH is about 6.0.
[0038] Performance Testing (1) Hemolytic evaluation Figure 4 This is the result of hemolytic test. Figure 4 a is Bacillus subtilis Hemolytic test results chart, Figure 4 b is E. coli DH5α hemolytic test results chart, Figure 4 c is the hemolytic test result of Lactobacillus amylovora ZC01, and the results show that Lactobacillus amylovora ZC01 is not hemolytic.
[0039] (2) Detection of azoreductase production capacity Figure 5 Schematic diagram of the ability to produce azoreductase. Figure 5 a is a schematic diagram of the azoreductase production ability of Lactobacillus amylovora ZC01. Figure 5 b is Bacillus subtilisSchematic diagram of azoreductase production ability. The results show that there is no obvious hydrolysis zone around the colony of Lactobacillus amylovora ZC01, but there is an obvious hydrolysis zone around the colony of B. Subtilis. Figure 5 a and Figure 5 The culture medium in b was prepared by coating Direct Blue 71 on the surface of LB solid culture medium.
[0040] (3) Biogenic amine capacity detection Figure 6 This is a schematic diagram of the ability of Lactobacillus amylovora ZC01 to produce bioamines. Figure 6 a is a schematic diagram of the tyrosine production ability of Lactobacillus amylovora ZC01. Figure 6 b is a schematic diagram of the histidine production ability of Lactobacillus amylovora ZC01. The results showed that after Lactobacillus amylovora ZC01 was subcultured for 10 days and cultured in tyrosine detection medium and histidine detection medium for three days respectively, the color was not much different from that of the blank group. Lactobacillus amylovora ZC01 does not have the ability to produce azoreductase.
[0041] (4) Antibiotic resistance genes The results of whole genome sequencing resistance gene screening showed that Lactobacillus amylovora ZC01 had no resistance genes.
[0042] (5) Detection of nitroreductase production capacity Figure 7 This is a schematic diagram of the nitroreductase production ability of Lactobacillus amylovora ZC01. Figure 7 a is a schematic diagram of the nitroreductase production capacity of the blank control (LB liquid culture medium), Figure 7 b is a schematic diagram of the ability of starch-eating bacteria ZC01 to produce nitroreductase. Figure 7 c is a schematic diagram of the nitroreductase production ability of E. coli DH5α. The results show that the color of the culture medium inoculated with E. coli DH5α changed from yellow to red, while the color of the culture medium inoculated with strain Lactobacillus amylovora ZC01 did not change. The strain Lactobacillus amylovora ZC01 does not have the ability to produce nitroreductase and cannot reduce the nitrate in the environment to the harmful metabolite nitrite.
[0043] Application Examples Animal experiments were conducted on 817 broiler chickens. The experimental period was 21 days, standard broiler chicken feed was used, and feeding and management were carried out according to standard broiler feeding and management. Amoxicillin was added to the drinking water of the positive control group; only drinking water was added to the negative control group. Lactobacillus amylovora was added to the drinking water of the experimental group. The number of chicks in each group was 96.
[0044] Lactobacillus amylovora was added to the drinking water of 817 broilers, and the final addition amount after dilution was 10 6 -10 7 CFU / mL. The water was replaced every day.
[0045] The experimental results are shown in Table 1. The results show that the mortality rate of the experimental group with added Lactobacillus amylovora is the same as that of the positive control group (amoxicillin), and the mortality rate is lower. The mortality rate of the negative control group is higher. In addition, after adding Lactobacillus amylovora, the chicks are in good condition and their feed intake increases.
[0046] Table 1 Statistics of broiler chicken mortality The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials used in the present invention, addition of auxiliary raw materials, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A Lactobacillus amylovora ZC01, characterized in that The preservation date of the Lactobacillus amylovora ZC01 is July 1, 2024, the preservation location is General Microbiology Center of China Microorganism Collection Administration, and the preservation number is CGMCC No.31126.
2. The Lactobacillus amylovora ZC01 according to claim 1, characterized in that The Lactobacillus amylovora ZC01 is activated and cultured in MRS culture medium.
3. The Lactobacillus amylovora ZC01 according to claim 1, characterized in that The Lactobacillus amylovora ZC01 is cultured at 34°C.
4. The Lactobacillus amylovora ZC01 according to claim 1, characterized in that The amylovorax Lactobacillus ZC01 is cultured in an environment of pH=6.
5. The Lactobacillus amylovora ZC01 according to claim 1, characterized in that The Lactobacillus amylovora ZC01 is cultured under an anaerobic environment.
6. The use of Lactobacillus amylovora ZC01 according to any one of claims 1 to 4, characterized in that: The amylovorax lactobacillus ZC01 is used in the fields of animal husbandry, food or medicine.