CN110982737A

CN110982737A - Biofilm lactic acid bacteria agent for dogs and preparation method thereof

Info

Publication number: CN110982737A
Application number: CN201911219339.XA
Authority: CN
Inventors: 陈则东; 朱建平; 刘蕾; 马艳梅; 卢炜
Original assignee: Jiangsu Agri Animal Husbandry Vocational College
Current assignee: Jiangsu Agri Animal Husbandry Vocational College
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-04-10

Abstract

The invention relates to the technical field of lactic acid bacteria, in particular to a biofilm-state lactic acid bacteria agent for dogs and a preparation method thereof, wherein the preparation method comprises the following steps: inoculating lactobacillus into an improved MRS culture medium for activated culture, transferring the culture solution, flatly spreading the culture solution into a cell culture plate, and standing for culture to obtain an enveloped bacterial solution; and preparing the tunica membranaceus bacterial liquid into a biofilm-state lactic acid bacteria agent for dogs. The film-coated lactic acid bacteria agent prepared by the preparation method can obviously improve the average daily gain of dogs, reduce the material-weight ratio and have obvious regulation effect on canine intestinal flora.

Description

Biofilm lactic acid bacteria agent for dogs and preparation method thereof

Technical Field

The invention relates to the technical field of lactic acid bacteria, in particular to a biofilm-state lactic acid bacteria agent for dogs and a preparation method thereof.

Background

Dogs are one of the earliest domesticated animals and are currently the most prevalent companion animals in the world. The scale of feeding is extremely large worldwide. Such mass raising is due to great value of dogs, such as high intellectual level, social behavior, more developed sense organs than human, playing various roles in human society, often trained as working dogs, etc. Thus, the health of dogs is receiving increasing attention.

The health of canines is largely dependent on their gastrointestinal microorganisms, the composition and activity of their gut microflora being associated with several diseases. This microbial ecosystem plays a role in a variety of aspects, such as influencing the absorption and metabolism of nutrients, the nutrition and the protective function of the host. Any disturbance of the gastrointestinal flora may lead to the development of a number of diseases, such as diarrhoea, allergy, obesity and stress syndrome. Different treatments are being developed in order to balance the flora structure and to combat infections, among which probiotic therapy is of great interest.

Biofilm refers to a mass of bacterial biofilm-like material formed by bacteria adhering to a contact surface, secreting polysaccharide matrices, fibrin, lipoprotein, etc., and wrapping themselves around them. Microorganisms in the natural state often live in the form of a biofilm state, and have the advantages which are incomparable with the traditional culture state. At present, research on harmful microbial biofilms is more, research on probiotics, particularly lactobacillus biofilms is very rare, the probiotics and lactobacillus biofilms are suitable for dogs, average daily gain of dogs can be effectively improved, and no biofilm with an obvious regulation effect on canine intestinal flora is reported.

Disclosure of Invention

In view of the above, the present invention aims to provide a biofilm lactic acid bacterial agent for dogs and a preparation method thereof. The biomembrane state lactic acid bacteria agent provided by the invention can effectively improve the average daily gain of dogs and has an obvious regulation effect on canine intestinal flora.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a preparation method of a biofilm lactic acid bacteria agent for dogs, which comprises the following steps:

inoculating lactobacillus into an improved MRS culture medium for activated culture, transferring the culture solution, flatly spreading the culture solution into a cell culture plate, and standing for culture to obtain an enveloped bacterial solution; preparing the tunicate bacteria liquid into a biofilm lactic acid bacteria agent for dogs;

the improved MRS culture medium comprises a liquid improved MRS culture medium and a solid improved MRS culture medium, wherein the liquid improved MRS culture medium comprises the following components: 10g/L of peptone, 5g/L of yeast extract, 2g/L of diammonium citrate, 20g/L of glucose and MgSO₄·7H₂0.58g/L of O, 10g/L of beef extract and K₂HPO₄·3H₂O2 g/L, sodium acetate 5g/L, Tween 801 g/L, MnSO₄·4H₂O0.25 g/L, 0.1-1% signal factor AI-2; the solid modified MRS culture medium consists of the components and agar of 16 g/L.

In some embodiments, the Lactobacillus is Lactobacillus plantarum LR-1.

In some embodiments, the liquid modified MRS medium consists of: 10g/L of peptone, 5g/L of yeast extract, 2g/L of diammonium citrate, 20g/L of glucose and MgSO₄·7H₂0.58g/L of O, 10g/L of beef extract and K₂HPO₄·3H₂O2 g/L, sodium acetate 5g/L, Tween 801 g/L, MnSO₄·4H₂O0.25 g/L, 0.5% signal factor AI-2; the solid modified MRS culture medium consists of the components and agar of 16 g/L.

In some embodiments, the activated culture is specifically that lactobacillus is inoculated in a solid modified MRS culture medium for streak culture, and then a single colony is taken to be cultured in a liquid modified MRS culture medium.

In some embodiments, the streaking culture is a three-zone streaking culture.

In some embodiments, the transferring is specifically: and adding the liquid modified MRS culture medium into the culture solution according to the volume ratio of 1: 100.

In some embodiments, the temperature of the static culture is 30-37 ℃, and the static culture is carried out until the viable count is more than or equal to 1 × 10⁸CFU/mL. In some embodiments, the temperature of the static culture is 30 ℃, and the static culture is carried out until the viable count is 1 × 10⁸CFU/mL。

In some embodiments, the preparation of the biofilm bacteria solution into the biofilm lactic acid bacteria agent for dogs specifically comprises: and (3) taking the membrane-state bacterium liquid, and performing refrigerated centrifugation, enrichment, heavy suspension and freeze drying to obtain the biofilm-state lactic acid bacteria agent for dogs.

Further, the temperature of the freezing centrifugation is 4-8 ℃, the time is 5-10 min, and the rotating speed is 4000-6000 g. In some embodiments, the temperature of the refrigerated centrifugation is 4 ℃, the time is 5min, and the rotation speed is 5000 g.

In some embodiments, the resuspension is performed with skim milk powder. In some embodiments, the volume ratio of the skim milk powder to the enriched liquid in the enveloped state is 1: 10. In some embodiments, the number of viable bacteria after resuspension is greater than or equal to 1X 10⁹CFU/mL. In some embodiments, the number of viable bacteria after resuspension is 1X 10⁹CFU/mL。

In some embodiments, the temperature of the freeze drying is-80 ℃, and the time of the freeze drying is 36-48 h.

The invention also provides the biofilm state lactic acid bacteria agent for dogs prepared by the preparation method.

In some specific embodiments, the formulation of the biofilm lactic acid bacteria agent for dogs is freeze-dried powder.

The invention provides a dog biofilm lactic acid bacteria agent and a preparation method thereof, wherein the preparation method of the dog biofilm lactic acid bacteria agent comprises the following steps: inoculating lactobacillus into a solid modified MRS culture medium for streak culture, taking a single colony to culture in a liquid modified MRS culture medium, and then transferring and spreading a culture solution into a cell culture plate for standing culture to obtain an enveloped bacterial solution; and (3) taking the membrane-state bacteria liquid for freezing and centrifuging, enriching, resuspending and freeze-drying to obtain the biological membrane-state lactic acid bacteria agent for dogs. The invention adopts a specific process combined with an improved MRS culture medium to culture the lactobacillus, and the prepared tunica mucosa lactic acid bacteria agent can obviously improve the average daily gain of dogs, reduce the material-weight ratio and have obvious regulation effect on canine intestinal flora

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows canine blood IgG content in example 3;

FIG. 2 shows canine blood sIgA in example 3;

FIG. 3 shows the blood IL-6 levels in dogs of example 3;

FIG. 4 shows the canine blood TNF- α levels in example 3;

FIG. 5 shows the blood SOD levels in dogs according to example 3;

FIG. 6 shows the malondialdehyde content in blood of dogs in example 3;

FIG. 7 shows the performance of the canine fecal microflora at the phylum level in example 4;

FIG. 8 shows the performance of the canine fecal microbiota at class level in example 4;

FIG. 9 shows the behavior of the canine fecal microbial flora at the genus level in example 4.

Detailed Description

The invention discloses a biofilm lactic acid bacteria agent for dogs and a preparation method thereof, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

The invention is further illustrated by the following examples:

EXAMPLE 1 Lactobacillus plantarum LR-1 activation and preparation of biofilm-State lyophilized powder

Preparing a liquid modified MRS culture medium, a solid modified MRS culture medium and 0.85% of normal saline, sterilizing corresponding articles, drying and sterilizing an aseptic operation table.

The liquid modified MRS culture medium consists of the following components: 10g/L of peptone, 5g/L of yeast extract, 2g/L of diammonium citrate, 20g/L of glucose and MgSO₄·7H₂0.58g/L of O, 10g/L of beef extract and K₂HPO₄·3H₂O2 g/L, sodium acetate 5g/L, Tween 801 g/L, MnSO₄·4H₂O0.25 g/L, 0.5% signal factor AI-2; the solid modified MRS culture medium consists of the components and agar of 16 g/L.

Activated Lactobacillus plantarum LR-1: taking Lactobacillus plantarum LR-1 from a glycerol tube a little, standing and culturing at 30 ℃ until a single colony grows out, selecting the single colony in a liquid modified MRS culture medium, standing and culturing at 30 ℃ for 24h, then transferring the single colony with the liquid modified MRS culture medium according to the volume ratio of 1:100, and spreading the single colony in a cell culture plate, standing and culturing at 30 ℃ until the viable count reaches 10⁸CFU/mL, biofilm formation.

Collecting 10mL of the bacterial liquid, centrifuging at 4 ℃ for 5min at 5000g, discarding the supernatant, washing with sterile physiological saline once, centrifuging to collect bacterial sludge, suspending in 1mL of sterile skim milk, placing in a sterile penicillin bottle, and sealing with sterile gauze. Placing the penicillin bottle containing the bacterial liquid and a vacuum freeze dryer tray together in a refrigerator at the temperature of 80 ℃ below zero for pre-freezing for 2 to 3 hours, and then placing the penicillin bottle and the vacuum freeze dryer tray into a vacuum freeze dryer for freeze drying for 36 hours to obtain bacterial powder, namely the biofilm peptide lactic acid bacteria agent.

Example 2 Effect of the biofilm-state lactic acid bacteria agent of the present invention on body weight and digestibility

1. Feeding with lactobacillus

Lactic acid prepared in example 1The microbial inoculum is diluted by 1mL of sterile normal saline, and the total bacterial count is 10⁹CFU/mL, the total volume is 1mL, a 4-month-old beagle dog is fed by adopting an injector, 5 dogs in a control group, a floating state treatment group and a capsule state treatment group respectively and a floating state treatment group are adopted, and the preparation method of the floating state is as follows: inoculating lactobacillus into a solid modified MRS culture medium for streak culture, taking a single colony to culture in a liquid modified MRS culture medium, transferring a culture solution into a test tube, and performing shake culture at 30-37 ℃ until the viable count is at least 10⁸CFU/mL to obtain planktonic bacteria liquid; taking the floating state bacterial liquid for refrigerated centrifugation, enrichment, heavy suspension and freeze drying until the viable count is at least 10⁹CFU/mL, which is the planktonic lactic acid bacteria agent for dogs. The control group was fed the same volume of sterile normal saline.

2. Body weight determination

The body weights of the tested dogs are detected and recorded, and the average daily gain and the material weight ratio are calculated, and the results are shown in table 1.

TABLE 1

Note: the different letters of the shoulder marks in the same row show significant differences (P < 0.05).

From the results in table 1, it can be seen that the modal treated group of the invention significantly increased the average daily gain of dogs (P <0.05) and significantly decreased the feed-to-weight ratio (P <0.05) compared to the control group and the planktonic treated group.

3. Determination of digestibility

Dog faeces of the control and test groups were collected at 50g per sample and 7 items of moisture, crude protein, crude fat, crude fibre, calcium, total phosphorus and ash were measured separately and the total raw composition of the feed was compared to calculate the digestibility of 6 of these items as shown in table 2.

TABLE 2

Note: the shoulder marks in the same row show significant differences (P <0.05) in different lower case letters, and show significant differences (P < 0.01) in different upper case letters.

As can be seen from Table 2, the biofilm freeze-dried lactic acid bacteria for dogs can remarkably improve the digestibility of crude protein, crude fat, crude fiber and total phosphorus, and has a remarkable difference (P is less than 0.05) compared with the planktonic treatment group; significant difference in crude protein digestibility compared to the control group (P < 0.05); the total phosphorus digestibility of the fat, the fiber and the phosphorus were significantly different from that of the control group (P < 0.05).

Example 3 Effect of the biofilm lactic acid bacteria agent of the present invention on blood indices

1. Feeding with lactobacillus

The same procedure as in example 2 was used for feeding the lactic acid bacteria.

2. Blood index measurement

ELISA kit is used for detecting the content of IgG, sIgA, IL-6 and TNF- α in the canine serum, and biochemical index detection kit is used for detecting the content of SOD and malondialdehyde.

The results are shown in FIGS. 1 to 6, which indicate that the mean IgG content of the treated group was increased.

Example 4 microbial diversity in feces after dogs received biofilm lactic acid bacteria

1. Feeding with lactobacillus

2. Feces collection

Feces were collected at 28 days after feeding, 50g of each sample, and frozen in liquid nitrogen.

3. Genome extraction and library construction

And extracting a genome of the collected sample, constructing a library of the qualified sample for detection, performing cluster preparation and sequencing on the qualified library, and performing corresponding biological information analysis on data obtained by off-machine analysis.

4. Data analysis

The data of the off-line terminal is filtered to remove low-quality reads and leave high-quality Clean data; sample species complex analysis as well as inter-group species difference analysis were performed based on OUT and species annotation results. Diversity was determined based on the sequence of the 16SrDNA variable region. The results are shown in FIGS. 7 to 9. As can be seen from the graphs in FIGS. 7 to 9, the microbial diversity in the dog feces of the membrane-state treated group is remarkably changed, wherein the content of lactobacillus is remarkably increased, which indicates that the lactobacillus of the membrane-state treated group has more field planting numbers, and thus the membrane-state treated group has a better regulation effect on dog intestinal flora.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. a preparation method of biofilm state lactic acid bacteria agent for dogs, is characterized in that, comprises the following steps:

The lactobacillus is inoculated in the modified MRS medium for activation and culture, and the culture solution is transferred and flattened into a cell culture plate for static culture to obtain a membrane state bacterial solution; the membrane state bacteria solution is made into a canine biofilm state Lactic acid bacteria agent;

The modified MRS medium includes a liquid modified MRS medium and a solid modified MRS medium, and the liquid modified MRS medium is composed of the following components: peptone 10g/L, yeast extract 5g/L, diammonium citrate 2g/L L, glucose 20g/L, MgSO ₄ 7H ₂ O 0.58g/L, beef extract 10g/L, K ₂ HPO ₄ 3H ₂ O 2g/L, sodium acetate 5g/L, Tween 80 1g/L, MnSO ₄ ·4H ₂ O 0.25g/L, 0.1%-1% signal factor AI-2; the solid modified MRS medium consists of the above components and agar 16g/L.

2 . The preparation method according to claim 1 , wherein the lactobacillus is Lactobacillus plantarum LR-1. 3 .

3. preparation method according to claim 1, is characterized in that, described liquid MRS medium is made up of following components: peptone 10g/L, yeast extract 5g/L, diammonium citrate 2g/L, glucose 20g /L, MgSO ₄ 7H ₂ O 0.58g/L, beef extract 10g/L, K ₂ HPO ₄ 3H ₂ O 2g/L, sodium acetate 5g/L, Tween 80 1g/L, MnSO ₄ 4H ₂ O 0.25g/L, 0.1%-1% signal factor AI-2; the solid modified MRS medium consists of the above components and agar 16g/L.

4. preparation method according to claim 1, is characterized in that, described activation culture is specifically inoculating lactobacillus in solid improved MRS medium for streak culture, then gets single colony and cultivates in liquid improved MRS medium .

5. The preparation method according to claim 1, characterized in that, the transfer is specifically adding the liquid modified MRS medium to the culture medium according to a volume ratio of 1:100.

6 . The preparation method according to claim 1 , wherein the temperature of the static culture is 30° C. to 37° C., and the static culture is performed until the number of viable bacteria ≥ 1×10 ⁸ CFU/mL. 7 .

7. preparation method according to claim 1, it is characterised in that described filmed state bacterial liquid is made into canine biofilm state lactic acid bacteria agent is specifically: get described filmed state bacterial liquid through freezing centrifugation, enrichment, re-weighting. After suspension and freeze-drying, the lactic acid bacteria agent in biofilm state for dogs is prepared.

8. preparation method according to claim 7 is characterized in that, the temperature of described freezing centrifugation is 4～8 ℃, time is 5～10min, rotating speed is 4000～6000g; 1×10 ⁹ CFU/mL; the freeze-drying temperature is -80° C. and the time is 36-48 h.

9. The canine biofilm state lactic acid bacteria agent prepared by the preparation method of any one of claims 1-8.

10 . The biofilm-state lactic acid bacteria agent for dogs according to claim 9 , wherein the dosage form of the canine biofilm-state lactic acid bacteria agent is freeze-dried powder. 11 .