CN110016452B

CN110016452B - Butyric acid producing bacterium DG1 with probiotic activity and culture method and application thereof

Info

Publication number: CN110016452B
Application number: CN201910361921.3A
Authority: CN
Inventors: 李升康; 李忠贞; 孙再桥; 黎照熙
Original assignee: Shantou University
Current assignee: Shantou University
Priority date: 2019-04-30
Filing date: 2019-04-30
Publication date: 2022-12-20
Anticipated expiration: 2039-04-30
Also published as: CN110016452A

Abstract

The invention relates to a butyric acid producing bacterium DG1 with probiotic activity, a culture method and application thereof, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2019072. The butyric acid producing bacteria (Proteocatella sphenisci DG 1) with probiotic activity and the synbiotic PSGS obviously improve the growth performance of fish, improve the activity of intestinal digestive enzyme, change the composition and diversity index of intestinal flora, enrich a plurality of SCFA producing bacteria and increase the content of SCFAs in fish intestines. In addition, the immune system of the light-colored spotted maigre can be activated by regulating the serum complement and cytokine levels, the lysozyme activity and the intestinal antioxidant capacity. The antibiotic testing solution is hemolysis-free, sensitive to tested antibiotics, free of drug resistance and high in safety; can be used as feed additive for light color spotted maigre to promote growth and prevent and treat diseases.

Description

Butyric acid producing bacterium DG1 with probiotic activity and culture method and application thereof

Technical Field

The invention belongs to the field of butyric acid producing bacteria, and particularly relates to a butyric acid producing bacteria DG1 with probiotic activity, and a culture method and application thereof.

Background

Light-colored spotted maigre (Nibea Coibor), popular name is kaempferia galamga, golden thread salamander belongs to the family of stone-headed fish, the genus spotted maigre, is an important mariculture economic fish in China, and a swim bladder product of the spotted maigre, namely fish glue, is a high-grade tonic in the market and is favored by consumers. However, the high-density cultivation of the light-color spotted maigre is greatly damaged by bacterial diseases, so that the survival rate of adult fish cultivation is low, and the development of the cultivation industry of the light-color spotted maigre is directly influenced. Antibiotics and chemical drugs play an important role in preventing and treating diseases of aquatic animals, but the negative effects of bacterial drug resistance, drug residues and the like caused by the antibiotics and the chemical drugs are increasingly obvious, and meanwhile, due to the high concern of people on the quality safety of aquatic products, the ecological culture is increasingly paid attention to. At present, probiotics have obvious effects on promoting the growth of aquatic animals, improving the culture environment and enhancing the disease resistance, but the probiotics used in aquatic products only comprise lactobacillus, streptococcus, bacillus, bifidobacterium, saccharomycetes and the like. Therefore, the development of new probiotic strains is of far-reaching importance for the sustainable development of aquaculture.

Butyric acid producing bacteria as new probiotics become a leading edge and a hot spot of intestinal probiotic research in recent years. However, the number of butyric acid producing strains which are successfully separated is rare, only one kind of clostridium butyricum is applied to aquaculture, the systematic research on the clostridium butyricum is not well known and is not thorough, and the basic research work in the future needs more attention.

Many studies refer to the M2GSC medium isolation method reported by Barcenella et al (2000), and judge whether butyric acid producing bacteria are butyric acid producing bacteria by using a threshold value of butyric acid production in vitro of more than 2mM. However, the culture medium is rich in nutrition, satisfies the growth of most microorganisms in intestinal tracts, causes large separation workload, has certain blindness, is not convenient to make judgment in a short time, and limits deep understanding of the group. Barcenella et al (2000) isolated 313 butyric acid producing bacteria from fresh feces of 3 healthy persons (infants, adult vegetarians and adult omnivores) using anaerobic roller tube technology, wherein the butyric acid producing bacteria isolated from the feces of infants had the highest concentration of butyric acid, the largest variety of butyric acid producing bacteria, and the second to vegetarians, the feces of omnivores had significantly fewer varieties of butyric acid producing bacteria than the first two. Studies have shown that the synthesis of intestinal butyrate depends on the number and type of butyric acid producing bacteria in the gut, as well as on the number and type of carbohydrates entering the posterior gut. This is also the focus of the present invention, where it is desirable to enrich butyric acid producing bacteria in vitro with oligosaccharides and sugar alcohols in order to isolate probiotic strains with potential applications.

The safety problem of probiotics is always concerned, and researchers at home and abroad propose various safety evaluation indexes and methods, such as hemolysis, harmful metabolites, virulence factor detection, antibiotic resistance, in vitro evaluation of the decomposition capacity of gastrointestinal mucosal protein and animal model evaluation (von girl, 2017). Whether the strain has hemolysis or not is a prerequisite condition for screening probiotics, and the conventional hemolysis evaluation method is mainly carried out by blood plate culture. On the other hand, the widespread of bacterial resistance and the emergence of multidrug-resistant strains have become serious public health problems, which seriously affect human health and deteriorate the breeding environment if resistance genes carried by probiotics are transferred in the breeding environment and food.

Disclosure of Invention

The invention aims to provide a butyric acid producing bacterium DG1 with probiotic activity and a culture method and application thereof, so as to solve the problems in the prior art.

The invention respectively carries out in-vitro anaerobic fermentation experiments with intestinal contents of animals, and fermentation liquor with higher butyric acid yield is used for diluting and plating isolated single bacteria. After each strain is cultured in vitro for 24 hours, the butyric acid yield is more than 2mM, and the butyric acid producing bacteria are obtained. In a culture test of the light-colored spotted maigre, the growth performance and the immune response of the light-colored spotted maigre can be improved by adding the strain Proteocarella spenisci DG1 and the synbiotics thereof into the feed, and the host health and the intestinal microecology are improved. A butyric acid bacterium DG1 (a proteotella spinesci DG 1) is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 1 month and 22 days, wherein the preservation address is Wuhan university in Wuhan City, hubei province in China, and the preservation number is CCTCC NO. M2019072.

Further, the butyric acid producing bacterium DG1 with probiotic activity is sensitive to antibiotics.

Further, the antibiotics include piperacillin, carbenicillin, ampicillin, oxacillin, penicillin, ceftazidime, cefuroxime, cephradine, cefazolin, cephalexin, kanamycin, gentamicin, amikacin, cefoperazone, ceftriaxone, erythromycin, minocycline, doxycycline, tetracycline, neomycin.

The culture method of the butyric acid bacteria DG1 with probiotic activity adopts a PYAG culture medium which mainly comprises 5g of peptone, 5g of tryptone, 10g of yeast extract and Na ₂ CO ₃ 4g, 5mg of hemin ₂ HPO ₄ 0.4g，KH ₂ PO ₄ 0.04g，NaHCO ₃ 0.08g，NaCl 0.04g，CaCl ₂ 8mg，MgSO ₄ ·7H ₂ O1.9 mg, vitamin K1 mg, sodium acetate 4.49g and glucose 5g, adjusting pH to 6.8, adding double distilled water to reach the volume of 1L.

The application of the butyric acid bacteria DG1 with probiotic activity is applied to feed.

Further, a feed for light-colored spotted maigre, which comprises 10 ⁷ cfu/g DG1 or 10 ⁷ cfu/g DG1+0.5% D-sorbitol +0.5% galacto-oligosaccharide.

Compared with the prior art, the butyric acid bacteria DG1 with probiotic activity and the synbiotics PSGS thereof obviously improve the growth performance of fish, improve the activity of intestinal digestive enzymes, change the composition and diversity index of intestinal flora, enrich a plurality of SCFA producing bacteria and increase the content of SCFAs in fish intestines. In addition, the immune system of the light-colored spotted maigre can be activated by regulating the serum complement and cytokine levels, the lysozyme activity and the intestinal antioxidant capacity. The antibiotic testing solution is hemolysis-free, sensitive to tested antibiotics, free of drug resistance and high in safety; can be used as feed additive for light color spotted maigre to promote growth and prevent and treat diseases.

Drawings

FIG. 1 shows that the initial sample butyric acid content and the butyric acid content produced by fermentation of fructo-oligosaccharide and resistant starch are lower than the detection limit in the case of producing butyric acid by fermentation of 10 oligosaccharides or sugar alcohols from intestinal contents of two kinds of spotted maigre;

FIG. 2 shows butyric acid production by fermentation of 5 oligosaccharides or sugar alcohols from intestinal contents of two species of spotted maigre, wherein P is <0.05 and P is <0.01;

FIG. 3 shows colony morphology (A), scanning electron micrograph (B), and gram stain (C) of DG 1;

FIG. 4 shows the growth curve and SCFA production law of DG1 strain;

FIG. 5 is a graph of the effect of DG1 strain on β -diversity of gut flora of spotted maigre, a non-metric multidimensional scaling (NMDS) plot based on Weighted-unifrac distances of gut flora fed on 3 different feeds (PS, PSGS and Control, respectively); each point represents a sample;

FIG. 6 is the effect of DG1 strain on the abundance of dominant intestinal bacteria (phylum level) of light color spotted maigre; lower case letters without identity indicate significant differences from each other (P < 0.05);

FIG. 7 is an intestinal differential bacteria analysis of light-colored spotted maigre fed with three different feeds; in the LEfSe analysis, when the alpha value of Kruskal-Wallis and Wilcoxon tests is less than 0.05 and the logarithmic LDA score reaches 3.5, the relative abundance of the bacterial groups among the treatment groups is considered to have significant change;

FIG. 8 is an analysis of the intestinal differential pathway of light-colored spotted maigre fed with three different feeds;

FIG. 9 is the effect of DG1 strain on the intestinal short-chain fatty acid content of light-colored spotted maigre: (a) acetic acid, (B) propionic acid, (C) butyric acid, (D) SCFAs; data are mean ± sem (n = 3); lower case letters without identity indicate significant differences from each other (P < 0.05);

FIG. 10 shows the indexes of DG1 strain on digestive enzyme activity, immunity and oxidation resistance of light-colored spotted maigre; lower case letters without identity indicate significant differences from each other (P < 0.05).

Detailed Description

To make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Example 1

1. Preparing an anaerobic PBS buffer solution, a liquid culture medium and a solid culture medium:

PBS buffer: naCl 8g, KCl 0.2g, na ₂ HPO ₄ 1.42g，KH ₂ PO ₄ 0.24g or NaHPO ₄ ·12H ₂ And 3.58g of O, adjusting the pH value to 7.4, and adding double distilled water to reach the constant volume of 1L.

PY medium: peptone 5g, trypticasePeptone 5g, yeast extract 10g, na ₂ CO ₃ 4g, hemin 5mg, K ₂ HPO ₄ 0.4g，KH ₂ PO ₄ 0.04g，NaHCO ₃ 0.08g，NaCl 0.04g，CaCl ₂ 8mg，MgSO ₄ ·7H ₂ O1.9mg and Vitamin K1 1mg, adjusting the pH value to 6.8, and adding double distilled water to reach the constant volume of 1L. PYA culture medium is PY culture medium added with 4.49g of sodium acetate; the PYAG culture medium is PY culture medium added with 4.49g of sodium acetate and 5g of glucose; adding 15g of agar into the solid culture medium; the stock solution concentration of oligosaccharide and sugar alcohol was 0.5g/mL; .

Preparing an anaerobic culture medium: accurately weighing each component for preparing a culture medium (comprising PY, PYA, PYAG culture medium, oligosaccharide or sugar alcohol stock solution), adding water according to a certain sequence to dissolve in a narrow-mouth flask, heating and magnetically stirring while introducing high-purity nitrogen, adding 0.1% resazurin solution according to the proportion of 1.

2. In vitro anaerobic fermentation of oligosaccharides or sugar alcohols with intestinal contents

Firstly, wiping the whole body of the light-colored spotted maigre by using absorbent paper, then spraying 75% alcohol on the whole body, transferring the whole body to an ultra-clean workbench for dissection, taking the intestinal tract of the spotted maigre, extruding contents into a weighed centrifugal tube by using a pair of sterilization forceps, and quickly transferring the weighed contents to an anaerobic operation platform after weighing. Diluting and homogenizing by using sterilized deoxyPBS buffer solution according to the mass-volume ratio of 1. The results show that the butyric acid production of the intestinal contents of two spotted maigre is different under the same prebiotic fermentation, as shown in figure 1. For light-colored spotted maigre, the prebiotics with the highest butyric acid yield in the fermentation liquor are inulin, and then D-sorbitol and galactooligosaccharide GOS are added; for the spotted maigre, the prebiotics of the first three butyric acid yields in the fermentation liquor are xylo-oligosaccharide XOS, GOS and resistant starch RS respectively.

Therefore, the prebiotics (three for each fish) with better primary screening effect are selected for secondary screening, the inoculum concentration is increased to 10 percent, the operation is the same as the operation, 2mL fermentation liquor is taken to measure the concentration of the SCFA after the culture is carried out for 24 hours at 28 ℃ by using sterilized deoxidized PBS buffer solution. The results show that the tested prebiotics both significantly promote the capacity of the intestinal contents of two spotted maigre to produce acetic acid and propionic acid through in-vitro fermentation, thereby causing the total SCFAs to be also significantly increased, as shown in figure 2. For light-colored spotted maigre, inulin and GOS have obvious promotion effect on butyric acid generation; for the spotted maigre, the yield of butyric acid can be obviously improved by XOS, GOS and RS.

The rest fermentation liquor is sterilized deoxidized PBS buffer solution according to the mass-to-volume ratio of 10 ^-4 -10 ^-7 Diluting and plating (PYA + oligosaccharide or sugar alcohol culture medium), culturing at 28 deg.C for 48 hr, selecting single colony with different morphology, liquid culturing, culturing at 28 deg.C for 24 hr, and performing chromatography to determine SCFA yield, wherein the butyric acid yield of strain numbered DG1 is 4.69 + -0.08 mM, and the butyric acid yield is>A strain at 2mM can be considered a butyric acid producing bacterium. And D-sorbitol is enriched, so that DG1 butyric acid producing bacteria are separated from the light-color spotted maigre intestinal flora fermentation liquid. The DG1 strain was inoculated into two media, one was PYAG and the other was PY +0.5% D-sorbitol. DG1 produced butyric acid in the amount of 27.76. + -. 0.28mM on PYAG medium and 4.35. + -. 0.22mM on PY +0.5% D-sorbitol medium. On the one hand, the reason is probably that more butyric acid is produced within 24h because glucose is a carbon source which is easily utilized by bacteria; on the other hand it may be that part of the butyric acid producing bacteria can be produced by butyryl COA: the acetyl COA transferase pathway produces butyrate, which requires the consumption of acetate to synthesize butyrate, and the addition of acetate to PYAG accelerates butyrate production.

3. Strain identification and characterization

The 16S rDNA sequence of the strain was obtained using the universal primers 27F (5-.

Colonies of DG1 on PYAG were milky circular, 1-2mm in diameter, smooth edged, as shown in FIG. 3A. Under the scanning of an electron microscope, the diameter is 0.6 to 0.8 μm, the length is 3.0 to 10.0 μm, the shape of a straight rod or a little bending, and the shape of a single or a pair, or an irregular bending chain is formed, as shown in figure 3B. The gram-positive bacteria are blue-purple in gram stain, as shown in FIG. 3C. The growth curve of DG1 and the production pattern of SCFA are shown in FIG. 4. The culture medium used by DG1 is PY + GS (GOS + D-sorbitol), the OD value of the strain DG1 does not change greatly after 24 hours, which indicates that the growth of the bacteria is slow and enters a stable period, and the yield of SCFA is basically stabilized after 36 hours.

4. Enzyme activity and hemolytic assay

Amylase, cellulase, protease, vitellophospholipase, lipase and catalase activities were tested. The reaction is negative to enzyme activity. The hemolytic assay found that DG1 is not hemolytic.

5. Antibiotic resistance

According to the explanation standard of the bacteriostasis range of the drug sensitive test paper method matched with the antibacterial drug sensitive paper, the drug sensitive test result is divided into the following three stages: drug resistance (resistance), intermediaries (intermediaries), and sensitivity (susceptable). As shown in Table 1, the strain DG1 is sensitive to the tested antibiotics, has no drug resistance and is high in safety.

TABLE 1 antibiotic resistance of Strain DG1

Note: r, resistance (resist); i, intermediaries (intermediaries); s, sensitive (susceptable).

6. Butyric acid production pathway key enzyme gene

The butyric acid producing bacteria can metabolize pyruvic acid to generate butyryl CoA, and finally produce butyric acid through two metabolic pathways: butyryl-CoA acetyl-CoA transferase pathway and butyrate kinase pathway. Thus, having tested the key enzymes of these two butyrate-producing pathways, DG1 only detected butyryl CoA: acetyl CoA transferase gene.

7. Feeding experiment

The breeding experiment site is a south Australia Linhai test station of Shantou university, and the experimental juvenile fish are purchased from Shangping county. The commercial basal diet (crude protein 45.0%, crude fat 7%) was fed for 2 weeks in a marine large net cage (2.0X 2.0 m). The young light color spotted maigre with healthy physique and consistent specification (the initial weight is 13.68 +/-0.07) is selected for breeding experiments, randomly divided into 3 treatment groups and fed with the following three feeds respectively: PS (10) ⁷ cfu/g DG1)、PSGS(10 ⁷ cfu/g DG1+0.5% D-sorbitol +0.5% galacto-oligosaccharide) and basal feed group Control. 3 replicates per treatment group, one cage (1.0 × 1.0 × 1.5 m) per replicate, 20 fish per cage. Feeding was performed by satiation during the breeding experiment, twice daily in the morning of 6 00 and in the evening of 18. During the test, the temperature of the seawater is 20-30 ℃, the salinity is about 36 per mill, the pH is about 8.7, and the dissolved oxygen is more than 5.5mg/L. After 8 weeks of culture test, respectively measuring the weight and body length of the tested fish, then collecting blood samples and intestinal tissues and collecting intestinal contents by a sterile technology, and finishing the determination of intestinal microorganisms, short-chain fatty acids, digestive enzyme activity, immunoenzyme activity and serum immunoenzyme activity as soon as possible.

7.1 survival and growth Performance

Growth data from 8-week breeding experiments are shown in table 2. As can be seen from table 2, the weight gain and specific growth rate of the spotted maigre was increased in both the PS and PSGS groups relative to the control group, with significant changes being achieved in the PSGS group.

TABLE 2 influence of Strain DG1 on growth index of light-colored spotted maigre

Note: data are mean ± sem (n = 3); the absence of identity in the lower case notation indicates significant differences (P < 0.05).

7.2 intestinal flora analysis

(1) Basic information and diversity index of sequencing

Sequencing the variable region of the V4 gene of 16S rRNA of 12 intestinal content samples gives 1,052,520reads, and the number of OTU is from 762 to 1,698. The three treatment groups were found to be clustered into three clusters by NMDS analysis, respectively, as shown in fig. 5.

The results of the α -diversity index calculation are shown in table 3, and the diversity of the intestinal flora in the PSGS group is highest.

TABLE 3 Effect of Strain DG1 on gut flora alpha-diversity index

Note: data are mean ± sem (n = 3); lower case letters without identity indicate significant differences from each other (P < 0.05).

The predominant bacteria at phylum level in the gut of spotted maigre are Proteobacteria (Proteobacteria), firmicutes (Firmicutes) and Bacteroidetes (Bacteroidetes), as shown in fig. 6. Compared with a control group, PSGS obviously reduces the relative abundance of proteobacteria and improves the relative abundance of firmicutes and bacteroidetes; and PS can remarkably improve the relative abundance of firmicutes and bacteroidetes. On the genus level, the dominant bacterium is Acinetobacter Acinetobacter (Control: 9.47%; 5.07% of PS, 1.55% of PSGS), lawaonia (Control: 7.51%), unidentified Chloroplast (Control: 6.37%; 3.07% of PS, 1.42% of PSGS), staphylococcus staphylocccus (Control: 5.73%; 8.36% of PS, 1.52% of PSGS), bacteroides (Control: 4.27%; 6.36% of PS, 16.15% of PSGS), clostridium Faecalibacterium (Control: 0.55%; 3.04% of PS, 4.96% of PSGS).

The different species between treatment groups can be obtained by LEfSe analysis, as shown in figure 7. For comparison of the three groups of Control, PS and PSGS, the Control group is significantly enriched in Rhizobium (proteobacteria) and Corynebacterium1 (actinomycetales), the PS group is significantly enriched in Lactobacillus (firmicutes) and rhodobacter (α -proteobacteria), and the PSGS are significantly enriched in Faecalibacterium, blautia, clostridium sensu strict1, ruminococcus sp 5 3 \ u 39bfaa, ruminococcus UCG _014 (firmicutes), prevotellaceae _ UCG _003, aligenes (bacteroidetes), bifidobacterium (actinomycetes), and the like.

And (3) predicting the intestinal flora function of the light-color spotted maigre by utilizing Tax4Fun analysis, obtaining the relative abundance information of 3 levels and ko of KEGG, and screening out a passage with obvious difference through variance analysis. The PSGS group can significantly reduce the relative abundance of the Bacterial invasion pathway (epithelial cells) of epithelial cells, while PS and PSGS can significantly reduce the relative abundance of the Vibrio cholerae infection pathway Vibrio cholerae infection, as shown in fig. 8.

The bacteria enriched in PS and PSGS are mostly SCFA producing bacteria, so the content of SCFA in intestinal tract was also detected, and it was found that the total content of SCFA in intestinal tract was higher in PS and PSGS groups than in control group, where PS group significantly increased the content of acetate and propionate, and PSGS significantly increased the content of acetate and butyrate, as shown in fig. 9.

Compared with the control group, the PS and PSGS groups have the advantages that the C3, C4, igM, IL-1 beta, IL-6, IL-10, TNF-alpha, TGF-beta, LZM contents and Amylase Amylase enzyme activities are obviously increased, and the MDA content is obviously reduced, as shown in figure 10. In addition, the PS group obviously increases the content of T-AOC and T-SOD, and the PSGS obviously improves the enzymatic activity of catalase CAT.

In conclusion, the P.sphanisci DG1 and the synbiotics PSGS thereof remarkably improve the growth performance of the fish, improve the intestinal digestive enzyme activity, change the composition and diversity index of intestinal flora, enrich a plurality of SCFA producing bacteria and increase the content of SCFAs in intestinal tracts of the fish. In addition, the immune system of the light-colored spotted maigre can be activated by regulating the serum complement and cytokine levels, the lysozyme activity and the intestinal antioxidant capacity. The results show that the P.sphanisci DG1 and the synbiotics thereof can be used as feed additives of the light-colored spotted maigre to play roles in promoting growth and preventing and treating diseases.

Claims

1. Butyric acid bacterium DG1 (with probiotic activity)Proteocatella sphenisciDG 1) is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2019072.

2. The method for cultivating a probiotic activity-producing strain DG1 according to claim 1, wherein the culture medium used isThe PYAG culture medium mainly comprises peptone 5g, tryptone 5g, yeast extract 10g, na ₂ CO ₃ 4g, 5mg of hemin ₂ HPO ₄ 0.4 g，KH ₂ PO ₄ 0.04 g，NaHCO ₃ 0.08 g，NaCl 0.04 g，CaCl ₂ 8 mg，MgSO ₄ ▪7H ₂ O1.9 mg, vitamin K1 mg, sodium acetate 4.49g, glucose 5g, adjusting pH to 6.8, adding double distilled water to reach volume of 1L.

3. Use of the probiotic activity-producing bacterium DG1 according to claim 1, as feed additive for light-colored spotted maigre.

4. Use according to claim 3, in a feed for light-coloured spotted maigre, the feed comprising 10 ⁷ cfu/gDG1 or 10 ⁷ cfu/gDG1+0.5% D-sorbitol +0.5% galacto-oligosaccharide.