CN116998593A - Application of lactobacillus plantarum in preparation for improving fish sugar tolerance - Google Patents
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- CN116998593A CN116998593A CN202311194904.8A CN202311194904A CN116998593A CN 116998593 A CN116998593 A CN 116998593A CN 202311194904 A CN202311194904 A CN 202311194904A CN 116998593 A CN116998593 A CN 116998593A
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- 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
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- 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
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- 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
- C12R2001/25—Lactobacillus plantarum
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Abstract
The invention discloses application of lactobacillus plantarum in a preparation for improving fish sugar tolerance. The probiotic is Lactobacillus plantarum. The probiotics provided by the invention can regulate the PPAR signal path and the tricarboxylic acid circulating path of the liver by maintaining the intestinal flora steady state of the largemouth black bass, thereby relieving the liver glycolipid metabolic disorder of the largemouth black bass caused by high-sugar diet feeding and improving the antioxidant capacity of the liver. The invention provides a new solution for improving the sugar tolerance capability and healthy cultivation of carnivorous fishes.
Description
Technical field:
the invention belongs to the field of aquaculture, and particularly relates to application of lactobacillus plantarum preparations in improving fish sugar tolerance.
The background technology is as follows:
carbohydrates, which are one of the essential nutrients in aquatic feeds, are a cheaper source of energy than proteins and fats. The proper amount of carbohydrate is added into the aquatic feed to promote the protein utilization of fish. As a common carbohydrate, starch can act as a binder and granule expander in feed production. Under the background of rising prices of fish meal and fish oil, more carbohydrate is added into the aquatic feed, so that the feed cost can be reduced, the production benefit can be improved, and the green sustainable development of the aquatic product aquaculture industry in China can be ensured.
Because of the difference of the digestion and utilization capacity of different edible fishes on carbohydrate, especially the carnivorous fishes have the worst sugar utilization capacity. Thus, excessive ingestion of saccharides leads to an increase in blood glucose levels of the black bass, excessive deposition of liver glycogen and lipids, and ultimately, metabolic disorders of the liver. Enteric microorganisms have proven useful against obesity and metabolic disorders. Research has shown that beneficial microorganisms promote growth and immune responses in fish by improving intestinal morphology and altering intestinal microbial flora. Lactobacillus plantarum (Lactobacillus plantarum) has multiple physiological functions and is one of the most potential feeding microecological preparations. Lactobacillus plantarum has been demonstrated to regulate lipid metabolism and alleviate liver injury in mammals, however its functional studies on fish remain scarce.
Lateolabrax japonicus (Micropterus salmoides), commonly known as California perch, belongs to the genus Lateolabrax of the order Lateolabrax, family Sun-fish, and genus Lateolabrax, is a carnivorous fish. The fish feed has the advantages of high growth speed, rich nutrition and great economic value, and gradually becomes one of the most important commercial fishes in China and the most extensive cultivation. In order to meet the rapid development of the largemouth bass breeding industry, compound feed is increasingly used for feeding and breeding in production. In the preparation of the puffed floating feed granule for the largemouth bass, the addition amount of starch is generally not less than 20 percent. Many studies have shown that when the starch level of the feed exceeds 10%, the liver fat content of the micropterus salmoides increases, the antioxidant capacity decreases, and the intestinal health and the growth performance of the micropterus salmoides are also damaged, so that the cultivation income is finally affected. Therefore, the negative effect caused by the high-starch feed is a problem to be solved in the development of the largemouth bass breeding industry.
The invention comprises the following steps:
a first object of the present invention is to provide the use of Lactobacillus plantarum in a formulation for increasing the glucose tolerance of fish.
Preferably, the lactobacillus plantarum is derived from the Guangdong province microorganism strain collection, and the number is GDMCC1.140.
The fish is Lateolabrax japonicus.
Further, the Lactobacillus plantarum was cultured with MRS broth for 48 hours at 37℃and washed three times with sterile water and dissolved in sterile water at a concentration of 10 8 -10 9 CFU/mL。
Further, lactobacillus plantarum is uniformly sprayed into the largemouth bass feed, and the final concentration is about 10 7 CFU/g feed. It is further preferable to spray new bacterial liquid every 48 hours.
After the Lactobacillus plantarum is used for treating the micropterus salmoides for 9 weeks, the microbial composition structure in the intestinal canal of the micropterus salmoides can be improved, the microbial homeostasis of the intestinal canal can be maintained, and the PPAR signal path and the tricarboxylic acid circulating path of the liver can be regulated, so that the abnormal glycolipid metabolism of the micropterus salmoides can be regulated, and the glucose tolerance can be improved.
The invention has at least the following advantages:
1. the invention discloses a lactobacillus plantarum which regulates glycolipid metabolic disturbance by regulating a liver PPAR signal path and a tricarboxylic acid circulating path, so as to relieve liver fat deposition of micropterus salmoides.
2. The invention has important guiding significance for understanding the probiotic function of lactobacillus plantarum and the application of lactobacillus plantarum in healthy cultivation of the black perch.
Drawings
FIG. 1 is the effect of Lactobacillus plantarum on liver mass ratio and liver fat content of micropterus salmoides. (A) Liver volume ratio bar graphs (n=6) of largemouth black bass fed with different groups of feeds; (B) Liver crude fat content bar graph (n=6) of largemouth bass fed with different group feed. LS: a low starch group; HS: a high starch group; HSP's: high starch + lactobacillus plantarum group. * P <0.05, < P <0.01, < P <0.001..
FIG. 2 is the effect of Lactobacillus plantarum on liver health of Lateolabrax japonicus. (A) The liver oxidation resistance index (superoxide dismutase, malondialdehyde and catalase) bar graph (n=6) of the largemouth bass fed with different groups of feeds; (B) Liver section of largemouth black bass fed with different groups of feeds (hematoxylin-eosin staining), red arrow indicates the deviation of the nuclei from the center of the hepatocytes, black arrow indicates lipid vacuoles (n=3). LS: a low starch group; HS: a high starch group; HSP's: high starch + lactobacillus plantarum group. * P <0.05.
Fig. 3 is the effect of lactobacillus plantarum on the composition structure of the intestinal flora of micropterus salmoides (n=3). (A) Feeding intestinal flora OTU Venn diagrams of largemouth bass by different groups of feeds; (B) Intestinal flora diversity bar graphs of largemouth bass fed with different groups of feeds; (C) Intestinal flora distribution at Phyum (Phylum) and Genus level of large-mouth black bass fed with different groups of feeds. LS: a low starch group; HS: a high starch group; HSP's: high starch + lactobacillus plantarum group. * P <0.01.
Fig. 4 is a liver transcriptome KEGG pathway map (Top 30) of different groups of feed fed micropterus salmoides (n=3). LS: a low starch group; HS: a high starch group; HSP's: high starch + lactobacillus plantarum group. .
Fig. 5 is the effect of lactobacillus plantarum on liver gene expression of micropterus salmoides (n=6). (A) Feeding liver lipolysis related gene expression level bar graphs of largemouth black bass with different groups of feeds; (B) And feeding liver fat synthesis related gene expression level bar graphs of largemouth black bass by different groups of feeds. LS: a low starch group; HS: a high starch group; HSP's: high starch + lactobacillus plantarum group. ppar alpha, peroxisome proliferator activated receptor alpha; hsl, hormone sensitive lipase; atgl, fatty triglyceride lipase; pepck, phosphoenolpyruvate carboxykinase; ppar gamma, peroxisome proliferator-activated receptor gamma; srebp1, sterol regulatory factor binding protein 1; fas, fatty acid synthase; accα, acetyl-CoA carboxylase α. * P <0.05, < P <0.01, < P <0.001.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
The Lactobacillus plantarum used in the examples below was derived from the Guangdong province microorganism strain collection (GDMCC) and was numbered GDMCC1.140.
In the present invention, the culture medium used for culturing lactobacillus plantarum is as follows:
the liquid MRS broth culture medium formula is as follows: 10.0g of casein enzyme digest, 10.0g of beef extract powder, 4.0g of yeast extract powder, 2.0g of triammonium citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 20.0g of glucose, 1.0g of tween-80 and pH 5.7+/-0.2. Distilled water 1000mL. Autoclaving at 121℃for 15min.
The solid MRS broth medium was supplemented with 15g agar per liter.
In the invention, the recovery and preservation of lactobacillus plantarum GDMCC 1.140:
(1) Resuscitates the lactobacillus plantarum freeze-dried powder on an ultra-clean workbench. mu.L of MRS liquid culture medium was sucked up with a sterile gun head, dripped into a freeze-dried tube, and gently shaken until it was dissolved.
(2) All bacterial suspension was aspirated, inoculated on MRS solid medium plates and incubated at 37℃for 48h at rest.
(3) After the colony of the monoclonal bacteria had grown, the monoclonal bacteria were picked up by a sterile gun head into a 1.5mL centrifuge tube containing 500. Mu.L of medium and were subjected to stationary culture at 37℃for 48h. 500 μl of sterile 30% glycerol was then added.
(4) The selected monoclonal colonies were confirmed to be Lactobacillus plantarum by sequencing and comparison with NCBI (https:// blast. NCBI. Lm. Nih. Gov/blast. Cgi). Lactobacillus casei with 30% glycerol was deposited as a seed in a-80℃freezer.
In the invention, lactobacillus plantarum is subjected to amplification culture, collection and concentration:
(1) In an ultra clean bench, 20. Mu.L of Lactobacillus plantarum strain was inoculated into 15mL of liquid medium with a sterile gun head.
(2) The inoculated liquid culture medium is placed in a constant temperature incubator at 37 ℃ for standing for 48 hours.
(3) Centrifuging 4000g of the bacterial liquid at 4deg.C for 10 min, discarding the supernatant on an ultra-clean bench, washing with sterile water for 3 times, and suspending in sterile water to a concentration of about 10 8 -10 9 CFU/mL was placed in a refrigerator at 4deg.C for use.
Cultivation of experimental fish
Prior to the test, the largemouth bass was domesticated for 14 days and fed with low starch feed twice daily. The micropterus salmoides are cultivated in glass cylinders, all the glass cylinders are kept in a natural light/dark state, the dissolved oxygen is more than 6.04mg/L, the temperature is 28+/-1 ℃, the pH is about 7.2, and the nitrite and ammonia nitrogen concentration is lower than 0.1mg/L.
Example 1
The influence of the lactobacillus plantarum on liver health of the micropterus salmoides is detected, and the specific experimental operation steps are as follows:
(1) 225 healthy and similar largehead jewfish (initial body weight of 12.54.+ -. 0.02 g) were randomly divided into 3 groups (low starch group LS, high starch group HS, high starch+Lactobacillus plantarum group HSP), each group was set with 3 replicates, and allocated to 9 glass jars, 25 fish per jar. The cultivation period is 9 weeks, and different groups of lipid feeds such as nitrogen and the like are fed by feeding at 9 am and 5 pm every day, wherein the lipid feeds comprise low-starch group feed (LS group, 10% starch level), high-starch group feed (HS group, 20% starch level), high-starch+lactobacillus plantarum group feed (HSP group, 20% starch level evenly sprayed lactobacillus plantarum, and the final concentration is about 10) 7 CFU/g feed).
(2) After 9 weeks, the test micropterus salmoides are fasted for 1 day, and then sampled, dissected and weighed to obtain liver tissues, and the liver tissues are used for measuring indexes such as liver components, liver antioxidant enzyme activity, liver sections and the like.
As shown in fig. 1 and 2, the liver volume ratio of the micropterus salmoides in the HS group is significantly higher than that in the LS group, while the liver volume ratio of the micropterus salmoides in the HSP group is significantly lower than that in the HS group (fig. 1A). The liver fat content was significantly higher in the HS group than in the LS group, while the liver fat content was significantly lower in the HSP group than in the HS group (fig. 1B). The activities of superoxide dismutase and catalase of the HSP group micropterus salmoides are obviously higher than those of the HS group; the malondialdehyde content of the HS group is significantly higher than that of the LS group, whereas the malondialdehyde content of the HSP group is significantly lower than that of the HS group (fig. 2A). As can be seen from the results of liver sections, the livers of the HS group had a significant fat infiltration, more nuclei were extruded to the cell edges, severe nuclei disappeared and lipid vacuoles occurred, compared to the LS group, while the HSP group had significantly reduced lipid droplets, and significantly improved conditions of fat infiltration and hepatocyte damage, compared to the HS group (fig. 2B). The results show that the addition of the lactobacillus plantarum can obviously reduce the liver body ratio and liver fat deposition of the high-starch group, improve the liver oxidation resistance of the micropterus salmoides, relieve the liver cell injury caused by the high-starch feed and improve the starch tolerance of the micropterus salmoides.
Example 2
The influence of lactobacillus plantarum on intestinal microorganisms of the largemouth black bass is detected, and the specific experimental operation steps are as follows:
after 9 weeks of cultivation as described in example 1, the test micropterus salmoides were fasted for 1 day, and then sampled, dissected, and weighed to obtain intestinal contents of the intestinal tissue for intestinal microbiome determination. After the DNA extraction was completed, 16S V3-V4 region was selected and PCR amplification was performed using universal primers 338F and 806R, the amplified region being about 450bp in length. After quality detection of the extracted DNA, 3 samples per group were randomly selected for IlluminaMiSeq library preparation and sequencing. And splicing reads of each sample by using FLASH, filtering to obtain high-quality Tags data, and comparing the Tags with a database to remove a chimeric sequence to finally obtain effective data. The effective sequences were clustered into OTUs (Operational Taxonomic Units) using Uparse software, and then species annotation analysis and Alpha diversity analysis were performed using the Mothur method with the SSUrRNA database of SILVA.
The experimental results are shown in fig. 3, with the highest number of OTUs of intestinal microorganisms in HSP group, followed by LS group and HS group (fig. 3A); LS, HS and HSP groups contained 162, 113 and 236 unique OTUs, respectively. The Simpson index was significantly reduced for both the LS and HSP groups compared to the HS group, with higher averages of Shannon, chao and Ace indices for both the LS and HSP groups, but no significance was achieved (FIG. 3B). At the phylum level, the ratio of the fusobacterium in the LS group and the HSP group is the largest, while the ratio of the Proteus in the HS group is the largest, which is about 2 times that of the LS group and the HSP group. In the HS group, the proportion of the Thick-walled bacteria was only 1/7 of the LS group and the HSP group (FIG. 3C). At the genus level, the intestinal microbiota mainly comprises Cetobacterium (Cetobacterium), achromobacter (Achromobacter), romboutsia and Plasiomonas. Furthermore, the relative abundance of Achromobacter was significantly reduced in LS and HSP groups compared to HS group, while the relative abundance of romiboutsia was significantly increased (fig. 5B). The results prove that the lactobacillus plantarum can restore the decrease of Alpha diversity of intestinal microorganisms of the micropterus salmoides caused by high-starch feed, change the composition of intestinal flora structures of the micropterus salmoides, maintain the steady state of the intestinal microorganisms and improve the sugar tolerance capability of the micropterus salmoides. .
Example 3
The method for detecting the influence of the lactobacillus plantarum on the liver glycolipid metabolism of the largemouth black bass comprises the following specific operation steps: after 9 weeks of culture of the black perch according to example 1, the experimental black perch was fasted for 1 day, followed by sampling and dissection to obtain liver tissue for liver transcriptome and QPCR assays.
Transcriptome assay: total RNA was extracted from liver samples using TRIzol. RNA quality and quantity were measured, cDNA libraries were synthesized, and the libraries were pooled and sequenced on an Illumina NovaSeq 6000 platform. The expression level of the transcripts was calculated according to the per million reads Transcript (TPM) method. Differentially Expressed Genes (DEGs) were identified using DESeq2 software and set to |log2FC|+.1 and FDR+.0.05. KEGG enrichment analysis of DEGs is based on P.ltoreq.0.05.
QPCR assay: total RNA was extracted from liver samples using the total RNA extraction kit (BioFlux, china). Reverse transcription was performed using PrimeScript TMRT kit (Takara, japan). PCR amplification was performed by SYBR Green Master Mix (Toyobo, japan) in the CFX Connect Real-Time system (Bio-Rad Laboratories, USA), reaction program: 3min at 95 ℃;95 ℃ for 30s,60 ℃ for 30s,40 cycles; 72 ℃ for 1min. Relative expression level of genes according to 2 -ΔΔCT The method performs the calculation.
The results of liver transcriptome KEGG enrichment analysis are shown in fig. 4, and in comparison of HSP group and HS group, DEGs are significantly enriched in PPAR signaling pathway, tricarboxylic acid cycle, peroxisome, interconversion of pentose and glucuronic acid, ascorbic acid and aldehyde acid metabolism, unsaturated fatty acid synthesis and other pathways. The QPCR assay results are shown in fig. 5, where mRNA expression levels of HSP group lipolytic related genes (ppar a, hsl and atgl) were significantly up-regulated compared to HS group (fig. 5A). Furthermore, the expression of the genes involved in lipid synthesis (pepck, pparγ, srebp1 and fas) was significantly up-regulated in the HS group compared to the LS group, whereas the expression levels of these genes were significantly down-regulated in the HSP group compared to the HS group; mRNA expression level trends for the three groups of acc α were the same as described above, but did not reach significant levels (fig. 5B). The results show that the lactobacillus plantarum can promote liver lipolysis and inhibit high sugar-induced fat synthesis by regulating the liver PPAR signal pathway, tricarboxylic acid circulation and other pathways of the largehead jewflower.
Claims (7)
1. Use of lactobacillus plantarum formulation for improving fish carbohydrate tolerance.
2. The use according to claim 1, wherein the lactobacillus plantarum is derived from the collection of microorganisms of the cantonese province under the designation GDMCC1.140.
3. The use according to claim 1, wherein the lactobacillus plantarum is cultivated with MRS broth at 37 ℃ for 48h, washed three times with sterile water and dissolved in sterile water at a concentration of 10 8 -10 9 CFU/mL。
4. The use according to claim 1, wherein the fish is micropterus salmoides.
5. The use according to claim 4, wherein the lactobacillus plantarum is uniformly sprayed into the feed of the largemouth bass at a final concentration of about 10 7 CFU/g feed.
6. The method according to claim 5, wherein the new bacterial liquid is sprayed every 48 hours.
7. The use according to claim 1, wherein the lactobacillus plantarum formulation maintains intestinal microbial homeostasis of jewfish, regulates the hepatic PPAR signaling pathway and the tricarboxylic acid circulating pathway, thereby regulating glycolipid metabolic disorders, and achieves relief of jetfish hepatic fat deposition.
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