CN117568194B - Saccharomyces cerevisiae and application - Google Patents
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
- C12N1/185—Saccharomyces isolates
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- 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
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- 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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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
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Abstract
The invention belongs to the technical field of biology, and discloses a strain of saccharomyces cerevisiae, which is classified as saccharomyces cerevisiae 12-3 (Saccharomyces cerevisiae-3) with the preservation number of: CCTCC NO: M20231714; preservation date: 2023, 9, 18, deposit unit: china center for type culture Collection. The lipase activity of the saccharomyces cerevisiae is 126.24U/mL, which is far superior to that of similar engineering bacteria, and can effectively promote animal fat deposition. Meanwhile, the invention also provides application of the saccharomyces cerevisiae.
Description
Technical Field
The invention relates to the technical field of biology, in particular to saccharomyces cerevisiae and application thereof.
Background
Fat is one of the main nutrients in animal foods and is also one of the important constituent components of animal organisms. The dietary fat provides energy and maintains various physiological functions of the body, such as growth and development, cell signaling, cell membrane regulation, and the like. Insufficient dietary fat intake can lead to a deficiency of essential fatty acids and fat-soluble vitamins, thereby inducing a variety of diseases including growth retardation, immune dysfunction, cardiovascular disease, vision problems, retinopathy and inflammation. In addition, fat deposition is closely related to the flavor and quality of livestock and poultry products.
Lipase (EC 3.1.l.3), also known as triacylglycerol acyl hydrolase, is a class of esterases, and has the ability to hydrolyze triglycerides to glycerol and fatty acids, the most basic enzyme for fat digestion and utilization. Lipase is widely available in nature, and animals, plants and microorganisms all have the ability to produce lipase. Lipase is commonly present in nature, but lipase produced by microorganisms has the characteristics of wide source, short production period, high yield, easy extraction, mild acting condition, high catalytic efficiency and the like, and is a main source of industrial lipase.
Fungi are the preferred microorganisms for industrial lipase production, since they generally produce extracellular enzymes, which facilitate the extraction of lipases from fermentation media. Yeast is a large class of lipase-producing fungi, and has simple culture conditions and is the only fungus microorganism in a feed additive catalog. Therefore, the screening of lipase-producing strains with high enzyme activity, low production cost and novel characteristics has important practical significance in the aspects of fermenting the strains by the lipase, improving the fat digestion and absorption efficiency of intestinal tracts, improving the quality of livestock and poultry products and the like.
CN114058526a discloses a saccharomyces cerevisiae engineering bacterium with alkaline lipase displayed on the cell surface, a preparation method and a use method thereof, wherein the preservation name of the saccharomyces cerevisiae engineering bacterium is saccharomyces cerevisiae EBY-BSR, and the preservation unit is: china center for type culture Collection, accession number: cctccc NO: m2021887. The optimal induction time of the saccharomyces cerevisiae recombinant strain EBY-BSR is 36 hours, and the enzyme activity reaches 79.4338U/g stem cell weight.
The saccharomyces cerevisiae belongs to recombinant bacteria, and aims to improve the enzyme activity of alkaline lipase; however, the enzyme activity still does not meet the expected demand.
CN112094763A discloses a strain of Saccharomyces cerevisiae, which is Saccharomyces cerevisiae
PLBS012 (Saccharomyces cerevisiae PLBS 012), deposited in China general microbiological culture Collection center (CGMCC) with a deposit number of CGMCC No.16952 in 12 months 17 of 2018;
the description is as follows: saccharomyces cerevisiae PLBS012 is added into basic ration of fattening pigs, so that the richness of bacteroides in excrement of fattening pigs can be reduced, the richness of the armillaria mellea can be increased, and the richness of spirochete and armillaria mellea can be increased. The thick-walled bacteria can promote fat metabolism of fattening pigs and promote intramuscular fat deposition; the phylum spirochete and phylum tenella can influence the secretion of intestinal hormone of fattening pigs, increase the absorption of nutrient substances by small intestines, and contribute to weight gain. Therefore, the saccharomyces cerevisiae PLBS012 can improve the richness of microorganisms in fattening pigs, improve the digestion and utilization of the fattening pigs to feeds, and improve the production performance of the fattening pigs.
In this invention, it is achieved by increasing fat deposition, mainly by increasing the abundance of the firmicutes.
As can be seen from the description of the two schemes, there are a number of ways to increase fat deposition, and increasing lipase activity is an alternative.
The technical problems existing in the prior art are as follows: naturally screened strains have very low lipase activity and are often genetically engineered to increase lipase activity.
But the naturally screened strain is superior to the genetically engineered strain in stability, safety, environmental adaptability, production cost, social acceptance and the like.
The technical problem that the scheme should solve lies in: how to find a naturally occurring saccharomyces cerevisiae with high lipase activity.
Disclosure of Invention
The invention aims to provide a saccharomyces cerevisiae, the lipase activity of which is 120.96U/mL, which is far superior to that of similar engineering bacteria, and can effectively promote animal fat deposition.
Meanwhile, the invention also provides application of the saccharomyces cerevisiae.
In order to achieve the above purpose, the present invention provides the following technical solutions: a strain of Saccharomyces cerevisiae, classified under the name of Saccharomyces cerevisiae 12-3 (Saccharomyces cerevisiae 12-3), has the accession number: CCTCC NO: M20231714; preservation date: 2023, 9, 18, deposit unit: china center for type culture Collection.
In the saccharomyces cerevisiae, the whole genome sequence of the saccharomyces cerevisiae is shown as SEQ ID No. 1.
The invention also discloses application of the saccharomyces cerevisiae in preparing feed additives.
In general, it is recommended that the Saccharomyces cerevisiae of the present invention be added to the feed in an amount of 10≡8-10≡9cfu/kg of the feed during the raising of fattening pigs.
Compared with the prior art, the invention has the beneficial effects that:
the lipase activity of the saccharomyces cerevisiae is 126.24U/mL, which is far superior to that of similar engineering bacteria, and can effectively promote animal fat deposition.
Drawings
FIG. 1 is a primary screening media strain orange fluorescent ring;
FIG. 2 shows lipase activities of different strains;
FIG. 3A is a graph showing the effect of Saccharomyces cerevisiae 12-3 on mouse weight;
FIG. 3B is a bar graph showing the effect of Saccharomyces cerevisiae 12-3 on mouse body weight;
in fig. 3A and 3B, CTRL is a control group, and s.cerevisiae is a test group;
FIG. 4A is a graph showing the effect of Saccharomyces cerevisiae 12-3 on the weight of subcutaneous adipose tissue in mice;
FIG. 4B is a graph showing the effect of Saccharomyces cerevisiae 12-3 on the weight of epididymal adipose tissue in mice;
FIG. 4C is a graph showing the effect of Saccharomyces cerevisiae 12-3 on the weight of the perirenal adipose tissue in mice;
FIG. 4D is a graph showing the effect of Saccharomyces cerevisiae 12-3 on the ratio of subcutaneous adipose tissue weight and body weight of mice;
FIG. 4E is a graph showing the effect of Saccharomyces cerevisiae 12-3 on the weight of epididymal adipose tissue and the ratio of body weight of mice;
FIG. 4F is a graph showing the effect of Saccharomyces cerevisiae 12-3 on the weight and weight ratio of the perirenal adipose tissue in mice;
in fig. 4A to 4F, the results of the effects of adipose tissue weight and body fat rate are plotted. Wherein CTRL is the control group and S.cerevisiae is the test group.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: isolation and identification of high-yield lipase saccharomyces cerevisiae
1. Isolation of strains
1G of fresh Ningxiang pig intestinal tract content sample is inoculated in 9mL of YPDA culture medium (yeast extract 10.0g/L; peptone 20.0g/L; glucose 20.0g/L; adenine sulfate 0.03 g/L), then 2 particles of 6mm sterile glass beads are added, shaking is carried out on a vortex machine for 3 minutes, so that the sample is homogenized, and then the sample is placed in a constant temperature incubator at 30 ℃ for culturing for 48 hours for enrichment. The culture solution after enrichment is diluted to 10 -7 step by step according to 10 -1, then the culture solution with each concentration gradient is coated on a solid separation culture medium (peptone 10.0g/L, yeast extract powder 5.0g/L, glucose 20.0g/L, chloramphenicol 0.1g/L, agar 15 g/L), cultured for 48 hours at 30 ℃, and monoclonal colonies are picked up in YPDA culture medium and cultured for 48 hours at 30 ℃. Then 5uL of bacterial liquid is inoculated on a primary screening culture medium (peptone 10.0g/L, yeast extract powder 5.0g/L, glucose 20.0g/L, olive oil emulsion 5% (v/v), rhodamine B0.5 g/L and agar 15 g/L) at 30 ℃ for culturing for 72 hours, whether orange fluorescence rings appear on a flat plate is observed under 350nm ultraviolet light, and a colony 12-3 with the largest diameter of the fluorescence rings is selected as an alternative strain (figure 1), so that subsequent strain identification and fat fermentation determination are carried out.
2. Determination of Lipase producing Capacity
The candidate strains 12-3, 12-3 and 14-2 obtained by primary screening are respectively inoculated in YPDA culture medium, cultured for 48 hours at 30 ℃, inoculated in fermentation culture medium (yeast extract 10g/L; peptone 20g/L; olive oil 1% (v/v; glucose 20 g/L) at 30 ℃ for 120 hours, the fermentation broth is centrifuged for 10 minutes at 4 ℃ and 10000r/min, the supernatant is taken to obtain crude enzyme liquid, and the lipase activity is measured by adopting lipase measuring kit (A054-2-1) produced by Nanjing biological engineering research. As shown in FIG. 2, the lipase activity of the alternative strain 12-3 was 126.24U/mL, and the lipase activities of the other same species of bacteria selected were 109.76U/mL and 56.68U/mL, respectively.
3. Identification of species
Genomic DNA of the strain was extracted using a fungal genomic DNA extraction kit from Beijing Soxhaust technologies Co., ltd, and the extracted DNA sample was diluted in an appropriate amount to be used as a PCR template, and the universal primer of fungal ITS was amplified with 1×TSE101 gold plate mix of the family Praeparata.
The ITS1 primer sequence is as follows: TCCGTAGGTGAACCTGCGG;
the ITS1 primer sequence is as follows: TCCTCCGCTTATTGATATGC.
The amplification system components are shown in Table 1 below:
Table 1 amplification System formulation
| 1×TSE101 gold medal mix | 45ul |
| 27F(10P) | 2ul |
| 1492R(10P) | 2ul |
| DNA template | 1ul |
The above amplification system was amplified according to the following amplification procedure, see in particular table 2:
TABLE 2 amplification program Table
After PCR amplification, the sample is identified by 2% agarose gel electrophoresis (2 ul sample+6 ul bromophenol blue, under 300V voltage for 12 minutes) and then sent to Beijing qing biological science and technology Co., ltd for sequencing, and the whole genome sequence of the saccharomyces cerevisiae 12-3 is shown as SEQ ID No. 1; sequence results were aligned in NCBI database and showed strain 12-3 to be Saccharomyces cerevisiae. The strain is preserved in China Center for Type Culture Collection (CCTCC) No. M20231714 at 9 months and 18 days in 2023, and the preservation address is eight-path 299 of Wuhan university in Wuhan district of Wuhan, hubei province.
Example 2 Saccharomyces cerevisiae 12-3 promotes fat deposition in animals
1. Preparing bacterial liquid:
Saccharomyces cerevisiae 12-3 is inoculated in YPDA culture medium for activation, and after activation, the strain is inoculated in a new YPDA culture medium for amplification according to 10 percent, and after culturing for 48 hours at 30 ℃, bacterial liquid is counted, and then the bacterial liquid is subjected to resuspension concentration by using sterile PBS, and the bacterial liquid concentration is adjusted to be 5 multiplied by 10 8 cfu/mL.
2. Treatment of test animals
Healthy male C57BL/6J SPF mice of 7 weeks old were purchased from Gekko Biotechnology Inc. in Changsha, and were served 1 week in SPF mouse rooms. At 8 weeks of age, 24 healthy mice with similar body conditions were selected and randomly divided into two groups of 12 mice each. The control group was filled with 0.2mL of PBS per stomach, the test group was filled with 0.2mL of 5X 10 8 cfu/mL of Saccharomyces cerevisiae 12-3 bacteria solution per stomach (10 8 cfu of Saccharomyces cerevisiae 12-3 were obtained per mouse), and the filling was performed three times per week for 16 weeks. During the test period, the mice eat and drink water freely, the illumination period is 12 hours, the darkness period is 12 hours, the test environment temperature is kept between 22 and 24 ℃, and the humidity is 40 to 60 percent.
3. Index measurement
3.1. Weight of body
Mice weekly weights were recorded during the trial.
3.2. Fat mass and body fat percentage
After the end of the test, the living body weight of the mice was weighed, and then cervical dislocation was sacrificed, and subcutaneous adipose tissue, epididymal adipose tissue and perirenal adipose tissue of the mice were immediately isolated and weighed. The body fat rate calculation formula is: body fat rate = fat weight/body weight x 100%.
The data were subjected to outlier rejection using SPSS26.0, and then subjected to significance analysis using independent sample t-test, the results were expressed as mean ± standard error, wherein P <0.001; * Represents P <0.01; * Represents P <0.05.
The effect of Saccharomyces cerevisiae 12-3 on mouse body weight results are shown in FIGS. 3A and 3B, and supplementation with Saccharomyces cerevisiae 12-3 significantly increased the mouse body weight (P < 0.05).
The effect of Saccharomyces cerevisiae 12-3 on the fat weight and body fat rate of mice results are shown in FIGS. 4A-4F, and supplementation of Saccharomyces cerevisiae 12-3 significantly increases the weight of subcutaneous adipose tissue, epididymal adipose tissue, and perirenal adipose tissue of mice and their percentage of weight (P < 0.05), indicating that Saccharomyces cerevisiae 12-3 can promote fat deposition in animals.
From the above experiments, it can be found that:
1. The saccharomyces cerevisiae 12-3 is taken as a strain which is obtained by natural separation, and lipase in a fermentation product of the saccharomyces cerevisiae has enzyme activity exceeding that of lipase produced by an engineering strain.
2. The saccharomyces cerevisiae 12-3 of the invention can significantly improve the weight and fat deposition of the feed objects.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (2)
1. The saccharomyces cerevisiae is characterized by being classified as saccharomyces cerevisiae (Saccharomyces cerevisiae) 12-3 with a preservation number of: CCTCC NO: M20231714; preservation date: 2023, 9, 25, deposit unit: china center for type culture Collection.
2. Use of the saccharomyces cerevisiae according to claim 1 for the preparation of feed additives.
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