CN109401990B - Saccharomyces cerevisiae HKB-36 with bacteriostatic activity and application thereof - Google Patents
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Abstract
The invention relates to a Saccharomyces cerevisiae HKB-36 and application thereof, wherein the Saccharomyces cerevisiae HKB-36 is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 11, 8 and has a preservation number of CGMCC No. 13261. The saccharomyces cerevisiae HKB-36 provided by the invention has the characteristics of vigorous growth and metabolism capacity, bacteriostatic activity of fermentation metabolites and the like, 200 mu L of strain fermentation liquor has the highest bacteriostatic ring diameter of 7mm, can be applied to the production of novel feed raw material yeast cultures, and can be added into livestock and poultry feeds to inhibit the growth of harmful microorganisms in intestinal tracts of livestock and poultry, improve the intestinal health of the livestock and poultry, improve the growth performance and reduce the use of antibiotics in the feeds.
Description
Technical Field
The invention relates to the field of fungi, in particular to saccharomyces cerevisiae HKB-36 with bacteriostatic activity and application thereof.
Background
Saccharomyces cerevisiae, also known as Saccharomyces cerevisiae or Saccharomyces cerevisiae, belongs to the family of Saccharomyces in the subgenus Ascomycotina, is a eukaryotic microorganism with a relatively simple cellular morphological structure, and is a yeast most closely related to human. As early as 1963, Bevan et al discovered a yeast with antibacterial/bacteriostatic activity in the preserved Saccharomyces cerevisiae for the first time, and secreted a toxin protein to the outside during the growth and reproduction process, and the toxin protein could kill or inhibit other microorganisms. With the progress of research, the bacteriostatic yeast has a plurality of purposes and has wide development prospects in the aspects of fermentation, medicine, environment, food, agriculture and the like.
The application of saccharomyces cerevisiae source biological feed in livestock breeding has been known for many years, but most of the prior livestock breeding industry is yeast single cell protein or primary processed products, and the high added value of yeast is not fully utilized. With the development of the restriction and even forbidding of the action of the feed antibiotics in China, for example, a veterinary antibacterial use reduction action test point working scheme (2018 and 2021) is published by the rural part of agriculture in 4 months in 2018, and the scheme indicates that the veterinary antibacterial use reduction action test point working in the breeding link is tried to pass through 3 years, and the drug feed additive is completely withdrawn in 2020. The method has great impact on the production efficiency of livestock breeding industry in China, and the full development of feed resources capable of promoting the health level of livestock and poultry and improving the production performance is urgent, so that the saccharomyces cerevisiae and biological products thereof have important application prospects in the form.
However, in the prior art, the content of protein in saccharomyces cerevisiae and its biological products are emphasized, and there is no report on yeast bacteriostatic biological products. How to fully utilize the saccharomyces cerevisiae, improve the quality and the effect of the yeast bacteriostatic biological product, and reduce the production cost is a problem to be solved urgently in domestic research at present.
Disclosure of Invention
The invention aims to solve the problems and provides a saccharomyces cerevisiae HKB-36 with bacteriostatic activity and application thereof.
According to the first aspect of the invention, a Saccharomyces cerevisiae (HKB-36) with bacteriostatic activity is provided, and is preserved in China general microbiological culture Collection center (CGMCC) at 11-8 th of 2016, wherein the preservation number is CGMCC No. 13261.
According to a second aspect of the present invention, there is provided a fermentation metabolite that is an aerobic fermentation metabolite of the HKB-36 of Saccharomyces cerevisiae.
According to a third aspect of the invention, there is provided a bacteriostatic biological product comprising the fermentation metabolite.
According to a fourth aspect of the present invention, there is provided a method for producing the fermentation metabolite, the method comprising the step of aerobic fermentation:
wherein the liquid culture medium comprises 145g/L malt extract powder and 0.08g/L chloramphenicol, and the pH value of the liquid culture medium is 5.8-6.8; the fermentation temperature is 28-36 ℃, the fermentation time is 24-36h, and the ventilation volume is 1-4L/min.
Among them, the fermentation temperature is preferably 32 ℃ and the pH of the liquid medium is preferably 6.4.
Wherein, the preparation method also comprises a seed culture step before the aerobic fermentation step:
the liquid culture media comprise 145g/L of malt extract powder and 0.08g/L of chloramphenicol, and the pH value of the liquid culture media is 5.8-6.8; the culture temperature is 28-36 ℃, the shaking culture is carried out at a constant temperature, the culture time is 18-30h, and the rotating speed is 120-.
Wherein, in the aerobic fermentation step, the inoculation amount of the seed liquid is 10 to 20 percent.
According to the fourth aspect of the invention, the bacteriostatic biological product is provided, and the preparation method comprises the steps of slant strain activation, seed liquid preparation, liquid aerobic fermentation and solid anaerobic fermentation:
wherein in the solid anaerobic fermentation step, the fermentation culture medium comprises 2-4 parts of vinasse, 2-4 parts of corn bran, 1-3 parts of bran and 1-3 parts of apple pomace, the carbon-nitrogen ratio of the culture medium is 2-7:1, and the water content is 35-70%; the fermentation temperature is 28-36 ℃, and the fermentation time is 18-48 h.
The saccharomyces cerevisiae HKB-36 provided by the invention has the characteristics of vigorous growth and metabolism capability, bacteriostatic activity of fermentation metabolites and the like, and the diameter of a bacteriostatic ring of 200 mu L of strain fermentation liquor can reach 7mm at most. The saccharomyces cerevisiae HKB-36 can be applied to the production of a novel feed raw material yeast culture, and can be added into livestock and poultry feed to inhibit the growth of harmful microorganisms in intestinal tracts of livestock and poultry, improve the intestinal health and growth performance of livestock and poultry, and reduce the use of antibiotics in the feed.
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Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:
FIG. 1 shows a growth profile of Saccharomyces cerevisiae HKB-36 according to an embodiment of the present invention.
FIG. 2 shows a colony morphology electron microscope picture of Saccharomyces cerevisiae HKB-36 according to an embodiment of the invention;
FIG. 3 shows a picture of a developmental tree of Saccharomyces cerevisiae HKB-36 according to an embodiment of the invention;
FIG. 4 is a graph showing bacteriostatic effects of fermentation metabolites of different Saccharomyces cerevisiae according to an embodiment of the present invention;
fig. 5 shows a bacteriostatic effect picture of bacteriostatic biological products of different saccharomyces cerevisiae according to an embodiment of the present invention.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
EXAMPLE 1 isolation screening of HKB-36 from Saccharomyces cerevisiae
(1) Culture medium: wort medium. Taking 500g of malt, crushing the malt, putting the malt into a beaker, adding 2000mL of distilled water, stirring, carrying out water bath at 45 ℃ for 30min, adjusting the temperature to 70 ℃, and keeping the temperature for 1 h; filtering with 8 layers of gauze, washing the beaker and the filter tank with regular Liu tree to make the filtrate reach 2000mL, boiling, cooling, and storing at 10 ℃ for later use.
(2) Separating the yeast: taking beer, wine, pickle, fermented fruit and vegetable juice, dough, mare's milk wine and the like as raw materials, respectively collecting 400mL or 400g of raw material samples (solid mashing), placing the raw material samples in a sterile conical flask, and covering 8 layers of gauze to naturally ferment for 2-3 days at 25-30 ℃. Shaking the fermentation culture solution sample under aseptic condition, sucking appropriate amount of fermentation liquid with aseptic pipette into sterilized wort culture solution, and culturing at 25 deg.C. Diluting with gradient dilution method until the concentration is 10 when there is alcohol taste-4And 10-5Sucking 0.1-0.2mL diluted culture solution, uniformly coating on a wort culture medium (wort culture solution + 2% agar), inverting for 48h at 25 ℃, after bacteria grow out, selecting a typical single bacterial colony (yeast bacterial colony) for further streaking and purifying for 2-3 times, respectively transferring the pure culture obtained by microscopic examination onto a wort solid slant culture medium, and storing at 4 ℃ for later use.
(3) Screening yeast: and (3) culturing by using a wort culture medium, observing the growth condition in the period, and selecting the bacteria with the highest survival rate and the most vigorous growth for the next step of screening the bacteriostatic test. The method comprises the steps of respectively adding yeast for standby preservation into culture media for culturing escherichia coli, salmonella and staphylococcus aureus by adopting an oxford cup method, wherein the inhibiting and killing effects of different yeasts on three harmful bacteria are shown in table 1, selecting a single strain with the best antibacterial/bacteriostatic activity, and naming the single strain as HKB-36.
Table 1: inhibition and killing test of different yeasts on escherichia coli, salmonella and staphylococcus aureus
Growth metabolic capacity and acid resistance assay:
the HKB-36 was cultured in wort medium at different pH values by the shake flask method, and the growth of the yeast HKB-36 was observed at different time points, and the growth curve is shown in FIG. 1. It is shown that HKB-36 reaches the stationary phase of growth in less than 20min under appropriate conditions, and grows well in the medium at pH 3, 4, 5, 6, and only shows slow growth at pH 2. Therefore, the saccharomyces cerevisiae HKB-36 has luxuriant growth and metabolism capability and better acid resistance.
And (3) strain identification:
1. colony characteristics: culturing in malt wort liquid culture medium at 25 deg.C for three days to obtain spherical, elliptical and sausage-shaped cells with size of (5.6-9.3) × (4.3-5.2) μm, and precipitate. The malt extract agar slant culture is 1 month, the colony is cheese-like, milk white, smooth in surface and regular in edge. The plates of corn agar Dalmau produced no pseudohyphae, as shown in FIG. 1.
2. Phylogenetic analysis of strains
Selecting purified HKB-36 single colony, inoculating the single colony in a screening culture medium with the pH value of 5.8, and carrying out shake culture at the constant temperature of 28 ℃ at the rotating speed of 120 r/min; culturing for 30h, then taking the bacterial liquid as a template to serve as an ITS sequence of a PCR amplification strain, sequencing, and taking a sequencing result as a development tree of the strain by using molecular evolution genetic analysis software, wherein as shown in figure 3, the HKB-36 has the highest sequence homology with the registered Saccharomyces cerevisiae, and reaches 99 percent, so that the strain is determined to be the Saccharomyces cerevisiae.
3. And (3) molecular identification: ITS sequences determined for HKB-36 as SED ID NO: 1, the HKB-36 is a new member of the genus Saccharomyces cerevisiae and is named as the Saccharomyces cerevisiae HKB-36 based on homology angle analysis by carrying out sequence alignment on http:// archive-dtd.ncbi.nlm.nih.gov/website.
Example 2 preparation of fermentation metabolites of different Saccharomyces cerevisiae HKB-36
(1) Preparing a seed solution: inoculating the activated yeast strain into a liquid culture medium containing 145g/L of malt extract powder, 0.08g/L of chloramphenicol and pH value of 5.8-6.8 by using ring 1, culturing by shaking at the constant temperature of 28-36 ℃ at the rotation speed of 100-200r/min for 18-30 h.
(3) Liquid aerobic fermentation: inoculating the prepared seed liquid with 10-20% of inoculation amount into a fermentation tank filled with a new liquid culture medium containing 145g/L of malt extract powder, 0.08g/L of chloramphenicol and 5.8-6.8 (5.8, 6.0, 6.2, 6.4, 6.6 and 6.8) of pH value for carrying out amplification culture, controlling the ventilation amount to be 1-4L/min and the temperature to be 28-36 ℃ (28 ℃, 30 ℃, 32 ℃, 34 ℃ and 36 ℃ respectively), carrying out fermentation time to be 24-36h, and carrying out inactivation at 65 ℃ for 20min after the fermentation is finished to obtain fermentation metabolites of different saccharomyces cerevisiae HKB-36 without live bacteria.
Example 3 fermentation metabolite in vitro bacteriostasis test of HKB-36 of Saccharomyces cerevisiae
And (3) clamping the sterilized Oxford cup by using sterile forceps, putting the sterilized Oxford cup on the flame of an alcohol burner, quickly passing a fire, vertically putting the sterilized Oxford cup on the surface of the LB culture medium inoculated with the Escherichia coli, and slightly pressing the sterilized Oxford cup to ensure that no gap exists between the bottom of the Oxford cup and the culture medium. 200 μ L of the metabolites fermented by the yeast Saccharomyces cerevisiae HKB-36 under different pH and temperature conditions of example 2 were injected into each Oxford cup, and 3 replicates of each metabolite were run with physiological saline as a blank. Culturing at 37 deg.C for 24h, measuring and recording the diameter of the zone of inhibition.
The specific experimental results are shown in FIG. 4. In FIG. 4, pH 1 to pH6 indicate pH values of 5.8, 6.0, 6.2, 6.4, 6.6 and 6.8, respectively, and temperatures 1 to 5 indicate fermentation temperatures of 28 ℃, 30 ℃, 32 ℃, 34 ℃ and 36 ℃, respectively. The fermentation process parameters of the yeast culture are optimized by a response surface method, and the diameter of the antibacterial ring is the largest and the antibacterial effect of the yeast fermentation metabolite is the strongest when the pH of the fermentation medium is 6.4 and the fermentation temperature is 32 ℃.
Example 4 preparation of bacteriostatic biologicals (yeast cultures) of the Saccharomyces cerevisiae HKB-36 (1) slant strain activation: inoculating the frozen Saccharomyces cerevisiae HKB-36 to a slant culture medium containing 145g/L of malt extract powder, 0.08g/L of chloramphenicol and 2% of agar, standing at a constant temperature of 30 ℃, and culturing for 48h to obtain an activated yeast strain.
(2) Preparing a seed solution: inoculating the activated yeast strain to the malt extract powder containing 145g/L and 0.08g/L chloramphenicol by using a 1-ring inoculation method; carrying out shake culture at constant temperature of 28-36 ℃ in a liquid culture medium with the pH value of 5.8-6.8 at the rotation speed of 120-; culturing for 18-30 h.
(3) Liquid aerobic fermentation: inoculating the prepared seed liquid into a fermentation tank filled with a new liquid culture medium containing 145g/L of malt extract powder, 0.08g/L of chloramphenicol and pH 5.8-6.8 at 15% of inoculation amount for amplification culture, controlling the ventilation amount to be 1-4L/min, the temperature to be 28-36 ℃ and the fermentation time to be 24-36 h. Obtaining liquid strain liquid for enlarged culture.
(4) Solid anaerobic fermentation: inoculating liquid strain liquid with the inoculation amount of 25% into a sterile solid culture medium which comprises 2-4 parts of distiller's grains, 2-4 parts of corn bran, 1-3 parts of bran and 1-3 parts of apple pomace in parts by mass, adding urea to control the carbon-nitrogen ratio to be 2-7:1, performing anaerobic fermentation in a temperature-controllable fermentation tank, controlling the water content to be 35-70%, the fermentation temperature to be 28-36 ℃, and the fermentation time to be 18-48 h. Obtaining a solid fermentation product.
(5) Obtaining of yeast culture product: after the solid fermentation is finished, adjusting the temperature of the fermentation tank to 60-75 ℃, inactivating and breaking the walls of the yeast in the culture to fully release mannan, nucleic acid and other substances in yeast cell contents, inactivating and breaking the walls for 1h, and then transferring the culture to a jet milling dryer for milling and drying to obtain a finished product of the yeast culture.
Example 5 in vitro bacteriostasis assay of Yeast cultures of Saccharomyces cerevisiae HKB-36
Different yeast culture products were prepared according to the procedure of example 4 (wherein the fermentation medium was selected to have a pH of 6.4, a fermentation temperature of 32 ℃ and a fermentation time of 24h) using HKB-36 of the present invention and the other 5 strains of yeast isolated in example 1, respectively, as fermentation strains. An oxford cup method is adopted for carrying out bacteriostatic tests, the bacteriostatic effects of each strain of yeast on escherichia coli, staphylococcus aureus and salmonella are respectively observed, and except that physiological saline is used as a blank control, ampicillin is added as a positive control.
The results of the experiment are shown in FIG. 5. In FIG. 5, d3 represents the yeast culture resulting from fermentation of HKB-36, a yeast of the invention, d3H, d5, d5H, d10 and d10H are the yeast cultures resulting from fermentation of HKB-6, HKB-10, HKB-27, HKB-33 and HKB-46, respectively. The neutral ampicillin is not marked below a physiological saline blank.
As can be seen from FIG. 5, ampicillin has a good bacteriostatic effect on E.coli, Staphylococcus aureus and Salmonella. The yeast culture fermented by the saccharomyces cerevisiae HKB-36 can also obviously inhibit the growth of escherichia coli, staphylococcus aureus and salmonella, the antibacterial effect is slightly weaker than that of ampicillin, and the antibacterial effect on the 3 strains is that escherichia coli is greater than salmonella is greater than staphylococcus aureus according to the diameter of the antibacterial ring. And the yeast culture produced by fermenting other 5 strains of saccharomyces cerevisiae has almost no bacteriostatic activity on escherichia coli, staphylococcus aureus and salmonella.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
SEQUENCE LISTING
<110> Beijing Zhongnong Hongkong Biotechnology Co., Ltd
<120> saccharomyces cerevisiae HKB-36 and application thereof
<130> 20180705
<160> 1
<170> PatentIn version 3.5
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<211> 554
<212> DNA
<213> unknown
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Claims (7)
1. A Saccharomyces cerevisiae HKB-36 is characterized by being preserved in China general microbiological culture Collection center (CGMCC) at 11, 8 and 2016, and the preservation number is CGMCC No. 13261.
2. A method for preparing fermentation metabolites of Saccharomyces cerevisiae HKB-36, wherein the strain is the Saccharomyces cerevisiae HKB-36 of claim 1, the method comprises the following liquid aerobic fermentation steps:
wherein the liquid culture medium comprises 145g/L malt extract powder and 0.08g/L chloramphenicol, and the pH value of the liquid culture medium is 5.8-6.8;
the fermentation temperature is 28-36 ℃, the fermentation time is 24-36h, and the ventilation volume is 1-4L/min.
3. The method for producing a fermentation metabolite according to claim 2, wherein the production method further comprises a seed culture step prior to the aerobic fermentation step:
wherein the liquid culture medium comprises 145g/L malt extract powder and 0.08g/L chloramphenicol, and the pH value of the liquid culture medium is 5.8-6.8;
the culture temperature is 28-36 ℃, the shaking culture is carried out at a constant temperature, the culture time is 18-30h, and the rotating speed is 120-.
4. The method for producing fermentation metabolites according to claim 3,
in the aerobic fermentation step, the inoculation amount of the seed liquid is 10-20%.
5. A preparation method of bacteriostatic biological products of Saccharomyces cerevisiae HKB-36 is characterized in that the strain is the Saccharomyces cerevisiae HKB-36 of claim 1, and the preparation method comprises the steps of slant strain activation, seed liquid preparation, liquid aerobic fermentation and solid anaerobic fermentation;
wherein in the solid anaerobic fermentation step, the fermentation culture medium comprises 2-4 parts of vinasse, 2-4 parts of corn bran, 1-3 parts of bran and 1-3 parts of apple pomace, the carbon-nitrogen ratio of the culture medium is 2-7:1, and the water content is 35-70%;
the fermentation temperature is 28-36 ℃, and the fermentation time is 18-48 h.
6. The method for preparing bacteriostatic biological products according to claim 5, which is characterized in that the method further comprises the following steps of preparing bacteriostatic biological product finished products after the solid anaerobic fermentation:
adjusting the fermentation temperature to 60-75 ℃, inactivating and breaking the walls of the saccharomyces cerevisiae HKB-36, and then crushing and drying to prepare the finished product of the bacteriostatic biological product.
7. A bacteriostatic biological product prepared by the preparation method of claim 6.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105441343A (en) * | 2015-11-24 | 2016-03-30 | 中国农业科学院饲料研究所 | Saccharomyces cerevisiae and Saccharomyces cerevisiae culture thereof |
| WO2018047123A1 (en) * | 2016-09-12 | 2018-03-15 | The New Zealand Institute For Plant And Food Research Limited | Biological control of plant pathogenic microorganisms |
| CN108085261A (en) * | 2017-12-29 | 2018-05-29 | 深圳市善成生物技术有限公司 | One Accharomyces cerevisiae and its culture and the application in feed |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105441343A (en) * | 2015-11-24 | 2016-03-30 | 中国农业科学院饲料研究所 | Saccharomyces cerevisiae and Saccharomyces cerevisiae culture thereof |
| WO2018047123A1 (en) * | 2016-09-12 | 2018-03-15 | The New Zealand Institute For Plant And Food Research Limited | Biological control of plant pathogenic microorganisms |
| CN108085261A (en) * | 2017-12-29 | 2018-05-29 | 深圳市善成生物技术有限公司 | One Accharomyces cerevisiae and its culture and the application in feed |
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