CN113801800A - Saccharomyces cerevisiae and application thereof - Google Patents
Saccharomyces cerevisiae and application thereof Download PDFInfo
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- CN113801800A CN113801800A CN202111119074.3A CN202111119074A CN113801800A CN 113801800 A CN113801800 A CN 113801800A CN 202111119074 A CN202111119074 A CN 202111119074A CN 113801800 A CN113801800 A CN 113801800A
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- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
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- A23L11/60—Drinks from legumes, e.g. lupine drinks
- A23L11/65—Soy drinks
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Abstract
The invention provides a strain of saccharomyces cerevisiae, wherein the strain is saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) stored in China general microbiological culture Collection center (CGMCC) at 16 months 7 and 2020, with the preservation number of CGMCC NO. 20379. The invention relates to wine brewingYeast SF1 (Saccharomyces cerevisiaeSF 1) has fermentation effect on bean clear water and bean dregs samples processed by bean curd, can produce fermented bean clear beverage by fermenting the bean clear water as a raw material, and can also produce fermented bean dregs by fermenting the bean dregs as a raw material, thereby providing a new fermentation bacteria source for the production and development of the fermented bean clear beverage and the fermented bean dregs.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a microorganism culture mediumSaccharomyces cerevisiae and its application are provided. The strain is saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1), the strain can be used for preparing fermented beverage and fermented bean dregs.
Background
The soybean is the fourth major food crop in China, and the processing of the soybean product is the most common edible method of the soybean. In the process of processing bean products, a large amount of byproducts can be generated, and the amount of bean clear water and bean dregs generated in the process of processing bean products in China is about 2100-3500 ten thousand tons and about 200 ten thousand tons respectively. The bean clear water is rich in nutrition and comprises a plurality of effective components such as soluble protein, soluble sugar, vitamins, lipids, trace elements, isoflavone, polysaccharide and the like, but the bean clear water is directly discharged by enterprises in a waste water mode due to heavy fishy smell, poor taste and lack of processing and utilizing technology. The bean dregs contain 50-55% of dietary fiber and 18-23% of protein, and are rich in various substances such as various vitamins, mineral substances, isoflavone and the like of a human body, but the utilization rate of the bean dregs is low because the bean dregs have poor taste, heavy bean smell, difficult storage and various resistance factors, and the protein has large molecular weight and is not easy to digest.
In order to solve the problems, the applicant aims to screen out a strain which can prepare the fermented soy bean milk beverage or the fermented soy bean dregs by taking the soy bean milk or the soy bean dregs as raw materials, and provide a new fermentation bacterium source for development and utilization of the soy bean milk or the soy bean dregs and production of the soy bean milk beverage and the fermented soy bean dregs.
Disclosure of Invention
The invention aims to provide saccharomyces cerevisiae and application thereof. The strain is saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) stored in China general microbiological culture Collection center (CGMCC) at 16.7.2020, with the storage address of No.3 of Xilu No. 1 on North Chen in the area of the rising of Beijing and the storage number of CGMCC NO. 20379. Saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) can produce the fermented drink of the bean whey by taking the bean whey as the main raw material, can also produce the fermented bean dregs by taking the bean dregs as the raw material, and provides a new fermentation bacteria source for the development and the utilization of the bean whey and the bean dregs as well as the production of the fermented drink of the bean whey and the fermented bean dregs.
The invention solves the technical problems through the following technical scheme:
taking Nanping farmhouse traditional home brew preserved rice koji cake 10g, adding 250ml of bean clear water obtained by the production process of Changting traditional sour pulp bean curd, performing domestication and fermentation at 28 ℃, continuously changing for several times with 7 days as a period until the bean clear water has no bean smell and has faint scent, inoculating the domesticated bean clear fermentation broth into sterilized bean dregs, performing solid state fermentation and domestication for 3 days, continuously inoculating and domesticating until macromolecular protein of a fermentation product is remarkably reduced, diluting the domesticated fermentation mixture to 10-4、10-5Respectively taking 0.1ml of the diluent with the concentration gradient, coating the diluent on a flat plate of a yeast solid culture medium, carrying out inverted culture at 28 ℃ for 3 days, selecting 4 single colonies of suspected yeast with similar colony morphology, carrying out streak purification culture on the yeast solid flat plate for 3 times, simultaneously carrying out static culture on each single colony in a bean clear water culture medium at 28 ℃ for 72 hours to obtain a bean clear fermentation culture solution, measuring and analyzing the alcohol content of the fermentation culture solution, and tasting the flavor and the taste of the fermentation culture solution; respectively inoculating the fermented culture solution of the bean whey into bean dregs for fermentation, finally selecting a strain SF1 which has unique fermented flavor and can degrade macromolecular proteins in the bean dregs, and finally identifying the strain as Saccharomyces cerevisiae (Saccharomyces cerevisiae) by performing morphological observation, 18S rDNA gene sequence determination and phylogenetic tree homology analysis on the strain.
Further, Saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) has the following characteristics:
a. the colony is round, and is milky white and opaque on a yeast culture medium; the thallus is oval, the width is 2-4 μm, and the length is 4-6 μm;
b. glucose, galactose, maltose, sucrose and fructose can be utilized for fermentation to produce acid and gas, and lactose, starch, xylose, arabinose and trehalose cannot be utilized;
c. can survive for 6 hours under the condition that the pH value is 2.0; when cultured for 2h and 6h under the condition of pH value of 3.0, the colony count can grow to 130.34% and 225.64% of the original value.
The saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) can be used for preparing fermented beverage and fermented bean dregs.
Saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) can ferment the bean clear water as raw material in a short time of 6h, remove beany flavor, and increase protein content in the bean clear water to 121.74% of that before fermentation, to produce a fermented beverage of the bean clear water without alcohol with unique taste, wherein the obtained fermented beverage of the bean clear water contains isoflavone (m/v) 0.92%, amino acids 254.9mg/100ml, protein 0.3% and polysaccharide 3.01 mg/ml.
Saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) can also take the bean dregs as raw materials for fermentation, degrade macromolecular protein substances with molecular weight more than 25kDa in the bean dregs into small molecular protein substances with molecular weight less than 25kDa, simultaneously improve the total protein content in the bean dregs to 114.05% when the bean dregs are not fermented, obviously improve the small molecular peptide protein content in the bean dregs, and the fermented bean dregs can be used for preparing fermented bean dregs food or fermented bean dregs feed, are applied to the fermentation treatment and utilization of the bean dregs, and can be used as feed raw materials for the breeding industry.
The invention has the beneficial effects that: providing Saccharomyces cerevisiae SF1Saccharomyces cerevisiaeSF 1), the strain can utilize the bean whey as a raw material to produce the bean whey fermented beverage in a short time, and can also use the bean dregs as a raw material to produce fermented bean dregs, thereby providing a new fermentation bacteria source for the development of the bean whey fermented beverage and the production and development of the fermented bean dregs.
Drawings
FIG. 1 is a colony morphology of SF 1.
FIG. 2 is a scanning electron micrograph of SF1 strain.
FIG. 3 shows the amplification result of the target fragment.
FIG. 4 shows a phylogenetic tree of SF1 strain.
FIG. 5 shows the optimum fermentation pH of SF2 strain.
FIG. 6 is a PAGE picture of Saccharomyces cerevisiae SF1 fermentation on okara and okara. Lane M is protein Marker; lane Z-CK is okara (unfermented); lane Z1 bean dregs (3 days fermentation with SF 1); lane Z2 for okara (fermentation product after molasses addition); lane S-CK is legume clear water (unfermented); lane S1 for fermented soy whey beverage (unfiltered); lane S2 shows a fermented soy whey beverage (filtration treatment).
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.
The invention relates to saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) is obtained from traditional fermented rice cake of Nanping farmhouse.
EXAMPLE 1 acclimatization, selection and isolation of bacteria
Taking Nanping farmhouse traditional home brew preserved rice koji cake 10g, adding 250ml of bean clear water obtained by the production process of Changting traditional sour pulp bean curd, domesticating and fermenting at 28 ℃, continuously changing for 3 times with 7 days as a period until the bean clear water has no bean smell, inoculating the domesticated bean clear fermentation broth into sterilized bean dregs according to the inoculation amount of 10% (v/w), fermenting and domesticating at 28 ℃ for 3 days in a solid state, continuously inoculating and domesticating until the macromolecular protein of a fermentation product is remarkably reduced, diluting the domesticated fermentation mixture to 10 percent-4,10-5Respectively taking 0.1mL of diluent, coating the diluent on a flat plate of a yeast solid culture medium, carrying out inverted culture at 28 ℃ for 24h, selecting single colony of suspected yeast with the most similar colony morphology, carrying out streak purification culture on the yeast solid flat plate for 3 times to obtain 4 single colonies, respectively carrying out standing culture at 28 ℃ for 7h in 200mL of bean clear water culture medium to obtain bean clear fermentation liquor, measuring the alcohol content of the bean clear fermentation liquor, and tasting the flavor and the taste of the bean clear fermentation liquor; and inoculating the fermentation culture solution of the bean whey into sterilized bean dregs according to the inoculation amount of 10% (v/w) respectively for fermentation. The fermentation result is shown in table 1, and finally, a strain 1 which is unique in fermentation flavor of the bean whey and can degrade macromolecular proteins in the bean dregs is selected, stored and named as SF 1. Finally identified as saccharomyces cerevisiae by morphological observation, 18S rDNA gene sequence determination and phylogenetic tree homology analysis of the strainsSaccharomyces cerevisiae）。
The formula of each culture medium is as follows:
(1) yeast medium (1L): 5.0g of peptone, 3.0g of malt extract, 3.0g of yeast extract and 3.0g of glucose are dissolved in distilled water, the volume is adjusted to 1000 mL, the pH value is adjusted to 6.2, and the components are packaged and sterilized at 121 ℃ for 20 min under high pressure. (Note: solid Medium on this basis 20.0g/L agar powder was added)
(2) Bean clear water culture medium: the conventional processing method of sour pulp bean curd comprises obtaining bean clear water, adding sucrose according to (w/v) ratio of 15:100, and autoclaving at 115 deg.C for 20 min.
(3) And (3) sterilizing the bean dregs: the bean dregs obtained by the traditional processing technology of the sour pulp bean curd are sterilized under high pressure at 115 ℃ for 15 min.
TABLE 1 analysis of fermented soy drink with various bacteria
EXAMPLE 2 identification of the species
2.1 morphological Observation of SF1 bacterium
The main morphological characteristics of the SF1 strain are as follows: round colonies, milky white and opaque on yeast medium (FIG. 1); the thallus is oval, 2-4 μm wide and 4-6 μm long (FIG. 2).
2.2 analysis of carbon Source utilization by SF1 bacterium
Preparing culture medium (each 1 liter of culture medium contains 10g of peptone, 3g of beef powder, 5g of sodium chloride and 0.04% of bromocresol purple), subpackaging the culture medium into test tubes according to 10 ml/tube, respectively adding 1wt% of glucose, galactose, maltose, sucrose, lactose, starch, fructose, xylose, arabinose and trehalose, reversely putting the test tubes into Dehan tubules, each sugar is 3, sterilizing at 121 ℃ for 20 min), inoculating saccharomyces cerevisiae SF1 bacterial liquid (the concentration of the SF1 bacterial liquid is 2.0 x 10) according to the inoculation amount of 1% (v/v)8cfu/mL) at 30 ℃ and produces acid and gas, the culture medium turns orange yellow (the culture medium is originally dark purple), the gas is produced, the result is shown in Table 2, and SF1 can utilize glucose, galactose, maltose, sucrose and fructose to produce acid and gas, and cannot utilize lactose, starch, xylose, arabinose and trehalose.
TABLE 2 utilization of SF1 for sugars
Note: "+" is positive and "-" is negative.
2.3 molecular biological identification of SF1 bacterium
Extracting bacterial genome DNA: extracted by adopting a yeast genome DNA extraction kit of TIANGEN company.
PCR amplification of 18S rDNA sequence: amplifying 18S rDNA gene sequence by using the following primers:
Au2-F（SEQ ID NO.1）：5’-TTTCGATGGTAGGATAGDGG-3’；
Au4-R（SEQ ID NO.2）：5’-RTCTCACTAAGCCATTC-3’。
and (3) PCR reaction system: 2 Mix 12.5. mu.L, primers and DNA 1. mu.L each, ddH20 9.5μL。
PCR amplification procedure: pre-denaturation at 93 deg.C for 4 min; then denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 60s, and extension at 72 ℃ for 90s for 35 cycles; finally, fully extending for 10min at 72 ℃, and storing at 4 ℃.
PCR product detection and sequencing analysis: taking 5 mu L of PCR product, carrying out gel electrophoresis separation detection in 1.0% agarose added with EB, and amplifying to obtain a 18S rDNA target fragment with the length of 1257bp (figure 3). And (3) trusting a platfon biological sequencing company to complete sequencing on a product containing the target fragment, and obtaining an actual effective nucleotide sequence of 18S rDNA, wherein the nucleotide sequence is shown in SEQ ID NO.3 and the size is 1257 bp.
Phylogenetic analysis: performing Blast comparison analysis on each 18S rDNA sequence in NCBI data to obtain a sequence andSaccharomyces cerevisiaeper. Ident is 99.92%, Query Cover is 100%, construction and analysis of development tree are carried out by Maximum Parsimony in MEGA 4.1, and SF1 and Query are finally determinedSaccharomyces cerevisiae (NR 132207.1) on the same development branch (see FIG. 4), combined with morphological observation of SF1 bacteria, physiological and biochemical tests of bacteria, and homology analysis in DNA phylogenetic tree, the SF1 bacteria were determined to beSaccharomyces cerevisiae (Saccharomyces cerevisiae) And (5) strain.
Example 3 Saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) growth at different pH values
Respectively adjusting the pH of the yeast fermentation culture medium to 1.5-3.5, inoculating Saccharomyces cerevisiae SF1 bacterial liquid (SF 1 bacterial liquid concentration is 2.0 × 10) according to the inoculation amount of 1% (v/v)8cfu/mL), culturing for 2h and 6h respectively at 30 ℃ and 150 r/min, then taking 1mL of bacterial suspension for colony counting, and the result is shown in Table 3, and the bacterium SF1 can survive for 6h under the condition that the pH value is 2.0; the growth and propagation can be realized under the condition of pH value of 3.0, the colony number grows to 130.34% of the original value when the yeast is cultured for 2h on a yeast culture medium with pH value of 3.0, and grows to 225.64% of the original value when the yeast is cultured for 6 h. To show that the SF1 strain can realize positive growth under the acidic condition with the pH value of 3.0, the optimum fermentation pH value of SF1 is determined to be between 6.0 and 6.5 (figure 5).
TABLE 3 growth of bacterium SF1 under acidic conditions
Example 4 Saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) analysis of fermented beverages produced whey during different fermentation times
1mL of saccharomyces cerevisiae SF1 bacterial liquid (SF 1 bacterial liquid concentration is 2.0 multiplied by 10)8cfu/mL) was inoculated into 200mL of aqueous media of soy bean curd, fermentation samples were taken at different fermentation times, and the colony count, pH, total sugar, protein concentration, alcohol content and mouthfeel were analyzed and tested, and the results are shown in Table 4, which shows that after 6 hours of fermentation with SF1, the beany flavor in the soy bean curd was completely disappeared, and an excellent mouthfeel was exhibited, and at this time: the pH value is 5.1; polysaccharide 3.00 mg/mL; no alcohol content; the protein content is 0.298mg/mL, which is increased by 21.74 percent compared with the unfermented protein (0 h). Based on the above, 6h is selected as the optimal fermentation time to produce the fermented soy bean whey beverage.
TABLE 4 analysis of fermentation of soy bean whey with SF1 at different fermentation times
Example 5 Saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) in fermented beverage of bean whey
The analysis of the amino acid content, isoflavone and protein in the fermented soy whey beverage specifically comprises the following steps: 1mL of saccharomyces cerevisiae SF1 bacterial liquid (SF 1 bacterial liquid concentration is 2.0 multiplied by 10)8cfu/mL) was inoculated into 200mL of a media of whey, and the media was subjected to static fermentation at 28 ℃ for 6 hours to prepare a whey fermented beverage, the parameters of which are shown in Table 5: after fermentation with Saccharomyces cerevisiae SF1, a fermented soy whey beverage free of beany flavor and giving a light flavor was obtained, which contained (m/v) 0.92% isoflavones, 254.9mg/100ml total amino acids, 0.3% protein, 3.01mg/ml polysaccharide, and a pH of 5.1. After the fermentation liquor is filtered to remove solid substances, the obtained liquid beverage can keep the fermentation liquor in a stable state for a long time without obvious precipitation due to the high content of soybean polysaccharide, so that the beverage does not need to be added with substances such as a stabilizer and the like. After the fermented soy drink was concentrated in the same ratio, PAGE protein detection was performed (fig. 6), and it was found that the protein content of the fermented soy drink was significantly increased compared to the unfermented soy drink (S-CK), and that the protein content of the fermented soy drink was not significantly changed by filtering the fermented soy drink, indicating that the increased protein was present in the form of soluble protein.
TABLE 5 Soy whey fermented beverage product parameter testing
Example 6 Saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) fermenting to produce fermented bean dregs
And (3) producing the fermented bean dregs by using the saccharomyces cerevisiae SF 1. Sterilizing bean dregs at 115 deg.C for 15min, inoculating Saccharomyces cerevisiae SF1 bacterial liquid (SF 1 bacterial liquid concentration is 2.0 × 10) at 5% (v: w)8 cfu/mL），28℃Fermenting at constant temperature for 3 days. Meanwhile, 10% of molasses is added into bean dregs according to the mass ratio, the sterilization treatment is carried out at 115 ℃ for 15min, and the saccharomyces cerevisiae SF1 bacterial liquid (the concentration of the SF1 bacterial liquid is 2.0 multiplied by 10) is inoculated according to the proportion of 5% (v: w)8cfu/mL), fermenting at 28 ℃ for 3 days. PAGE electrophoresis is carried out on the fermented bean dregs for protein size detection and analysis (figure 6), after SF1 fermentation, macromolecular proteins larger than 25kDa in the bean dregs are basically degraded into micromolecular proteins smaller than 25kDa, micromolecular polypeptides in the fermented bean dregs are remarkably increased, compared with carbon sources such as molasses and the like, more amino acids can be kept without adding a carbon source, and the SF1 can remarkably degrade the macromolecular proteins in the bean dregs into the micromolecular polypeptides on the basis of not adding other substances such as the carbon source and the like, and the bacterium can ferment the bean dregs to produce substances rich in the micromolecular polypeptides. Further, the results of the detection and analysis of the total protein in the bean dregs before and after fermentation by using Kjeldahl method GB/T6432-2018 are shown in Table 6, and after SF1 fermentation for 72h, the total protein in the bean dregs is increased from 23.525% before fermentation to 26.831%, and the protein in the fermented bean dregs is increased by 14.05%. The protein molecular weight of the fermented bean dregs obtained by adopting SF1 is lower than 25kDa, so that the protein content is obviously increased, and the fermented bean dregs are very suitable for being used as high-quality raw materials to be applied to the culture of foods, livestock and poultry and aquatic products.
TABLE 6 analysis of the Total protein content of fermented okara
SEQUENCE LISTING
<110> institute of agricultural engineering technology of agricultural academy of sciences of Fujian province
<120> saccharomyces cerevisiae and application thereof
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agggttcgat tccggagagg gagcctgaga aacggctacc acatccaagg aaggcagcag 60
gcgcgcaaat tacccaatcc taattcaggg aggtagtgac aataaataac gatacagggc 120
ccattcgggt cttgtaattg gaatgagtac aatgtaaata ccttaacgag gaacaattgg 180
agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta tattaaagtt 240
gttgcagtta aaaagctcgt agttgaactt tgggcccggt tggccggtcc gattttttcg 300
tgtactggat ttccaacggg gcctttcctt ctggctaacc ttgagtcctt gtggctcttg 360
gcgaaccggg acttttactt tgaaaaaatt agagtgttca aagcaggcgt attgctcgaa 420
tatattagca tggaataata gaataggacg tttggttcta ttttgttggt ttctaggacc 480
atcgtaatga ttaataggga cggtcggggg catcagtatt caattgtcag aggtgaaatt 540
cttggattta ttgaagacta actactgcga aagcatttgc caaggacgtt ttcattaatc 600
aagaacgaaa gttaggggat cgaagatgat cagataccgt cgtagtctta accataaact 660
atgccgacta gggatcgggt ggtgtttttt taatgaccca ctcggcacct tacgagaaat 720
caaagtcttt gggttctggg gggagtatgg tcgcaaggct gaaacttaaa ggaattgacg 780
gaagggcacc accaggagtg gagcctgcgg cttaatttga ctcaacacgg ggaaactcac 840
caggtccaga cacaataagg attgacagat tgagagctct ttcttgattt tgtgggtggt 900
ggtgcatggc cgttcttagt tggtggagtg atttgtctgc ttaattgcga taacgaacga 960
gaccttaacc tactaaatag tggtgctagc atttgctggt tatccacttc ttagagggac 1020
tatcggtttc aagccgatgg aagtttgagg caataacagg tctgtgatgc ccttagacgt 1080
tctgggccgc acgcgcgcta cactgacgga gccagcgagt ctaaccttgg ccgagaggtc 1140
ttggtaatct tgtgaaactc cgtcgtgctg gggatagagc attgtaatta ttgctcttca 1200
acgaggaatt cctagtaagc gcaagtcatc agcttgcgtt gattacgtcc ctgccct 1257
Claims (3)
1. A strain of saccharomyces cerevisiae is characterized in that: the strain is saccharomyces cerevisiae SF1 (Saccharomyces cerevisiaeSF 1) stored in China general microbiological culture Collection center (CGMCC) at 16.7.2020, with the storage address of No.3 of Xilu No. 1 on North Chen in the area of the rising of Beijing and the storage number of CGMCC NO. 20379.
2. Use of the saccharomyces cerevisiae according to claim 1 for the preparation of a fermented soy whey beverage.
3. Use of the saccharomyces cerevisiae according to claim 1 for the preparation of fermented okara.
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