CN112300950B - Acid-resistant saccharomyces cerevisiae and application thereof - Google Patents
Acid-resistant saccharomyces cerevisiae and application thereof Download PDFInfo
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- CN112300950B CN112300950B CN201910711549.4A CN201910711549A CN112300950B CN 112300950 B CN112300950 B CN 112300950B CN 201910711549 A CN201910711549 A CN 201910711549A CN 112300950 B CN112300950 B CN 112300950B
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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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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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Abstract
The invention discloses an acid-resistant saccharomyces cerevisiae and application thereof. The preservation number of the acid-resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) YR201705 is CGMCC No.15315. The invention also provides an application of the preserved strain in fermenting TMR. The acid-resistant saccharomyces cerevisiae can grow in an environment with pH of 2.5, and grow and propagate rapidly in a large quantity under the condition with pH of 4.0, and the saccharomyces cerevisiae strain grows on a potato glucose culture medium to generate smell of rice wine fragrance. The palatability of the fermented feed can be obviously improved under the high acidity environment of the fermented TMR.
Description
Technical Field
The invention relates to the fields of microbial fermentation and feed processing, in particular to acid-resistant saccharomyces cerevisiae and application thereof in fermenting TMR.
Background
With the development of the feed industry in recent years, the technology of total mixed ration (total mixed rations, TMR) has become the preferred feeding technology of large-scale dairy farms such as standardization, intensification and the like at home and abroad at present. Because TMR can better mix forage grass, grain, protein feed, mineral matters and feed additives, the nutritional requirements of animals can be met, therefore, TMR can obviously improve the feed conversion ratio, increase the milk yield and the quality, and can also effectively reduce the incidence of related diseases, thus being an ideal feed model in the dairy cow industry. However, the common TMR is a feed which is easy to deteriorate, the service time and the storage time are required to be paid attention, and the use of the TMR is limited to a wide extent due to the problem of labor cost. TMR is extremely easy to be subjected to aerobic spoilage in the aspect of storage and transportation, so that nutrition loss is caused, and certain diseases can be brought to cows. One way to effectively solve such problems is to use fermented TMR.
The fermented TMR is a complete fermented feed prepared by sealing and storing the prepared TMR feed for a period of time by adopting silage technologies such as stretch film wrapping or bagging and the like and performing lactic acid fermentation. The preparation principle is derived from silage principle, namely, under the condition of non-strict anaerobism, a large amount of lactobacillus on the feed raw material grows and breeds, soluble carbohydrate in the raw material is changed into lactic acid, and when the concentration reaches a certain value, the growth and the breeding of harmful microorganisms are inhibited, so that the purpose of preserving the feed is achieved. On the basis of effectively maintaining the nutritional value of TMR ration, fermentation TMR storage property and aerobic stability are obviously improved by fermentation, and commercialization and circulation of TMR are facilitated.
Yeast is a facultative anaerobic single-cell fungus, and the yeast is rich in various nutrients such as protein, B vitamins, fat, sugar and the like. The yeast has the capability of regulating and controlling the environment in rumen, relieving rumen acidosis and stimulating the growth activity of fibrinolytic bacteria, has important effects on the generation of rumen methane and the metabolism of nitrogen and hydrogen, has special flavor generated by yeast fermentation, and can improve the palatability of the feed.
The pH value generally decreases continuously along with the generation and accumulation of acidic substances such as lactic acid and the like in the preparation process of the fermented TMR. Saccharomyces cerevisiae is used as a main flora in fermentation production, and has strong ethanol production capacity in the early stage of the whole fermentation process, but after the acidity is reduced to pH less than or equal to 4.5 along with the fermentation process, the physiological metabolism of the saccharomycetes is gradually inhibited or even stopped. Therefore, the screening can still have vigorous metabolic activity under the high acidity environmental condition in the later period of the fermentation TMR production. The acid-resistant saccharomyces cerevisiae flora with stronger ethanol fermentation production capacity is a technical key for improving fermentation quality in the production process of a fermentation TMR technology.
Disclosure of Invention
The invention aims to provide an acid-resistant saccharomyces cerevisiae which is suitable for a high-acidity environment in the later period of the production of the fermented TMR, and the prepared fermented TMR has better palatability.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides an acid-resistant saccharomyces cerevisiae (Saccharomyces cerevisiae), which is named YR201705 and classified as follows: saccharomyces cerevisiae Saccharomyces cerevisiae has been deposited in China general microbiological culture Collection center (CGMCC) (address: north Xielu No.1, 3, china academy of sciences of China, with a accession number of CGMCC No. 15315) at 25 days of 1 month of 2018.
The acid-resistant saccharomyces cerevisiae YR201705 is obtained by screening acid-resistant saccharomyces cerevisiae capable of growing and propagating under the condition of fermenting and producing acid pH4.0 in a fermentation TMR experiment by adding saccharomyces cerevisiae. The acid-resistant Saccharomyces cerevisiae is facultative anaerobic fungus, has strong acid resistance (can grow in pH 2.5 acidity environment and can rapidly reproduce in large quantity in pH4.0 acidity environment), and has high growth rate (the total number of colonies can reach 9.0X10 when cultured in MEB culture medium for 18 hr) 11 cfu/ml). As with other organisms, the acid-resistant Saccharomyces cerevisiae of the present invention is still susceptible to variation. Thus, mutant strains of the strain can be obtained using physical and chemical mutagenesis methods known in the art. These mutants are also part of the present invention, as long as they retain the acid-resistance of Saccharomyces cerevisiae.
The method for obtaining the acid-resistant saccharomyces cerevisiae comprises the following steps: adding Saccharomyces cerevisiae enriched and cultured by MEB culture medium into TMR feed added with lactobacillus starter, vacuum sealing and packaging, and fermenting at room temperature. Fermented TMR samples were collected at 24h, 48h, 72h, 5d, 7d, 10d, 15d, 20d, 30d, 40d, 50d, 60d of fermentation, respectively, for pH and total yeast count determinations. Under aseptic condition, 25g of fermented TMR is taken in an aseptic homogenizing bag, 225ml of sterilized distilled water is added for shaking and mixing uniformly, the final concentration is 0.1g/ml, then 1ml of diluted sample is taken, added in a test tube containing 9ml of sterilized distilled water for shaking and mixing uniformly, the final concentration is 0.01g/ml, the mixture is repeatedly diluted to the proper dilution according to the method, 1ml of the diluted sample is taken in a potato glucose plate, the potato glucose plate is coated by an aseptic coater, and the potato glucose plate is placed in a constant-temperature incubator at 28 ℃ for culturing for 5 days and counting. After fermentation at 20d, the pH of the TMR sample was reduced to around 4.2, and yeast was detected in only 2 samples. On a plate with saccharomycetes, picking single colony with an inoculating loop, inoculating to a wort broth culture medium, placing the wort broth culture medium in a 28 ℃ shaking incubator for culturing for 18 hours, picking bacterial liquid with the inoculating loop, streaking the bacterial liquid on a potato glucose culture medium plate, culturing at a constant temperature of 28 ℃, repeatedly separating and purifying for 3 times, streaking the purified wort bacterial liquid on a potato glucose slant culture medium, and storing in a refrigerator at 4 ℃. Wherein the potato dextrose medium is: 1.0L of potato leaching solution (200 g of peeled potato is taken, cut into small pieces, boiled with 1.0L of water for 30 minutes, potato pieces are filtered off, and the filtrate is less than 1.0L); glucose 20.0g; 15.0g of agar; chloramphenicol 0.1g,121 ℃, and sterilized for 20min. The wort medium was: malt wort 1.0L,121℃and 20min.
In the low pH environment of fermentation, acid-tolerant Saccharomyces cerevisiae strains are produced and present due to spontaneous mutation and natural selection. Preparing a wort culture medium with gradient difference of acidity according to the acidity of the low pH living environment of saccharomycetes in the TMR fermentation process, culturing an acid-resistant saccharomyces cerevisiae strain stored in a potato glucose slant culture medium, further screening and measuring the acid resistance of the acid-resistant saccharomyces cerevisiae strain. The preparation method of the acid wort selection medium comprises the following steps: after preparing wort medium according to conventional method, adjusting acidity of the medium with lactic acid under aseptic condition to form acidity of: the wort selection medium is an acidic gradient with a pH of 2.0-4.0. Extracting yeast genome DNA by using an Ezup column type yeast genome extraction kit, amplifying 26S rDNA D1/D2 region sequences by using PCR amplification primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3' shown as SEQ ID No.1 in a sequence table) and NL4 (5'-GGTCCGTGTTTCAAGACGG-3' shown as SEQ ID No.2 in the sequence table), and comparing related sequences on NCBI to determine strains. And finally, respectively adding the screened strain and the reference strain into the fermented TMR, fermenting the strain and the reference strain in a normal temperature environment, unsealing, analyzing the quality of the fermented TMR, and comparing the quality of the fermented TMR, so that the acid-resistant saccharomyces cerevisiae can improve the fermentation quality of the fermented feed in the normal temperature environment.
The invention also protects the application of the acid-resistant saccharomyces cerevisiae YR201705 in fermenting TMR.
The acid-resistant saccharomyces cerevisiae YR201705 can be used for preparing a fermented TMR feed, and the specific method comprises the following steps:
(1) Adding a bacterial suspension of acid-resistant saccharomyces cerevisiae YR201705 into TMR feed according to the addition amount of 5% of the total weight of TMR;
(2) Adding molasses into TMR feed according to the addition amount of 5% of total weight of TMR;
(3) The water content of TMR added with acid-resistant saccharomyces cerevisiae and molasses is adjusted to 45% -55%;
(4) Aerobic fermentation;
(5) After aerobic fermentation, 1.0X10 of lactobacillus is added into each kilogram of fermented material 8 More than one;
(6) And (5) vacuum sealing and storing to obtain the fermented TMR.
Optionally, in the step (1), the bacterial count in the bacterial suspension of the acid-resistant saccharomyces cerevisiae is 4×10 11 CFU/ml-8×10 11 CFU/ml。
Optionally, in the step (4), the aerobic fermentation conditions are: the temperature is 18-40 ℃ and the time is 0.1-24 hours.
Optionally, the material is turned over in the aerobic fermentation process.
Optionally, in the step (6), the temperature of sealed storage is 18-40 ℃ and the time is 2-21 days.
Compared with the prior art, the invention has the following beneficial effects:
(1) The acid-resistant yeast strain can improve the fermentation quality of TMR feed, and has low cost, safety and reliability.
(2) The fermented TMR is easy to preserve for a long time and convenient to transport.
(3) The saccharomyces cerevisiae strain grows on a potato dextrose culture medium, and generates smell of rice wine fragrance. The palatability of the fermented feed can be obviously improved under the high acidity environment of the fermented TMR.
Drawings
FIG. 1 shows a diagram of the cell morphology of acid-tolerant yeasts.
FIG. 2 shows the growth curve of acid-resistant Saccharomyces cerevisiae at 28℃and pH 4.0.
Detailed Description
The invention will be further illustrated with reference to the following specific examples, which are not intended to limit the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent suppliers.
Example 1 screening of acid-tolerant Saccharomyces cerevisiae
And separating yeast strain 2 from TMR feed with pH of 4.2 on fermentation day 20, performing colony morphology observation and cell morphology observation, and screening acid-resistant and fast-growing yeast according to experiments of fertility pH (2.0, 2.5, 3.0, 3.5 and 4.0). The results showed that the acid-fast yeast cells were spherical or oval in morphology, and the colonies were milky white, glossy, flat and clean in edge (see FIG. 1). Can grow at pH 2.5 and grow rapidly in large quantity at pH4.0 (see figure 2), and has strong acid resistance. Glucose, maltose and sucrose can be fermented, lactose cannot be fermented. Classifying and identifying the degradation strain by adopting morphological, physiological and biochemical and 26S rDNA sequence analysis and other classification methods
Physiological and biochemical test of acid-resistant yeasts: fermentation type and morphology observations are described in the handbook of characterization and identification of Yeast, by Inbanite, instroma (Hu Ruiqing).
The culture medium and the preparation method thereof are as follows:
(1) Potato dextrose medium: 1.0L of potato leaching solution (200 g of peeled potato is taken, cut into small pieces, boiled with 1.0L of water for 30 minutes, potato pieces are filtered off, and the filtrate is less than 1.0L); glucose 20.0g; 15.0g of agar; chloramphenicol 0.1g,121 ℃, and sterilized for 20min.
(2) The wort medium was: malt wort 1.0L,121℃and 20min.
(3) Acidic wort medium: malt wort 1.0L,121 ℃, sterilization for 20min, under aseptic condition, adjusting acidity of culture medium with lactic acid, forming acidity respectively as follows: an acidic gradient malt juice liquid medium with pH of 2.0-4.0.
Example 2 identification of acid-tolerant yeasts
1.0ml of the yeast liquid cultured overnight was added to a 1.5ml centrifuge tube, and the mixture was centrifuged at 10,000rpm for 1 minute at room temperature to collect the cells. Then extracting the microzyme genome DNA by using Ezup column type microzyme genome extraction kit, and measuring the absorbance at OD 600 nm. Then, PCR amplification is carried out on the primers NL1 and NL4 of 26S rDNA D1/D2 region sequence, wherein the PCR reaction is that the pre-denaturation is carried out for 4min at 94 ℃; (94 ℃ C. 45sec,55 ℃ C. 45sec,72 ℃ C. 1 min) 30 cycles; extending at 72 ℃ for 10min; the reaction was terminated at 4 ℃. Sequencing the amplified product in biological engineering (Shanghai) Co., ltd, comparing the result in NCBI gene library, analyzing with DNAman software, and analyzing 26S rDNA D1/D2 region sequence (about 592 bp) of the strain, as shown in SEQ ID No.3 in the sequence table, and performing similarity analysis with standard bacteria, wherein the similarity of the acid-resistant saccharomyces cerevisiae and saccharomyces cerevisiae is 100%, and the strain is the same bacteria.
The Saccharomyces cerevisiae (Saccharomyces cerevisiae) YR201705 of the invention is preserved in China general microbiological culture Collection center (CGMCC) (address: north Chen Xi Lu No.1, 3 of the area of Chaoyang in Beijing, and the post code 100101 of the institute of microorganisms of the national academy of sciences of China) in the 1 st month 25 of 2018, and the preservation number is CGMCC No.15315.
Example 3 Effect of addition to TMR feed
TMR fermentation and addition tests are carried out by taking TMR as a material. Adding acid-resistant saccharomyces cerevisiae suspension into TMR feed according to the addition amount of 5% by weight of TMR, and adding molasses according to the addition amount of 5% by weight of TMR to ensure that the water content of TMR is 45% -55%. After aerobic fermentation for 18h, 1.0X10 of Lactobacillus plantarum is added per kg of the above fermented material 8 More than one. And (3) carrying out vacuum sealing packaging on TMR added with strains, filling 2 bags in each treatment, wherein each bag is about 500g, and storing at room temperature for 60d.
The method for measuring the nutritional ingredients and the fermentation indexes comprises the following steps:
(1) And (3) microorganism detection: after the sample is unsealed, the microbial indicator is first measured. In an ultra clean bench, 25g of the sample is accurately weighed and added with 225ml of sterile physiological saline, and the sample is detected according to GB 4789.15-2016 food safety and sanitation standard food microbiology test mould and yeast counting method.
(2) And (3) nutrient component measurement: after the sample was dried at 65 ℃, the moisture, crude ash, crude protein, starch content were measured. The experiment uses Kjeldahl method to determine the content of CP in the sample; burning the sample in a muffle furnace at 550 ℃ for more than 4 hours to determine Ash; starch was assayed using Megazyme kit.
(3) And (3) fermentation index measurement: 10g of fresh sample was taken, 90ml of distilled water was added, homogenized by a juicer for 1min,4 layers of roving were filtered, 35ml of filtrate was taken, and the pH value was measured by a pH meter. Centrifuging the filtrate at 3500r/min for 15min, and collecting supernatant, and measuring ammonia nitrogen content by phenol-sodium hypochlorite method; the supernatant was filtered through a 0.45 μm filter membrane and measured by Agilent (model: 1260) high performance liquid chromatograph: chromatographic conditions: chromatographic column (KC-811) mobile phase: KH 0.02mol/L 2 PO 4 Adjusting the pH to 2.80 with H3PO 4; flow rate: 0.7ml/min, column temperature 35 ℃; detection wavelength: 210nm, and the sample injection amount is 10 mu L.
TABLE 1 influence of addition of acid-tolerant Saccharomyces cerevisiae on the chemical and microbial Components of fermented TMR at room temperature
| Detection index | Adding common commercial Saccharomyces cerevisiae | Adding acid-resistant saccharomyces cerevisiae |
| Moisture (%) | 57.06 | 58.07 |
| Crude protein (% DM) | 18.13 | 17.24 |
| Coarse ash (% DM) | 6.70 | 6.10 |
| Calcium (% DM) | 0.75 | 0.62 |
| Total phosphorus (% DM) | 0.46 | 0.42 |
| Starch (% DM) | 23.20 | 24.10 |
| Lactic acid bacteria CFU/g | 1.5×10 8 | 1.1×10 9 |
| Yeast CFU/g | 0 | 2.1×10 6 |
TABLE 2 influence of addition of acid-fast Saccharomyces cerevisiae on TMR fermentation quality at room temperature
Compared with the common commercial saccharomyces cerevisiae agent, the TMR fermented by the acid-resistant saccharomyces cerevisiae has slightly higher lactic acid content than that of the control group and obviously lower acetic acid, propionic acid, butyric acid and ammoniacal nitrogen content than that of the control group, so that the fermentation quality of the TMR is obviously improved. On day 60 of fermentation, no Saccharomyces cerevisiae was detected in the control group, while Saccharomyces cerevisiae still grew in the experimental group, which was significantly superior to the control group in terms of odor.
It is to be understood that the above examples of the present invention are provided by way of illustration only and not by way of limitation of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Not all embodiments are exhaustive. All obvious changes or modifications which come within the spirit of the invention are desired to be protected.
Sequence listing
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<120> an acid-resistant saccharomyces cerevisiae and application thereof
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ggccgttcct tgtctatgtt ccttggaaca ggacgtcata gagggtgaga atcccgtgtg 180
gcgaggagtg cggttctttg taaagtgcct tcgaagagtc gagttgtttg ggaatgcagc 240
tctaagtggg tggtaaattc catctaaagc taaatattgg cgagagaccg atagcgaaca 300
agtacagtga tggaaagatg aaaagaactt tgaaaagaga gtgaaaaagt acgtgaaatt 360
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aggggaatct cgcatttcac tgggccagca tcagttttgg tggcaggata aatccatagg 480
aatgtagctt gcctcggtaa gtattatagc ctgtgggaat actgccagct gggactgagg 540
actgcgacgt aagtcaagga tgctggcata atggttatat gccgcccgtc tt 592
Claims (6)
1. An acid-resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) YR201705 with a preservation number of CGMCC No.15315.
2. The use of the acid-resistant saccharomyces cerevisiae YR201705 of claim 1 in fermenting a fully mixed ration.
3. The use according to claim 2, characterized by the steps of:
(1) Adding the acid-resistant saccharomyces cerevisiae bacterial suspension of claim 1 into the total mixed ration feed according to the addition amount of 5 percent of the total weight of the total mixed rationThe method comprises the steps of carrying out a first treatment on the surface of the The bacteria number in the acid-resistant saccharomyces cerevisiae bacterial suspension is 4 multiplied by 10 11 CFU/ml-8×10 11 CFU/ml
(2) Adding molasses into the total mixed ration feed according to the addition amount of 5% of the total weight of the total mixed ration;
(3) The water content of the total mixed ration added with the acid-resistant saccharomyces cerevisiae and molasses is adjusted to 45-55%;
(4) Aerobic fermentation;
(5) After aerobic fermentation, 1.0X10 of lactobacillus is added into each kilogram of fermented material 8 More than one;
(6) Vacuum sealing and storing to obtain fermented mixed ration.
4. The use according to claim 3, wherein in step (4), the aerobic fermentation conditions are: the temperature is 18-40 ℃ and the time is 0.1-24 hours.
5. The use according to claim 4, wherein the material is turned during aerobic fermentation.
6. The use according to claim 3, wherein in step (6) the temperature of the sealed storage is 18 ℃ to 40 ℃ for 2 to 21 days.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008078878A1 (en) * | 2006-12-22 | 2008-07-03 | Deuk-Sik Lee | Fermented feeds for livestock farming using lactic acid bacteria and yeast and processing method thereof |
| CN102373160A (en) * | 2011-05-08 | 2012-03-14 | 南昌大学 | Screening and application of high glucose, high alcohol and high acid-resistant saccharomyces cerevisiae ATCC9763 |
| CN103074240A (en) * | 2012-12-06 | 2013-05-01 | 北京中科景明生物技术有限公司 | Yeast for high-activity feed, and its application |
| CN105524851A (en) * | 2015-09-18 | 2016-04-27 | 江南大学 | Saccharomyces cerevisiae and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008078878A1 (en) * | 2006-12-22 | 2008-07-03 | Deuk-Sik Lee | Fermented feeds for livestock farming using lactic acid bacteria and yeast and processing method thereof |
| CN102373160A (en) * | 2011-05-08 | 2012-03-14 | 南昌大学 | Screening and application of high glucose, high alcohol and high acid-resistant saccharomyces cerevisiae ATCC9763 |
| CN103074240A (en) * | 2012-12-06 | 2013-05-01 | 北京中科景明生物技术有限公司 | Yeast for high-activity feed, and its application |
| CN105524851A (en) * | 2015-09-18 | 2016-04-27 | 江南大学 | Saccharomyces cerevisiae and application thereof |
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| Title |
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| Effects of Saccharomyces cerevisiae Culture and Saccharomyces cerevisiae Live Cells on In Vitro Mixed Ruminal Microorganism Fermentation;H. A. Lynch et al.;《J. Dairy Sci. 》;第85卷;第2603-2608页 * |
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