CN117736871A - Screening method of heat-resistant Kluyveromyces marxianus strain - Google Patents
Screening method of heat-resistant Kluyveromyces marxianus strain Download PDFInfo
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Abstract
The invention discloses a screening method of heat-resistant Kluyveromyces marxianus strains, which comprises the steps of adding 1kg of vinasse sample into an enrichment medium, and carrying out solid state fermentation at 37 ℃ for 7-15 days; weighing 10g of distilled grain fermentation sample, adding 90mL of sterile water, magnetically stirring for 20min, and carrying out gradient dilution with the sterile water; 100 mu L of sample dilution is coated on YPD medium plates, yeast strains with consistent colony morphology and good growth are selected, the strains are purified by repeated streaking, and the strains are identified as Kluyveromyces marxianus by 18s rDNA sequencing. The yeast culture of the Kluyveromyces marxianus strain provided by the invention can improve the nutritive value of feed, promote the organism immunity and improve the growth performance of animals. Compared with the traditional yeast, the Kluyveromyces marxianus has high temperature tolerance and higher growth rate, can save production cost, reduce the risk of bacteria contamination, improve production efficiency and have higher industrial application value.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a screening method of a heat-resistant Kluyveromyces marxianus strain.
Background
Kluyveromyces marxianus belongs to the genus Kluyveromyces of the order Saccharomyces of the fungus Ascomycota and the subgenomycetales of the family Saccharomyces, and widely exists in plants and dairy products, and the produced lipid, ketone, aldehyde and alcohol can add special flavor to the fermented products such as dairy products, wines and the like. Kluyveromyces marxianus is GRAS-grade microorganism authenticated by the United states food and drug administration, passes the safety authentication of the European Union food safety administration, is approved as a new food raw material in 2013 by the China Commission of health and protection, and is approved as a new feed additive in 2023 by the China agricultural rural area.
Kluyveromyces marxianus as one kind of unconventional yeast has wide application in the fields of biofuel production, recombinant protein synthesis, sewage treatment, etc. In addition, kluyveromyces marxianus has great development potential in solid state fermentation of animal feed. The solid state fermentation has the advantages of wide sources of fermentation substrates, simple equipment requirements, low energy consumption, less waste liquid and the like, but also has the problems of long fermentation period, difficult complete sterility of a culture medium, easy local overheating caused by material accumulation and the like.
Therefore, based on the defects of the solid state fermentation, the application of the Kluyveromyces marxianus in the solid state fermentation of animal feed is still in a starting stage, and related research reports are few.
Disclosure of Invention
The invention aims to provide a screening method of a heat-resistant Kluyveromyces marxianus strain, which is used for screening and obtaining the heat-resistant Kluyveromyces marxianus strain and applying the heat-resistant Kluyveromyces marxianus strain to solid state fermentation of animal feed so as to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a screening method of heat-resistant Kluyveromyces marxianus strains comprises the following steps:
s1: adding 1kg of distillers' grains sample into enrichment medium, and performing solid state fermentation at 37 ℃ for 7-15 days;
s2: weighing 10g of distilled grain sample, adding 90mL of sterile water, magnetically stirring for 20min, and carrying out gradient dilution with the sterile water;
s3: coating 100 mu L of sample diluent on a YPD culture medium flat plate, standing and culturing at 40 ℃ for 48 hours, selecting a yeast strain with a colony form conforming to that of the strain and good growth, and purifying the strain by repeated streaking to obtain a pure strain for identification and preservation;
s4: by 18s rDNA sequencing, sequences were aligned on NCBI database and strain identified as Kluyveromyces marxianus strain designated TBY2.
Further, the YPD medium includes YPD solid medium and YPD liquid medium.
Further, the preparation process of the YPD solid medium comprises the following steps:
dissolving 10g of yeast extract, 20g of peptone and 20g of glucose in 1000mL of distilled water, adding 20g of agar powder, fully and uniformly mixing, sterilizing at 121 ℃ for 20min under high pressure, cooling the culture medium to about 45 ℃, subpackaging the culture medium into culture dishes in an ultra-clean workbench, cooling and solidifying, and inverting a culture medium plate for later use.
Further, the preparation process of the YPD liquid culture medium comprises the following steps:
taking 10g of yeast extract, 20g of peptone and 20g of glucose, adding 1000mL of distilled water for heating and dissolving, subpackaging the culture medium into test tubes, sterilizing at 121 ℃ for 20min, and cooling for later use.
Further, temperature tolerance test was performed on kluyveromyces marxianus strain:
kluyveromyces marxianus TBY2 is inoculated onto a YPD solid culture medium flat plate for activation, a single colony is obtained by streaking the flat plate, one colony is selected and inoculated into a YPD liquid culture medium, the liquid culture medium is placed in a shaking table at 30 ℃ for shake culture at 150rpm for 24 hours to obtain seed liquid, and the obtained seed liquid is transferred into a new YPD liquid culture medium in a proportion of 1 per mill and is cultured for 24 hours under different temperature gradients respectively.
Further, the solid state fermentation method of the Kluyveromyces marxianus strain comprises the following steps:
fermentation raw materials: taking 400 parts of bran, 500 parts of palm meal and 100 parts of bean meal, and uniformly mixing for later use;
activating strains: inoculating the frozen TBY2 strain to YPD solid culture medium, and culturing at 30deg.C in biochemical incubator for 48 hr to obtain activated strain;
preparing yeast seed liquid: selecting single colony of TBY2 strain, inoculating into YPD liquid culture medium, placing into a shaking table at 30deg.C, shake culturing at 150rpm for 24 hr to obtain TBY2 strain seed solution;
solid state fermentation: inoculating the TBY2 strain seed solution into the mixed fermentation raw material with an inoculum size of 2%, adding 740 parts of water, uniformly stirring, subpackaging into fermentation bags, and culturing for 48h in 37 to obtain a fermented feed;
yeast count: at the beginning of solid state fermentation, a proper amount of yeast seed liquid is taken, 10g of fermentation samples are taken respectively at 0h,24h and 48h of solid state fermentation, 90mL of sterile water is added, magnetic stirring is carried out for 20min, the yeast seed liquid and the fermentation sample aqueous solution are subjected to gradient dilution by the sterile water, and the number of yeast viable bacteria is determined by a plate colony counting method.
Compared with the prior art, the invention has the beneficial effects that:
1. kluyveromyces marxianus strains can generally grow at the temperature of 40 ℃, wherein part of strains can resist the temperature of more than 50 ℃, so that the Kluyveromyces marxianus strains are more convenient to use for high-temperature fermentation, on one hand, the cooling cost and the bacteria-dyeing risk can be greatly reduced by high-temperature fermentation, and on the other hand, the optimal action temperature of cellulase, xylanase, pectase and the like is generally 45-55 ℃, and the high-temperature fermentation is beneficial to improving the catalytic efficiency of the enzymes.
2. Kluyveromyces marxianus also has higher growth rate, the growth rate can reach OD 0.86-0.99/h at 40 ℃, which is far higher than that of other yeasts, a large number of cells can be obtained by rapid proliferation, and the production efficiency is improved.
3. In addition to glucose, kluyveromyces marxianus can also grow by using other saccharides as a single carbon source, including fructose, xylose, arabinose, galactose, lactose, inulin and the like, so that low-value agricultural and food industry byproducts with the saccharides as main components can be applied to fermentation of kluyveromyces marxianus as a carbon source. The byproducts are used as partial substrates for fermentation, which is helpful for reducing environmental pollution, reducing feed cost and promoting sustainable development of the aquaculture industry.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, 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.
A screening and identifying method of heat-resistant Kluyveromyces marxianus strains comprises the following steps:
adding enrichment medium into 1kg of vinasse sample, performing solid state fermentation at 37 ℃ for 7-15 days, weighing 10g of vinasse sample, adding 90mL of sterile water, performing magnetic stirring for 20min, performing gradient dilution with the sterile water, taking 100 mu L of sample diluent, coating on a YPD medium plate, performing stationary culture at 40 ℃ for 48 hours, selecting yeast strains with colony morphology conforming to that of the strain and good growth, and purifying the strain by repeated streaking, thereby obtaining pure strain for identification and preservation. By 18s rDNA sequencing, sequences were aligned on NCBI database and strain identified as Kluyveromyces marxianus strain designated TBY2.
YPD solid medium: 10g of yeast extract, 20g of peptone and 20g of glucose are taken and dissolved by 1000mL of distilled water, and then 20g of agar powder is added for fully and uniformly mixing, and the mixture is autoclaved for 20min at 121 ℃. Cooling the culture medium to about 45 ℃, subpackaging the culture medium into culture dishes in an ultra-clean workbench, cooling and solidifying, and inverting the culture medium plate for later use.
YPD liquid medium: taking 10g of yeast extract, 20g of peptone and 20g of glucose, adding 1000mL of distilled water for heating and dissolving, subpackaging the culture medium into test tubes, sterilizing at 121 ℃ for 20min, and cooling for later use.
Kluyveromyces marxianus TBY2 is inoculated on a YPD solid culture medium flat plate for activation, single colony is obtained by streaking the flat plate, one colony is selected and inoculated in a YPD liquid culture medium, and the culture medium is placed in a shaking table at 30 ℃ for shake culture at 150rpm for 24 hours to obtain seed liquid. The seed solution obtained was transferred to a new YPD liquid medium at a ratio of 1%o (V/V), cultured for 24 hours under different temperature gradients, and the viable count of different strains under different temperature culture conditions was measured by plate colony counting method using the same treatment method as a control for 3 commercial yeasts, and the results are shown in Table 1.
TABLE 1 viable count of strains cultured at different temperatures (10 7 CFU/mL)
The results show that the TBY2 strain has a much smaller reduction degree of viable count than other 3 strains under the two temperature conditions of 42 ℃ and 47 ℃, which indicates that the TBY2 strain has better high temperature tolerance than the other three strains.
The solid state fermentation method of the Kluyveromyces marxianus strain comprises the following steps:
fermentation raw materials: 400 parts of bran, 500 parts of palm meal and 100 parts of bean meal are uniformly mixed for standby.
Activating strains: inoculating the frozen TBY2 strain to YPD solid culture medium, and culturing at 30deg.C for 48 hr in biochemical incubator to obtain activated strain.
Preparing yeast seed liquid: and (3) selecting a single colony of the TBY2 strain, inoculating the single colony into a YPD liquid culture medium, and placing the single colony in a shaking table for shake culture at 30 ℃ and 150rpm for 24 hours to obtain a seed solution of the TBY2 strain.
Solid state fermentation: the seed solution of the TBY2 strain is inoculated into the mixed fermentation raw material with the inoculation amount of 2 percent, 740 parts of water is added, the mixture is stirred uniformly, and the mixture is packaged into fermentation bags and placed in 37 culture for 48 hours, thus obtaining the fermented feed.
Yeast count: at the beginning of solid state fermentation, a proper amount of yeast seed liquid is taken, 10g of fermented feed is taken in 0h,24h and 48h of solid state fermentation respectively, 90mL of sterile water is added, magnetic stirring is carried out for 20min, the yeast seed liquid and the fermented feed aqueous solution are subjected to gradient dilution by the sterile water, the number of yeast viable bacteria is measured by a plate colony counting method, and the number of all sample viable bacteria is shown in Table 2.
TABLE 2 Yeast viable count in samples
The fermented feed is placed in a 65 ℃ oven to dry water, a pulverizer is used for pulverizing the sample, the pulverized sample passes through a 0.42mm sample sieve, the crude protein content in the fermented sample and a control sample is measured by a Kjeldahl nitrogen method, the measuring method refers to GB/T6432-2018, and an unfermented sample is used as a control. The moisture content in the sample was determined by direct drying, from which the crude protein content of the fermented sample and the control sample under the same moisture condition was calculated as shown in table 3.
TABLE 3 crude protein content of samples (10% moisture conversion)
The results show that the crude protein content of the fermented feed is obviously increased in the fermentation process, and the crude protein content of the sample after 48h fermentation is increased by 12% compared with that of a control group.
A feeding test is carried out by using a fermented feed in a certain company, 60 fattening pigs with the same birthday and similar weight are selected, randomly divided into a test group and a control group, and 30 pigs in each group are fed in a housing every 5 pigs. The control group uses basic ration, and the experimental group uses 10% fermented feed to replace 4% bran, 5% palm meal and 1% soybean meal in the basic ration. During the test period, pigs only normally eat, drink water and move in the whole process, and regularly perform the work of disinfection, immunization, insect expelling, health care and the like. Recording initial weight and final weight of the test pigs, counting feeds consumed by the test group and the control group, and calculating average daily feed intake, average daily weight gain and feed-weight ratio of the test pigs. The effect of the fermented feed on the growth performance of fattening pigs is shown in table 4.
TABLE 4 influence of fermented feeds on the production performance of fattening pigs
The results show that the weight ratio of the test group is obviously reduced compared with that of the control group, which shows that the addition of Kluyveromyces marxianus fermented feed in daily ration is beneficial to improving the production performance of fattening pigs. The Kluyveromyces marxianus strain can be applied to the field of animal feed, has the characteristics of high temperature resistance, high growth rate, wide applicable substrate and the like, can overcome adverse conditions in solid state fermentation to a certain extent, reduces production cost, on one hand, the yeast degrades a part of macromolecular substances into micromolecular substances with higher bioavailability, and improves the nutritional value of the feed. On the other hand, active strains and some beneficial metabolites produced by the active strains, such as amino acids, vitamins, polysaccharides and the like, can also have positive effects on the growth and health of pigs.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should be covered by the protection scope of the present invention by making equivalents and modifications to the technical solution and the inventive concept thereof.
Claims (6)
1. The screening method of the heat-resistant kluyveromyces marxianus strain is characterized by comprising the following steps of:
s1: adding 1kg of distillers' grains sample into enrichment medium, and performing solid state fermentation at 37 ℃ for 7-15 days;
s2: weighing 10g of distilled grain fermentation sample, adding 90mL of sterile water, magnetically stirring for 20min, and carrying out gradient dilution with the sterile water;
s3: coating 100 mu L of sample diluent on a YPD culture medium flat plate, standing and culturing at 40 ℃ for 48 hours, selecting a yeast strain with a colony form conforming to that of the strain and good growth, and purifying the strain by repeated streaking to obtain a pure strain for identification and preservation;
s4: by 18s rDNA sequencing, sequences were aligned on NCBI database and strain identified as Kluyveromyces marxianus strain designated TBY2.
2. The method for screening a heat-resistant Kluyveromyces marxianus strain according to claim 1, wherein the YPD medium comprises YPD solid medium and YPD liquid medium.
3. The method for screening a heat-resistant Kluyveromyces marxianus strain according to claim 2, wherein the preparation process of the YPD solid medium comprises the following steps:
dissolving 10g of yeast extract, 20g of peptone and 20g of glucose in 1000mL of distilled water, adding 20g of agar powder, fully and uniformly mixing, sterilizing at 121 ℃ for 20min under high pressure, cooling the culture medium to about 45 ℃, subpackaging the culture medium into culture dishes in an ultra-clean workbench, cooling and solidifying, and inverting a culture medium plate for later use.
4. The method for screening a heat-resistant Kluyveromyces marxianus strain according to claim 2, wherein the preparation process of the YPD liquid medium comprises the following steps:
taking 10g of yeast extract, 20g of peptone and 20g of glucose, adding 1000mL of distilled water for heating and dissolving, subpackaging the culture medium into test tubes, sterilizing at 121 ℃ for 20min, and cooling for later use.
5. The method for screening a heat-resistant kluyveromyces marxianus strain according to claim 1, wherein the kluyveromyces marxianus strain is subjected to a temperature tolerance test:
kluyveromyces marxianus TBY2 is inoculated onto a YPD solid culture medium flat plate for activation, a single colony is obtained by streaking the flat plate, one colony is selected and inoculated into a YPD liquid culture medium, the liquid culture medium is placed in a shaking table at 30 ℃ for shake culture at 150rpm for 24 hours to obtain seed liquid, and the obtained seed liquid is transferred into a new YPD liquid culture medium in a proportion of 1 per mill and is cultured for 24 hours under different temperature gradients respectively.
6. The method for screening a heat-resistant Kluyveromyces marxianus strain according to claim 5, wherein the method for solid state fermentation of the Kluyveromyces marxianus strain comprises:
fermentation raw materials: taking 400 parts of bran, 500 parts of palm meal and 100 parts of bean meal, and uniformly mixing for later use;
activating strains: inoculating the frozen TBY2 strain to YPD solid culture medium, and culturing at 30deg.C in biochemical incubator for 48 hr to obtain activated strain;
preparing yeast seed liquid: selecting single colony of TBY2 strain, inoculating into YPD liquid culture medium, placing into a shaking table at 30deg.C, shake culturing at 150rpm for 24 hr to obtain TBY2 strain seed solution;
solid state fermentation: inoculating the TBY2 strain seed solution into the mixed fermentation raw material with an inoculum size of 2%, adding 740 parts of water, uniformly stirring, subpackaging into fermentation bags, and culturing for 48h in 37 to obtain a fermented feed;
yeast count: at the beginning of solid state fermentation, a proper amount of yeast seed liquid is taken, 10g of fermentation samples are taken respectively at 0h,24h and 48h of solid state fermentation, 90mL of sterile water is added, magnetic stirring is carried out for 20min, the yeast seed liquid and the fermentation sample aqueous solution are subjected to gradient dilution by the sterile water, and the number of yeast viable bacteria is determined by a plate colony counting method.
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