Trichosporon sp. For producing amylase and protease and application of trichosporon sp.

Abstract

The invention relates to the technical field of microorganisms, in particular to trichosporon for producing amylase and protease and application of the trichosporon. According to the invention, a strain of trichosporon is obtained from a liquor brewing process, can produce amylase and protease at the same time, has higher capability of degrading and utilizing protein, can adapt to a liquor production environment, can produce ketone and pyrazine flavor compounds, is an excellent liquor brewing functional strain, and can be widely applied to liquor brewing. The method can be safely and effectively applied to white spirit brewing and vinasse deep processing processes, can efficiently degrade neutral protein in fermentation raw materials and vinasse, so that the utilization rate of nutritional ingredients of the raw materials is increased, the flavor quality of food is improved, and the method can be used for protein feed processing and other production in the field of vinasse biological reutilization and has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Trichosporon producing amylase and protease and application thereof. Background Art

[0002] Traditional liquor is made from sorghum, barley, wheat, rice, corn and other raw materials, through solid fermentation or semi-solid fermentation, fermentation, distillation, storage and blending. The raw materials of Daqu are peas, wheat and barley. A large number of microorganisms and enzymes secreted by microorganisms grow on the qu, which provides a rich microbial community and a large number of enzymes for the fermentation of liquor, and plays an important role in the fermentation of liquor. Fermented grains are unfinished wine that has been saccharified and fermented, but not distilled and filtered. Fermented grains contain a large number of microorganisms and flavor substances. Due to the unique brewing method of saccharifying and fermenting Chinese liquor and the unique distillation method, the flavor of Chinese liquor is significantly different from that of the other five major international distilled liquors.

[0003] In recent years, in order to improve the nutritional value of grain protein such as sorghum, fermented food and feed manufacturing industries have added protease preparations to degrade alcohol-soluble proteins during production and processing to improve the digestibility of nutrients such as grain protein and starch; however, the activity of protease preparations is easily affected by changes in environmental factors during the production process, resulting in low efficiency in the degradation of alcohol-soluble proteins. Proteases are divided into neutral proteases, alkaline proteases and acidic proteases. Proteases can increase the wine production rate, and the product amino acids can participate in the synthesis of aroma substances and promote yeast growth. Neutral proteases can destroy the structure of proteins, make proteins lose their vitality, hydrolyze proteins into amino acid fragments, expose starch wrapped by proteins, and allow amylase to react with the wrapped starch to produce maltose, which eventually turns into glucose, increasing the carbon source that can be directly used by cells, promoting fermentation, and facilitating the growth of microorganisms. Proteins are also the precursors of some flavor substances. After being decomposed by neutral proteases, some proteins are more easily decomposed and transformed, increasing the types and content of flavor substances, and making a certain contribution to the flavor of liquor.

[0004] Trichosporon sp. As an important type of trichosporon yeast, it is widely present in the liquor brewing system. Because the types of yeast are different, the fermentation characteristics of yeast are also different, with obvious differences. Different strains will use different substrates, have different metabolites, and have different flavors. The characteristic aroma components of the products produced by solid-state fermentation will be significantly different.

[0005] At present, there are few reports on strains that can produce both amylase and protease in Daqu. Therefore, how to obtain Trichosporon yeast that can produce both amylase and protease and can adapt to the liquor production environment and produce ketone and pyrazine flavor compounds has become a technical problem that needs to be solved urgently in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a strain of Trichosporon ( Trichosporon sp.), which is Trichosporon cyw-2, and its deposit number is CGMCC No.31205.

[0007] The strain was isolated from the fermentation environment of light-fragrance liquor and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 8, 2024. The address of the depository unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Classification Name: Trichosporon Trichosporon sp. , the deposit number is CGMCC No.31205.

[0008] This strain can produce amylase and protease at the same time, adapt to the liquor production environment, and produce ketone and pyrazine flavor compounds. It is an excellent functional strain for brewing.

[0009] Furthermore, the present invention provides a microbial agent, which comprises the Trichosporon cyw-2.

[0010] In some embodiments, the microbial inoculant is a liquid inoculant or a solid inoculant.

[0011] In some embodiments, the microbial agent contains living cells of the Trichosporon cyw-2 bacteria, or freeze-dried Trichosporon cyw-2 bacteria, or immobilized Trichosporon cyw-2 cells; it can also be Trichosporon cyw-2 in other forms.

[0012] In some embodiments, the microbial agent also includes a protective agent.

[0013] In some embodiments, the protectant is a lyoprotectant.

[0014] In some embodiments, the lyoprotectant includes but is not limited to skim milk powder, dextrin, lactose, sucrose, and the like.

[0015] Furthermore, the present invention provides the use of the Trichosporon cyw-2 or the microbial agent in food production or protein feed processing.

[0016] Preferably, the food production is cereal fermentation production.

[0017] Preferably, the food production is fermentation production of fermented mash.

[0018] During the production process, the Trichosporon cyw-2 is cultured in a sorghum solid-state fermentation medium.

[0019] In some embodiments, the method for preparing the sorghum solid-state fermentation medium includes: mixing sorghum and water in a weight ratio of 1: (1-2), soaking at 60-80°C for 20-30 hours, then taking the drained sorghum, mixing it in a weight ratio of sorghum and water of (1-2): 1, and then sterilizing it.

[0020] During the production process, the Trichosporon cyw-2 simultaneously produces amylase and neutral protease, thereby degrading starch and protein in food production raw materials to generate ketone and pyrazine flavor compounds.

[0021] In some embodiments, the food production raw material is a cereal fermented food production raw material or residual grain fermented food mash.

[0022] In some embodiments, the Trichosporon cyw-2 is used for the degradation of starch and protein in the residual grains of fermented cereal foods, so as to produce a protein feed with flavor characteristics.

[0023] In some embodiments, the Trichosporon cyw-2 is added to Baijiu Daqu to obtain Baijiu with a directional flavor.

[0024] Preferably, the activity of the neutral protease is 37.654 U / mL.

[0025] Preferably, the use comprises inoculating the Trichosporon cyw-2 into a sorghum solid-state fermentation medium and culturing at 28-32° C. (most preferably 30° C.).

[0026] Compared with the prior art, the present invention has the following beneficial effects: The present invention obtains a strain of Trichosporon from the liquor brewing process. The strain can produce amylase and protease at the same time, has high ability to degrade and utilize protein, can adapt to the liquor production environment, and produce ketone and pyrazine flavor compounds. It is an excellent wine-making functional strain, can be safely and effectively applied to liquor brewing and lees deep processing, and can efficiently degrade neutral proteins in fermentation raw materials and lees, thereby improving the utilization rate of raw material nutrients and improving food flavor quality. It can be used in the field of lees biological recycling for protein feed processing and other production, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a photo of the colony morphology of Trichosporon cyw-2.

[0028] Figure 2 This is a photo of the transparent circle produced by the colony of Trichosporon cyw-2. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0031] The formula of the culture medium for producing amylase in the following examples is as follows: 2 g fusible starch, 5 g beef extract, 10 g peptone, 5 g sodium chloride, 20 g agar, 1000 mL distilled water, sterilize at 121℃ for 15 min.

[0032] The formula of the liquid culture medium with casein as the sole carbon source in the following examples is as follows: Casein 10 g, sodium chloride 10 g, distilled water 1000 mL, sterilize at 121℃ for 15 min.

[0033] Example 1 Screening and identification of Trichosporon cyw-2 The dilution plate method was used to isolate bacteria from Daqu and fermented grains: 20 g of Luzhou-flavor Daqu was weighed and sterilized with 200 mL of distilled water. The crushed Daqu was added into the sterilized distilled water and shaken at 30°C, 200 r / min, for 20 min. After that, 10 -1 Samples; 10 -1 The samples were serially diluted to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Sample solution. Select an appropriate concentration gradient, take 150 μL of the sample solution and spread it on YPD solid medium and WL solid medium, and culture it in a 30°C constant temperature incubator for 24 h to 48 h. Pick a single colony with clear colony morphology and good growth condition, and purify it again by the plate streak method.

[0034] The isolated and purified single strain was screened through the screening amylase-producing bacteria culture medium: the screening amylase-producing bacteria culture medium was prepared, and the activated single strain was inoculated by drawing a cross on the screening amylase-producing bacteria culture medium, and cultured in a 30°C constant temperature incubator for 24 to 48 hours, and the standard iodine solution (0.1 g / L) was dropped around the colony. If a transparent circle is produced around the colony, it means that the strain has the ability to produce amylase; if no transparent circle is produced around the colony, it means that the strain has no ability to produce amylase.

[0035] Prepare a liquid culture medium with casein as the sole carbon source, inoculate the activated single strain into the liquid culture medium with casein as the sole carbon source, and culture it in a constant temperature shaker at 30°C and 200 rpm for 48 to 72 hours. After the obtained bacterial solution is shaken well, 150 μL of the sample solution is spread on the solid YPD medium and WL medium, and cultured in a constant temperature incubator at 30°C for 24 to 48 hours. If there are colonies growing on the culture medium and the morphology is the same as the original strain, it proves that the strain has the ability to produce neutral protease. If there are no colonies growing on the culture medium, it proves that the strain has no ability to produce neutral protease.

[0036] Finally, a strain of Trichosporon that can produce both amylase and neutral protease was screened. Trichosporon sp. , named Trichosporon cyw-2, the colony morphology is as Figure 1 As shown, the transparent circle is Figure 2 According to SB / T 10317-1999 "Protease Activity Determination Method", the neutral protease activity of the strain was further tested using the Folin phenol method. The neutral protease activity of Trichosporon cyw-2 was 37.654 U / mL. The genomic DNA extracted from Trichosporon cyw-2 was subjected to molecular biological identification, and it was determined that Trichosporon cyw-2 was a Trichosporon. Trichosporon sp. , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.31205.

[0037] Example 2 Function of Trichosporon cyw-2 in Metabolizing Ketone and Pyrazine Flavor Compounds Sorghum solid fermentation medium: Take an appropriate amount of sorghum, wash it, mix it in a weight ratio of sorghum: water = 3:4, soak it in a 75℃ oven for 24 h, drain it, weigh 50 g of sorghum into a 500 mL Erlenmeyer flask, add 25 mL of water, and sterilize it at 121℃ for 20 min.

[0038] Fermentation culture: The screened Trichosporon cyw-2 strain was subjected to solid-state simulated fermentation using the triangular flask culture method. The steps are as follows: take a 250 mL triangular flask, pour 100 g of sorghum solid-state fermentation medium into the triangular flask, take the Trichosporon cyw-2 strain seed liquid, mix well, and then inoculate 5 mL of the strain seed liquid into the sorghum solid-state fermentation medium, and culture it in a constant temperature box at 30°C for 10 days, and then detect the volatile aroma components produced by fermentation by headspace solid phase microextraction gas chromatography.

[0039] The detection method is as follows: Pretreatment step (HS-SPME): Mix the solid fermentation product, take 3 g of solid fermentation product and place it in a 20 mL headspace extraction bottle, quickly seal it, and balance it in a 35°C constant temperature water bath for 5 minutes. Insert the aged extraction head into the bottle, push out the fiber head for adsorption for 30 minutes, and analyze it at the injection port for 10 minutes. Gas chromatography-mass spectrometry (GC-MS): Program temperature rise: initial temperature 35°C, maintain for 10 minutes, increase the temperature to 250°C at 10°C / minute, and maintain for 10 minutes. Carrier gas: high-purity helium (He), purity 99.999%; carrier gas flow rate 1 mL / min; flow control mode: constant linear velocity mode; injection mode: non-split injection. Ion source: EI source; electron energy 70 eV; Ion source temperature: 200 °C; interface temperature: 250 °C; detector voltage: 1.00 KV; mass analyzer: single quadrupole; acquisition mode: scan; mass scanning range: 35~500 amu; column oven temperature: 35 °C; vaporization chamber: 250 °C; chromatographic column: DB-5MS capillary column (30 m×0.25 mm×0.25 μm); injection port temperature: 250 °C.

[0040] The test results are shown in Table 1.

[0041] Table 1

[0042] It can be seen that the fermentation of Trichosporon cyw-2 produces 17 volatile aroma components: 1 alcohol, 1 phenol, 2 esters, 2 amines, 2 pyrazines, 3 acids, and 6 ketones. It has the function of metabolizing ketone and pyrazine flavor compounds and can produce flavors such as cream, yogurt, cheese, malt, chocolate, and nuts.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Trichosporon ( Trichosporon sp.), characterized in that It is Trichosporon cyw-2, and its deposit number is CGMCC No.31205.

2. A microbial agent, characterized in that: The method comprises the Trichosporon cyw-2 according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent is a liquid agent or a solid agent.

4. The microbial agent according to claim 3, characterized in that: The microbial agent also includes a protective agent.

5. Use of the Trichosporon cyw-2 described in claim 1 or the microbial agent described in any one of claims 2 to 4 in food production or protein feed processing.

6. The use according to claim 5, characterized in that: The food production is cereal fermentation production.

7. The use according to claim 5, characterized in that: The food production is fermentation production of fermented mash.

8. The use according to any one of claims 5 to 7, characterized in that: During the production process, the Trichosporon cyw-2 simultaneously produces amylase and neutral protease.

9. The use according to any one of claims 5 to 7, characterized in that: During the production process, the Trichosporon cyw-2 produces ketone and pyrazine flavor compounds.

10. The use according to any one of claims 5 to 7, characterized in that: include: The Trichosporon cyw-2 was inoculated into a sorghum solid fermentation medium and cultured at 28-32°C.

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