Fungus for producing amylase and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a fungus for producing amylase and application thereof. The fungus capable of producing amylase and efficiently degrading starch is obtained by screening from a fen-flavor liquor fermentation environment, has higher starch degradation and utilization capabilities, is an excellent liquor brewing functional strain, can be safely and effectively applied to liquor brewing and vinasse deep processing processes, and has a wide application prospect. Starch in fermentation raw materials and vinasse is efficiently degraded and alcohol and ester flavor compounds are generated, so that the utilization rate of nutritional ingredients of the raw materials is increased, the flavor quality of food is improved, and the method has a wide application prospect.

Description

Technical Field

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

[0002] As an important food crop, sorghum is widely used in industrial fields such as fermented food manufacturing, sugar making, feed preparation, building materials and papermaking. The chemical components of sorghum are mainly starch, protein, fat, cellulose, etc. Among them, starch has the highest content, accounting for about 60~80%. Therefore, adding enzyme preparations or screening microorganisms that efficiently degrade starch is a good choice to improve the nutritional value of sorghum in industries such as fermented food manufacturing and feed preparation. As an important saccharifying agent and fermentation agent in the production process of liquor, Daqu contains rich microorganisms and a variety of enzyme systems, and plays a key role in saccharification, fermentation, and aroma production in liquor brewing. Daqu adopts open production, with a complex microbial flora and rich and diverse microbial species. The microorganisms in Daqu are mainly bacteria, followed by molds, and yeasts are the least, and most of them are beneficial bacteria.

[0003] In recent years, in order to increase the production rate of liquor, fermented food and feed manufacturing industries have added amylase preparations to degrade starch during the production and processing process to improve the digestibility of nutrients such as starch and increase the yield of liquor. Amylase is widely used in grain processing, textile industry, paper manufacturing industry, detergent industry, brewing, fermentation and pharmaceutical industry. Animals, plants and microorganisms can produce amylase. Among them, amylase from microorganisms has the advantages of low acquisition cost, easy improvement and easy extraction, and is the main source of industrial enzymes. At present, the types of microorganisms reported to produce amylase mainly include Bacillus and Aspergillus oryzae, Aspergillus niger, Candida, and Lipozyme. Amylase can also promote the digestion of starch-containing foods in the digestive tract of marine organisms. Among them, yeast is a well-received feed additive. The use of biotechnology to develop and produce special flavor feed is an effective way to solve the current situation of insufficient feed production in the industry. Screening out domesticated microorganisms that can efficiently degrade starch from the fermented food brewing environment and then adding them to the fermented food, feed manufacturing and by-product deep processing production process is an excellent way to improve the nutritional value of grain raw materials.

[0004] In the solid-state fermentation process of traditional liquor, most of the starch in sorghum and other brewing raw materials is used. How to improve the utilization rate of starch in the brewing process and use the nutrients in the spent grains for deep processing such as feed preparation is of great significance to improving the utilization rate of cereal fermented foods, promoting industrial development and improving industrial benefits.

[0005] Apiotrichum montevideense Widely present in the liquor brewing system, this type of microorganism can secrete and produce a rich starch hydrolyzing enzyme system. Therefore, it is necessary to obtain a microorganism that can efficiently degrade starch and adapt to the liquor production environment. Apiotrichum montevideenseThis is particularly important for the fermentation production of liquor with mold as the main functional microorganism. Summary of the invention

[0006] The present invention screened and obtained a fungus capable of producing amylase and efficiently degrading starch from the fermentation environment of light-fragrance liquor. Apiotrichum montevideense cyw-1, its accession number is CGMCC No.31204.

[0007] The strain was isolated from the fermentation environment of light-fragrance liquor and was deposited in the General Microbiology Center of 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: Apiotrichum montevideense , the deposit number is CGMCC No.31204.

[0008] Furthermore, the present invention provides a microbial agent containing the fungus.

[0009] In some embodiments, the microbial inoculant is a liquid inoculant or a solid inoculant.

[0010] In some embodiments, the microbial agent contains living cells of the fungal body, or freeze-dried fungal bodies, or immobilized fungal cells; the fungus may also be in other forms.

[0011] In some embodiments, the protectant is a lyoprotectant.

[0012] In some embodiments, the lyoprotectant includes but is not limited to skim milk powder, dextrin, lactose, sucrose, and the like.

[0013] Furthermore, the present invention provides the use of the fungus or the microbial agent in food production or protein feed processing.

[0014] Preferably, the food production is cereal fermentation production.

[0015] Preferably, the food production is fermentation production of fermented mash.

[0016] Preferably, the fungus is cultured in a sorghum solid-state fermentation medium.

[0017] 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.

[0018] During the cultivation process, the fungus produces amylase to degrade starch.

[0019] Preferably, the use comprises inoculating the fungus into a sorghum solid-state fermentation medium and culturing at 28-32°C.

[0020] Preferably, the use comprises inoculating the fungus into a sorghum solid fermentation medium and culturing it statically at 30°C.

[0021] During cultivation, the fungus metabolizes to produce alcohol and ester flavor compounds.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention screened and obtained a fungus capable of producing amylase and efficiently degrading starch from a light-fragrance liquor fermentation environment. The fungus has a high ability to degrade and utilize starch, is an excellent wine-making functional strain, can be safely and effectively applied to liquor brewing and vinasse deep processing, efficiently degrades starch in fermentation raw materials and vinasse and produces alcohol and ester flavor compounds, thereby increasing the utilization rate of raw material nutrients, improving food flavor quality, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a photo of the transparent circle of the strain. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] The formula of the culture medium for screening amylase production bacteria 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.

[0027] Example 1 Amylase production Apiotrichum montevideense cyw-1 screening and identification 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 rpm, 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 a suitable concentration gradient, take 150 μL of sample solution and spread it on YPD solid medium and WL solid medium, and culture it in a constant temperature incubator at 30°C for 24 h to 48 h. Pick a single colony with clear colony morphology and good growth condition, and purify it again by plate streak method. The separated and purified single strain is screened through the screening amylase-producing bacteria culture medium. Prepare the screening amylase-producing bacteria culture medium, draw a cross on the screening amylase-producing bacteria culture medium with the activated single strain for inoculation, culture it in a constant temperature incubator at 30°C for 24 h to 48 h, and then drop the standard iodine solution (0.1 g / L) 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 does not have the ability to produce amylase.

[0028] Finally, a fungus that can produce high levels of amylase was screened out, as shown in the transparent circle photo. Figure 1 As shown. Genomic DNA was extracted for molecular biological identification. Apiotrichum montevideense , named Apiotrichum montevideense cyw-1, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC NO.31204.

[0029] Example 2 Function of strain metabolism to produce alcohol and ester 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.

[0030] Fermentation culture: The triangular flask culture method was used to culture the Apiotrichum montevideense The solid-state simulated fermentation of cyw-1 strain was carried out as follows: Take a 250 mL triangular flask, pour 100 g of sorghum solid-state fermentation medium into the triangular flask, take Apiotrichum montevideenseThe seed liquid of the fungus was mixed and 5 mL of the seed liquid was inoculated into the sorghum solid fermentation medium, and cultured in a constant temperature box at 30 °C for 10 days. Then, the volatile aroma components produced by the fermentation were detected by headspace solid phase microextraction gas chromatography.

[0031] The detection method is as follows: Pretreatment step (HS-SPME): Mix the solid fermentation product, take 3g of solid fermentation product and place it in a 20ml headspace extraction bottle, quickly seal it, and balance it in a 35℃ constant temperature water bath for 5min. Insert the aged extraction head into the bottle, push out the fiber head for adsorption for 30min, and analyze it at the injection port for 10min. Gas chromatography-mass spectrometry (GC-MS): Program temperature rise: initial temperature 35℃, keep it for 10min, increase the temperature to 250℃ at 10℃ / min, and keep it for 10min. Carrier gas: high-purity helium (He), purity 99.999%; carrier gas flow rate 1mL / 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 ℃; interface temperature 250 ℃; detector voltage: 1.00 KV; mass analyzer: single quadrupole; acquisition mode: scan; mass scanning range: 35~500 amu; column oven temperature 35 ℃; vaporization chamber 250 ℃; chromatographic column: DB-5MS capillary column (30 m×0.25 mm×0.25 μm); injection port temperature: 250 ℃.

[0032] The test results are shown in Table 1.

[0033] Table 1

[0034] visible, Apiotrichum montevideense cyw-1 fermentation produces 18 volatile aroma components, including 2 ketones, 3 acids, 3 phenols, 5 esters, and 5 alcohols. Apiotrichum montevideense cyw-1 has the characteristics of metabolizing to produce alcohol and ester flavor compounds, and can produce unique aromas such as light mint, fresh fruit, wine, ether, butter, etc.

[0035] 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 fungus, characterized in that Its Apiotrichum montevideense cyw-1, its accession number is CGMCC No.31204.

2. A microbial agent containing the fungus 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 fungus according to claim 1 or the microbial agent according to 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: The fungus produces amylase which degrades starch.

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

10. The use according to claim 9, characterized in that: The fungal metabolism produces alcohol and ester flavor compounds.

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