A fungus producing amylase and its use

Abstract

The present application relates to the field of microbial technology, and particularly relates to a strain of amylase-producing fungus and application thereof. The present application screens a strain of amylase-producing fungus from a fermentation environment of a light-flavor liquor, which can produce amylase and efficiently degrade starch, has a high ability of degrading and utilizing starch, is an excellent brewing functional strain, can be safely and effectively applied to liquor brewing and deep processing of distiller's grains, efficiently degrades starch in fermentation raw materials and distiller's grains and produces alcohol and ester flavor compounds, thereby improving the utilization rate of nutritional components of raw materials, improving the flavor quality of food, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a fungus that produces amylase and its applications. Background Technology

[0002] Sorghum, as an important grain crop, is widely used in industries such as fermented food manufacturing, sugar production, feed preparation, building materials, and papermaking. The main chemical components of sorghum are starch, protein, fat, and cellulose; starch is the most abundant, accounting for approximately 60-80%. Therefore, adding enzyme preparations or screening for highly efficient starch-degrading microorganisms is an excellent choice for improving the nutritional value of sorghum in fermented food manufacturing and feed preparation industries. Daqu, as an important saccharifying and fermenting agent in the production of baijiu (Chinese liquor), contains abundant microorganisms and various enzyme systems, playing a crucial role in saccharification, fermentation, and aroma development during baijiu brewing. Daqu employs open production, resulting in a complex microbial community with a rich diversity of microorganisms. The microorganisms in Daqu are primarily bacteria, followed by molds, with yeast being the least abundant; most are beneficial bacteria.

[0003] In recent years, to improve the yield of baijiu (Chinese liquor), fermented food and feed manufacturers have added amylase preparations during production and processing to degrade starch, thereby improving the digestibility of starch and other nutrients and increasing the alcohol yield. Amylases are widely used in grain processing, textiles, paper manufacturing, detergents, brewing, fermentation, and pharmaceuticals. Animals, plants, and microorganisms can all produce amylases, with microbial amylases having advantages such as low acquisition cost, ease of modification, and easy extraction, making them a major source of industrial enzymes. Currently reported amylase-producing microorganisms mainly include Bacillus, Aspergillus oryzae, Aspergillus niger, Candida, and Leptospira. Amylases can also promote the digestion of starchy foods in the digestive tract of marine organisms. Yeasts are widely praised feed additives, and developing and producing special flavored feeds using biotechnology is an effective way to address the current industry shortage of feed. Screening domesticated microorganisms capable of efficiently degrading starch from the fermentation environment of fermented foods and adding them to the production processes of fermented foods, feed, and by-products is an excellent way to improve the nutritional value of grain raw materials.

[0004] In the traditional solid-state fermentation process of baijiu (Chinese liquor), most of the starch in the brewing raw materials such as sorghum is utilized. Improving the utilization rate of starch during the brewing process, while simultaneously utilizing the nutrients in the spent grains for deep processing such as feed preparation, is of great significance for improving the utilization rate of fermented grain foods, promoting industrial development, and increasing industrial efficiency.

[0005] Apiotrichum montevideense These microorganisms are widely present in the baijiu brewing system and can secrete a rich system of starch-hydrolyzing enzymes. Therefore, obtaining microorganisms that efficiently degrade starch and can adapt to the baijiu production environment is crucial. Apiotrichum montevideenseThis is especially important for the fermentation production of baijiu (Chinese liquor) where molds are the main functional microorganisms. Summary of the Invention

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

[0007] This strain was isolated from the fermentation environment of light-aroma baijiu and deposited on July 8, 2024, at the China General Microbiological Culture Collection Center (CGC-CMEC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Classification and nomenclature: Apiotrichum montevideense The accession number is CGMCC No.31204.

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

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

[0010] In some embodiments, the microbial agent contains live cells of the fungal cells, or freeze-dried fungal cells, or immobilized fungal cells; it may also contain fungi in other forms.

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

[0012] In some implementations, the freeze-drying protectant includes, but is not limited to, skim milk powder, dextrin, lactose, sucrose, etc.

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

[0014] Preferably, the food production is a grain-based fermentation process.

[0015] Preferably, the food production is a fermentation process using wine mash.

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

[0017] In some embodiments, the preparation method of the sorghum solid fermentation culture medium includes: mixing sorghum and water at a weight ratio of 1:(1~2), soaking at 60~80 ℃ for 20~30 h, then taking the drained sorghum, mixing it with water at a weight ratio of (1~2):1, and then sterilizing it.

[0018] During cultivation, the fungus produces amylase that degrades starch.

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

[0020] Preferably, the application includes inoculating the fungus into a sorghum solid-state fermentation medium and incubating it statically at 30°C.

[0021] During cultivation, the fungi metabolize to produce alcohols and esters, which are flavor compounds.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention screened a fungus capable of producing amylase and efficiently degrading starch from the fermentation environment of light-aroma baijiu. It has a high ability to degrade and utilize starch and is an excellent functional strain for brewing. It can be safely and effectively applied to the brewing and deep processing of baijiu lees. It efficiently degrades starch in fermentation raw materials and lees and produces alcohol and ester flavor compounds, thereby improving the utilization rate of raw material nutrients and improving the flavor and quality of food. It has broad application prospects. Attached Figure Description

[0024] Figure 1 It is a photo of the transparent zone of the strain. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0026] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0027] The formula for the culture medium for screening amylase-producing bacteria is as follows:

[0028] 2 g of blendable starch, 5 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 20 g of agar, and 1000 mL of distilled water were sterilized at 121°C for 15 min.

[0029] Example 1: Amylase production Apiotrichum montevideense cyw-1 screening and identification

[0030] The dilution plating method was used to isolate microorganisms from Daqu (a type of starter culture) and fermented mash: 20 g of strong-aroma Daqu was weighed and sterilized in 200 ml of distilled water. The pulverized Daqu was then added to the sterilized distilled water, and the mixture was shaken at 30 °C, 200 rpm, and 20 min. After filtration, 10 microorganisms were obtained. -1 Samples; 10 samples were treated with sterile distilled water. -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 and spread 150 μL of sample solution onto YPD and WL solid media. Incubate at 30°C for 24-48 hours. Select single colonies with clear morphology and good growth, and purify them again using the streak plate method. Screen the purified single strains using amylase-producing bacteria screening medium. Prepare the amylase-producing bacteria screening medium, inoculate the activated single strain by drawing a cross on the medium, and incubate at 30°C for 24-48 hours. Then, drop standard iodine solution (0.1 g / L) around the colony. If a clear zone forms around the colony, it indicates that the strain has the ability to produce amylase; if no clear zone forms, it indicates that the strain does not have the ability to produce amylase.

[0031] Finally, a fungus capable of producing high levels of amylase was screened, as shown in the image of its clear zone. Figure 1 As shown. Genomic DNA was extracted and molecularly identified as... Apiotrichum montevideense Named Apiotrichum montevideense Cyw-1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.31204.

[0032] Example 2: Functions of the strain in producing alcohols and esters for flavor.

[0033] Sorghum solid-state fermentation medium: Take an appropriate amount of sorghum, wash it, mix it with water at a weight ratio of 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 at 121 ℃ for 20 min.

[0034] Fermentation culture: The Erlenmeyer flask culture method was used to screen the [products] selected in Example 1. Apiotrichum montevideense Solid-state fermentation of cyw-1 strain was performed as follows: Take a 250 mL Erlenmeyer flask, pour 100 g of sorghum solid-state fermentation medium into the flask, and take... Apiotrichum montevideenseAfter mixing the inoculum seed solution, 5 mL of the inoculum seed solution was inoculated into sorghum solid-state fermentation medium and cultured at 30 °C for 10 days in a constant temperature incubator. Then, the volatile aroma components produced by fermentation were detected by headspace solid-phase microextraction gas chromatography.

[0035] The detection method is as follows: Pretreatment step (HS-SPME): Mix the solid-state fermentation product thoroughly, take 3g of solid fermentation material and place it in a 20ml headspace extraction bottle, quickly seal the bottle, and equilibrate in a 35℃ constant temperature water bath for 5min. Insert the aged extraction head into the bottle, extend the fiber head for adsorption for 30min, and then desorb at the injection port for 10min. Gas chromatography-mass spectrometry (GC-MS): Temperature program: initial temperature 35℃, hold for 10min, increase to 250℃ at 10℃ / min, hold for 10min. Carrier gas: high-purity helium (He), purity 99.999%; carrier gas flow rate 1 mL / min; flow control mode: constant linear velocity mode; injection method: splitless 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 scan 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 ℃.

[0036] The test results are shown in Table 1.

[0037] Table 1

[0038]

[0039] 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 characteristic of producing alcohol and ester flavor compounds through metabolism, which can produce unique aromas such as a light mint scent, fresh fruit aroma, wine aroma, ether aroma, and buttery aroma.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fungus Apiotrichum montevideense characterized in that, It is Apiotrichum montevideense cyw-1, with the preservation number of CGMCC No. 31204, and the preservation address of China General Microbiological Culture Collection Center.

2. A microbial inoculant containing the fungus described in claim 1.

3. The microbial inoculant of claim 2, wherein, The microbial agent can be a liquid agent or a solid agent.

4. The microbial inoculant of claim 3, wherein, The microbial agent also includes a protectant.

5. The use of the fungus of claim 1 or the microbial agent of any one of claims 2 to 4 in food production or protein feed processing.

6. Use according to claim 5, characterized in that, The food production is a grain-based fermentation process.

7. Use according to claim 5, characterized in that, The food production process involves fermentation of rice wine mash.

8. The use according to any one of claims 5 to 7, characterized in that, The fungus produces amylase that degrades starch.

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

10. Use according to claim 9, characterized in that, The fungi metabolize to produce alcohols and esters, which are flavor compounds.

Images