Thermomyces lanuginosus capable of producing acid protease and application of thermomyces lanuginosus

Abstract

The invention relates to a thermomyces lanuginosus for producing acid protease and application of the thermomyces lanuginosus, the thermomyces lanuginosus for producing the acid protease is a thermomyces lanuginosus TL-S5 strain, the preservation number of the thermomyces lanuginosus TL-S5 strain is CCTCC (China Center for Type Culture Collection) NO: M 20241912, and the preservation date of the thermomyces lanuginosus TL-S5 strain is September 4, 2024. The strain has excellent capability of producing acid protease and good flavor producing capability, can produce various flavor substances such as esters, alcohols and ketones, and has a great application prospect in related fermentation processes of white spirit, starter propagation and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial culture, and relates to a thermophilic lanuginosus fungus producing acidic protease and application thereof. Background Art

[0002] Proteases produced by microorganisms are divided into acidic, neutral and alkaline proteases according to their acid and alkaline resistance. Acidic proteases refer to a class of enzymes that hydrolyze proteins to produce smaller peptides or amino acids under acidic conditions. These enzymes can maintain high activity and stability in an acidic environment, thereby effectively hydrolyzing proteins. As an important class of enzymes, acidic proteases have broad application prospects and important economic value in the fields of food, feed, and medicine.

[0003] Carbon dioxide produces a variety of organic acids, such as lactic acid and acetic acid. At the same time, microorganisms also produce acidic substances during growth and metabolism, making the brewing environment acidic. This acidic environment helps to inhibit the growth of certain harmful microorganisms and ensure the smooth progress of the fermentation process. Some of these microorganisms secrete various enzymes, including acid proteases. These enzymes can play a better role in an acidic environment and play an important role in liquor brewing and koji making by promoting protein hydrolysis, improving fermentation conditions, improving wine flavor, wine yield and raw material utilization. Therefore, it is very necessary to add an appropriate amount of acid protease during liquor brewing and koji making.

[0004] In current research, microorganisms with the ability to produce acidic proteases mainly include a variety of molds, yeasts, bacillus and thermophilic fungi. Molds mainly include Aspergillus niger, Aspergillus usami, Aspergillus oryzae in Aspergillus, Penicillium chrysogenum and Penicillium glaucum in Penicillium, Rhizopus oryzae in Rhizopus, etc. For example, CN109161489A discloses an Aspergillus niger strain with high acidic protease production; CN102676395A discloses a mutant strain of Aspergillus usami and its application in the preparation of acidic proteases. Yeasts, such as CN111286463A discloses a Pichia pastoris strain that produces acidic proteases. Bacillus, such as CN118086106A discloses a Potsdam Brevibacillus and its application in producing acidic protease; CN117603889B discloses a feed acidic protease-producing Bacillus subtilis and its application; CN114874942B discloses a protease-producing Bacillus cereus and its application in Daqu.

[0005] However, there are few reports on the production of acidic proteases by Thermomyces lanuginosus. Thermomyces lanuginosus is a widely distributed thermophilic fungus with an optimal growth temperature of about 50°C and a maximum growth temperature of 60°C, showing extremely strong heat resistance. The fungus can secrete a variety of enzymes with excellent thermal stability and a wide pH adaptability range, such as xylanase, chitinase, amylase, protease and lipase. At present, many researchers have isolated Thermomyces lanuginosus, but none of them have studied the production of acidic proteases by Thermomyces lanuginosus. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a lanuginosus thermophilic hyphomycete producing acidic protease and its application. The lanuginosus thermophilic hyphomycete improves the activity and diversity of the enzyme system in the fermentation process, makes up for the problems that the enzyme system in the current Daqu is not comprehensive or active enough, thereby improving the wine quality and wine yield, and is applied to the koji making of liquor and related food fermentation processes, and has broad application prospects and potential application value.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a lanuginosus thermophilic fungus that produces an acidic protease, wherein the lanuginosus thermophilic fungus that produces an acidic protease is the lanuginosus thermophilic fungus (Thermomyces lanuginosus) TL-S5 strain, with a preservation number of CCTCC NO: M 20241912 and a preservation date of September 4, 2024.

[0009] The present invention separates, screens and preserves a new strain of Thermomyces lanuginosus that can produce a high amount of acidic protease from a Daqu sample of Jinshiyuan Liquor Co., Ltd. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID No: 1. The sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is Thermomyces lanuginosus. The strain has excellent ability to produce acidic protease, good flavor production ability, and can produce a variety of flavor substances such as esters, alcohols, ketones, etc., and has great application prospects in related fermentation processes such as liquor and koji making.

[0010] In a second aspect, the present invention provides a culture of Thermomyces lanuginosus as described in the first aspect, wherein the culture is obtained by inoculating Thermomyces lanuginosus TL-S5 strain into a sterile culture medium and culturing at 45-60°C (e.g., 45°C, 50°C, 55°C, 60°C, etc.).

[0011] In a third aspect, the present invention provides a microbial preparation producing acidic protease, wherein the strain in the microbial preparation comprises the Thermomyces lanuginosus TL-S5 strain described in the first aspect.

[0012] Preferably, the microbial preparation is in the form of a solution or a powder.

[0013] Preferably, the microbial preparation further comprises a protective agent and / or a carrier material.

[0014] Preferably, the protective agent is selected from any one or a combination of at least two of skim milk, gelatin, gum arabic, dextran, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

[0015] Preferably, the carrier material is selected from any one of maltodextrin, starch, microcrystalline cellulose, calcium carbonate or calcium hydrogen phosphate, or a combination of at least two thereof.

[0016] In a fourth aspect, the present invention provides use of the Thermomyces lanuginosus described in the first aspect, a culture of the Thermomyces lanuginosus described in the second aspect, or the microbial preparation described in the third aspect in producing acidic protease.

[0017] In a fifth aspect, the present invention provides the use of the Thermomyces lanuginosus described in the first aspect, the culture of the Thermomyces lanuginosus described in the second aspect, or the microbial preparation described in the third aspect in liquor brewing or koji making.

[0018] In a sixth aspect, the present invention provides use of the Thermomyces lanuginosus described in the first aspect, a culture of the Thermomyces lanuginosus described in the second aspect, or the microbial preparation described in the third aspect in food fermentation.

[0019] In a seventh aspect, the present invention provides a method for screening Thermomyces lanuginosus according to the first aspect, the screening method comprising the following steps:

[0020] (1) A Daqu sample from Jinshiyuan Wine Co., Ltd. was crushed and added to sterile water for incubation to obtain a bacterial suspension;

[0021] (2) diluting the bacterial suspension in a gradient manner and spreading it on a sterile culture medium plate;

[0022] (3) Placing the plate in a constant temperature incubator, and after colonies grow, picking single colonies from different areas, and further isolating and purifying them on a new sterile culture medium plate;

[0023] (4) using casein medium to perform preliminary screening of bacterial strains, and selecting strains with a transparent circle around the colonies on the plate and a strong ability to produce acidic protease;

[0024] (5) The ability of the strain to produce acidic protease is then determined by the Folin phenol method, and the strains are rescreened to obtain strains with stronger ability to produce acidic protease.

[0025] Preferably, the incubation in step (1) is carried out in a constant temperature shaker at 25-35°C (e.g., 25°C, 27°C, 28°C, 30°C, 35°C, etc.) at 100-300 r / min (e.g., 100 r / min, 150 r / min, 200 r / min, 250 r / min, 300 r / min, etc.) for 10-50 min (e.g., 10 min, 20 min, 30 min, 40 min, 50 min, etc.).

[0026] Preferably, the culture medium in step (2) and step (3) is PDA culture medium, whose formula is: 10-14 g / L potato extract powder, 18-22 g / L glucose, 12-16 g / L agar powder, and the rest water, and the pH value is adjusted to 5.4-5.8 at 25°C.

[0027] Preferably, the temperature of the constant temperature incubator in step (3) is 30-50°C (e.g., 30°C, 35°C, 40°C, 45°C, 50°C, etc.).

[0028] Preferably, the formula of the casein culture medium in step (4) is: casein 8-12 g / L, beef extract 1-5 g / L, sodium chloride 3-7 g / L, disodium hydrogen phosphate 1-3 g / L, agar 15-25 g / L, and the rest water, and the pH value is 3.6-3.8.

[0029] Preferably, the culturing in step (4) is carried out at 45-55°C (e.g., 45°C, 48°C, 50°C, 52°C, 55°C, etc.) for 24-72h (e.g., 24h, 30h, 36h, 42h, 54h, 72h, etc.).

[0030] Preferably, the method for judging the acid-producing protease ability in step (4) is: the larger the ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d), the stronger the acid-producing protease ability.

[0031] Preferably, the method of rescreening in step (5) specifically comprises: inoculating the strain into a bran culture medium, culturing in an incubator at 45-55°C to prepare a seed strain; inoculating the seed strain into a wheat / barley / pea mixed culture medium, culturing in an incubator at 45-55°C for 24-72 days, and determining the acid protease content by the Folin phenol method. The higher the acid protease content, the stronger the ability to produce acid protease.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention separates, screens and preserves a new strain of Thermomyces lanuginosus that can produce high acidic protease from the Daqu sample of Jinshiyuan Liquor Co., Ltd. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID No: 1. The sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is Thermomyces lanuginosus. The strain has excellent ability to produce acidic protease, has good flavor production ability, and can produce a variety of flavor substances such as esters, alcohols, ketones, etc. The Thermomyces lanuginosus makes up for the problems that the enzyme system in the current Daqu is not comprehensive or active enough, thereby improving the wine quality and wine yield, and is applied to the koji making of liquor and related food fermentation processes, and has broad application prospects and potential application value.

[0034] The lanuginosus thermophilic hyphomycete involved in the present invention is classified and named as lanuginosus thermophilic hyphomycete TL-S5 Thermomyces lanuginosus TL-S5, the preservation time is September 4, 2024, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation address is: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20241912. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a transparent circle diagram of the hydrolysis of acidic proteases produced by each strain on casein solid medium;

[0036] Figure 2 is a schematic diagram of the phylogenetic tree of Thermomyces lanuginosus TL-S5;

[0037] Figure 3 This is a chromatographic detection chart of flavor substances produced by Thermomyces lanuginosus TL-S5. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0039] The formula of the PDA medium involved in the following examples is: 12 g / L potato extract powder, 20 g / L glucose, 14 g / L agar powder, and the rest water, adjusted to pH 5.6 at 25°C, and sterilized at 121°C for 20 min.

[0040] The formula of casein culture medium is: casein 10g / L, beef extract 3g / L, sodium chloride 5g / L, disodium hydrogen phosphate 2g / L, agar 20g / L, and the rest water, pH value is 3.7, and sterilization is carried out at 121℃ for 20min.

[0041] The formula of the bran culture medium is: bran and water are mixed evenly, the total water content is 48%, and sterilized at 121°C for 30 minutes.

[0042] The formula of the wheat / barley / pea mixed culture medium is as follows: wheat, barley and pea are weighed in a mass ratio of (7:2:1), crushed, and the crushed material is passed through a 40-mesh sieve, and the proportion of the sieve material is 20%; the sieve material and water are evenly mixed, and the total water content is 35%.

[0043] Example 1

[0044] Screening of Thermomyces lanuginosus TL-S5 strain and evaluation of its acidic protease production:

[0045] (1) A Daqu sample produced by Jiangsu Jinshiyuan Wine Co., Ltd. was selected and crushed by a grinder. 10 g of the crushed sample was weighed and added to 90 mL of sterile water. The sample was placed in a 30°C constant temperature shaker and incubated at 150 r / min for 30 min to obtain a bacterial suspension.

[0046] (2) Dilute the bacterial suspension in the flask with sterile water in a gradient manner (10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ), take 100 μL of each gradient and spread it on a PDA medium plate with glass beads, shake it well, and pour out the glass beads after the plate is dry.

[0047] (3) Place the plate in a 50°C constant temperature incubator. After colonies grow, pick single colonies evenly from different areas and separate and purify them on a new sterile PDA medium plate. Streak six times to obtain a single pure strain, which is then stored in a 4°C refrigerator for later use.

[0048] (4) According to the casein plate hydrolysis transparent zone method, a loop of the isolated and purified strain was scraped and inoculated on a casein solid medium. After culturing in a 50°C incubator for 48 h, 6 strains with strong acid protease production ability with transparent zones around the colonies on the plate were selected (the strains with strong acid protease production ability were determined based on the ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d), and the strains with small D / d values ​​were discarded). The culture results of each strain on the casein solid medium are shown in the following table. Figure 1 shown.

[0049] (5) In an ultra-clean workbench, a strain with a strong ability to produce acidic protease was inoculated into a bran culture medium, stirred evenly, and cultured in a 50°C incubator at an angle for 3 days, shaking once every 24 hours to prepare seed bacteria; the bran seed bacteria was inoculated into a wheat / barley / pea mixed culture medium at an inoculum rate of 3%, stirred evenly, and cultured in a 50°C incubator at an angle for 3 days; the acidic protease of the six strains was determined by the Folin phenol method, and each sample was repeated 3 times, as shown in Table 1, to obtain a pure strain No. 5 producing acidic protease.

[0050] Table 1

[0051]

[0052]

[0053] Example 2

[0054] Identification of Thermomyces lanuginosus TL-S5 strain:

[0055] (1) DNA lysis of the isolated and screened strain No. 5 was performed using the AB liquid lysis method. 45 μL of AB liquid was pipetted into a sterile PCR tube. A small amount of purified bacteria was picked up with a sterile toothpick and placed into the above tube. The tube was covered and marked, and then placed into a PCR instrument for lysis. After lysis, 5 μL of B liquid was added as a DNA template.

[0056] (2) Using ITS4 and ITS5 as primers and the DNA obtained in step (1) as a template for PCR amplification, the amplification reaction system is 25 μL, including 12.5 μL of 2×Taq PCR Master Mix, 10.5 μL of ddH2O, 0.5 μL of upstream primer, 0.5 μL of downstream primer, and 1 μL of DNA template; the PCR reaction conditions are 94°C for 4 min; 94°C for 40 s, 55°C for 50 s, 72°C for 40 s, 35 cycles; 72°C for 4 min;

[0057] (3) The PCR amplification product was detected by 1% agarose gel electrophoresis. 5 μL of PCR product was taken, 2 μL of loading buffer was added, mixed evenly and then spotted. The parameters of the electrophoresis instrument were adjusted to 130 V for 30 min. After the results were observed by the gel imaging system, the sequencing form was filled in and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The resulting strain sequence was shown in SEQ ID No: 1.

[0058] (4) The sequencing results were compared with the NCBI database using BLAST and a phylogenetic tree was constructed using MEGA. The results showed that the strain had the highest degree of identity with Thermomyces lanuginosus, so it was identified as Thermomyces lanuginosus. The phylogenetic tree results are shown in Figure 2. Figure 2 shown.

[0059] (5) The strain was deposited and classified and named Thermomyces lanuginosus TL-S5. The deposit date was September 4, 2024. The depository was China Center for Type Culture Collection (CCTCC). The deposit address was Wuhan University, Wuhan, China. The deposit number was CCTCC NO: M 20241912.

[0060] Example 3

[0061] Analysis of flavor substances produced by Thermomyces lanuginosus TL-S5 strain:

[0062] (1) The TL-S5 strain of Thermomyces lanuginosus was inoculated into a sterile wheat / barley / pea mixed medium and cultured at 50°C for 3 days. 10 g of the cultured strain was weighed and dried, 100 mL of water was added, the mixture was stirred and mixed, and ultrasonicated for 30 min to obtain the extract.

[0063] (2) Detection of volatile flavor substances using headspace solid phase microextraction and gas chromatography-mass spectrometry:

[0064] Weigh 2g of sodium chloride, add 5mL of supernatant extract and 20μL of 0.2 parts per million of three internal standards, 2 rotors, put into a 50℃ water bath for preheating for 5min, age the SPME extraction head at 210℃ for 30min and insert it into the sample bottle, extract for 45min, desorb the extraction head at 210℃ for 5min in the GC injection port and then pull it out; the temperature program of the detection instrument is 50℃ for 4min, increase from 4℃ / min to 80℃, maintain for 0min, increase from 8℃ / min to 210℃, maintain for 15min; the carrier gas (He, 99.999%) flow rate is 1.0mL / min, and the injection is non-split; the mass spectrometry conditions are electron bombardment ion source; the electron bombardment energy is 70eV; the transmission line temperature is 250℃; the ion source temperature is 200℃; the mass scanning range m / z is 35-500.

[0065] Chromatogram of flavor substances produced by Thermomyces lanuginosus Figure 3The results are shown in Table 2. The TL-S5 strain of Thermomyces lanuginosus of the present invention can produce a variety of flavor substances such as esters, alcohols, ketones, etc. Among them, ethyl hexanoate has complex and unique flavor characteristics such as sweet, fruity, irritating and astringent. At the same time, it is also one of the main flavor components of Luzhou-flavor liquor, with the cellar aroma of liquor, adding rich flavor to the liquor; allyl acetate usually has a fruit-like smell, and has a citrus-like fruity aroma like fresh bergamot and white lemon; methyl heptanoate has a sweet and berry fragrance, which is a fruity and sweet aroma feature; phenylethyl acetate has a floral, rose, honey, and fruity flavor; 2-methyl-3- Methyl hexenoate has a banana-like fruity and green aroma, with a green fruit flavor and a certain spicy taste; phenylethyl alcohol has a sweet rose scent and is an important aromatic substance in rice-flavored liquor; 3-octanol has earthy, mushroom, moldy, waxy aromas, and has woody, spicy and minty notes; 3-hexanol and 2-hexanol have similar odors, both with mellow and medicinal characteristics; 2,5-dimethylpyrazine has a strong smell of fried peanuts, chocolate and cream; this compound is widely used in food, medicine, spices, feed and other fields, 2-methyl-3-pentanone has a minty smell; acetophenone is considered to have the smell of hawthorn or a sweet fruit-like aroma

[0066] Table 2

[0067]

[0068] Therefore, the Thermomyces lanuginosus TL-S5 strain involved in the present invention has good flavor production ability and has great application prospects in related fermentation processes such as liquor and koji making.

[0069] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0070] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0071] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A Thermomyces lanuginosus producing acidic protease, characterized in that: The lanuginosus Thermomyces producing acidic protease is the lanuginosus Thermomyces lanuginosus TL-S5 strain, with a preservation number of CCTCC NO: M 20241912 and a preservation date of September 4, 2024.

2. The culture of Thermomyces lanuginosus according to claim 1, characterized in that The culture is obtained by inoculating Thermomyces lanuginosus TL-S5 strain into a sterile culture medium and culturing at 45-60°C.

3. A microbial preparation producing acidic protease, characterized in that: The strain in the microbial preparation includes the Thermomyces lanuginosus TL-S5 strain according to claim 1.

4. The microbial preparation according to claim 3, characterized in that The dosage form of the microbial preparation includes solution or powder.

5. The microbial preparation according to claim 3 or 4, characterized in that: The microbial preparation also includes a protective agent and / or a carrier material.

6. The microbial preparation according to claim 5, characterized in that The protective agent is selected from any one or a combination of at least two of skim milk, gelatin, gum arabic, dextran, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

7. The microbial preparation according to claim 5, characterized in that The carrier material is selected from any one of maltodextrin, starch, microcrystalline cellulose, calcium carbonate or calcium hydrogen phosphate, or a combination of at least two thereof.

8. Use of the Thermomyces lanuginosus according to claim 1, the culture of Thermomyces lanuginosus according to claim 2, or the microbial preparation according to any one of claims 3 to 7 in the production of acidic protease.

9. Use of the Thermomyces lanuginosus according to claim 1, the culture of Thermomyces lanuginosus according to claim 2, or the microbial preparation according to any one of claims 3 to 7 in liquor brewing or koji making.

10. Use of the Thermomyces lanuginosus according to claim 1, the culture of Thermomyces lanuginosus according to claim 2, or the microbial preparation according to any one of claims 3 to 7 in food fermentation.

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