Thermomucor spicatum vailantii producing acid protease and application thereof

Abstract

The present application relates to a kind of acid protease-producing thermomyces lanuginosus and its application, the acid protease-producing thermomyces lanuginosus is thermomyces lanuginosus (Thermomyces lanuginosus) TL-S5 strain, the preservation number is CCTCC NO: M 20241912, and the preservation date is September 4, 2024.The strain has excellent acid protease-producing capacity, has good flavor-producing capacity, can produce a variety of ester, alcohol, ketone and other flavoring substances, has great application prospect in related fermentation processes such as baijiu and koji making.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial culture, and relates to a thermomyces lanuginosus for producing acid protease and application thereof. BACKGROUND

[0002] Microbial proteases are divided into acid, neutral and alkaline proteases according to their acid and alkali resistance. Acid protease refers to a class of enzymes that can hydrolyze proteins into smaller peptide segments or amino acids under acidic conditions. Such enzymes can maintain high activity and stability in an acidic environment, thereby effectively hydrolyzing proteins. As an important class of enzymes, acid protease has wide application prospects and important economic value in the fields of food, feed, medical treatment, etc.

[0003] Carbon dioxide produces various 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, ensuring the smooth progress of the fermentation process. Some microorganisms secrete various enzymes, including acid protease. 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 liquor flavor and yield, and improving raw material utilization, etc. Therefore, it is necessary to add an appropriate amount of acid protease during liquor brewing and koji making.

[0004] In current research, microorganisms with acid protease-producing ability mainly include various molds, yeasts, bacillus and thermophilic fungi, etc. Molds mainly include Aspergillus niger, Aspergillus usamii, Aspergillus oryzae, Penicillium chrysogenum, Penicillium griseofulvum, Rhizopus oryzae, etc. For example, CN109161489A discloses a high-yield acid protease Aspergillus niger strain; CN102676395A discloses a mutant strain of Aspergillus usamii and its application in preparing acid protease. Yeasts, such as CN111286463A, disclose a Pichia pastoris strain for producing acid protease. Bacillus, such as CN118086106A, discloses a Brevibacillus pectinilyticus and its application in producing acid protease; such as CN117603889B, discloses a feed-use acid protease-producing Bacillus subtilis and its application; such as CN114874942B, discloses a protease-producing Bacillus cereus and its application in Daqu.

[0005] However, few studies have examined the production of acidic proteases by Thermomyces lanuginosus. Thermomyces lanuginosus is a widely distributed thermophilic fungus with an optimum growth temperature of approximately 50°C and a maximum growth temperature of 60°C, demonstrating exceptional heat tolerance. The fungus secretes a variety of enzymes with excellent thermostability and a broad pH range, such as xylanases, chitinases, amylases, proteases, and lipases. Many researchers have isolated Thermomyces lanuginosus, but none have investigated its production of acidic proteases. Summary of the Invention

[0006] To address the shortcomings of the prior art, the present invention aims to provide a Thermomyces lanuginosus strain that produces acidic proteases and its applications. This strain enhances the activity and diversity of enzymes in the fermentation process, addressing issues such as the incomplete or insufficient enzyme activity in current daqu (Chinese liquor) production, thereby improving liquor quality and yield. The strain is applicable to koji production and related food fermentation processes, demonstrating broad application prospects and potential 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 acidic protease, wherein the lanuginosus thermophilic fungus that produces 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 isolated, screened, and preserved a novel strain of Thermomyces lanuginosus that produces a high amount of acidic protease from Daqu samples obtained from Jinshiyuan Liquor Co., Ltd. Sequencing analysis revealed the 16S rDNA sequence of this strain as SEQ ID No: 1. Comparison of the sequenced sequence with the nucleotide sequence in GeneBank confirmed that the strain is Thermomyces lanuginosus. This strain exhibits excellent acidic protease production and flavor production, including the production of a variety of flavor compounds such as esters, alcohols, and ketones. It holds great promise for application in fermentation processes related to liquor production and koji making.

[0010] In a second aspect, the present invention provides a culture of Thermomyces lanuginosus 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 that produces acidic protease, wherein the strain in the microbial preparation includes 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 or a combination of at least two of maltodextrin, starch, microcrystalline cellulose, calcium carbonate or calcium hydrogen phosphate.

[0016] In a fourth aspect, the present invention provides 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 producing acidic protease.

[0017] In a fifth aspect, the present invention provides 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, the 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 Liquor Co., Ltd. was crushed and added to sterile water for incubation to obtain a bacterial suspension;

[0021] (2) Gradual dilution of the bacterial suspension was applied onto a sterile culture medium plate;

[0022] (3) Place the plate in a constant temperature incubator. After colonies grow, pick single colonies from different areas and further isolate and purify them on a new sterile culture medium plate.

[0023] (4) Use casein medium to perform preliminary screening of bacterial cultures, and select bacterial strains with a clear zone around the colonies on the plate and a strong ability to produce acidic protease;

[0024] (5) Then the ability of the strain to produce acid protease is determined by using the Folin phenol method, and the strain with stronger acid protease production ability is obtained.

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

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

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

[0028] Preferably, the formula of the casein 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 is water, and the pH value is 3.6-3.8.

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

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

[0031] Preferably, the method for re-screening in step (5) specifically comprises: inoculating the strain into bran medium, and preparing seed bacteria in a 45-55 DEG C incubator; inoculating the seed bacteria into wheat / barley / pea mixed medium, and culturing in a 45-55 DEG C incubator for 24-72 days, and determining the acid protease content by using the Folin phenol method. The higher the acid protease content, the stronger the acid protease production ability.

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

[0033] The present invention isolated, screened, and preserved a novel strain of Thermomyces lanuginosus that is capable of high acidic protease production from a Daqu sample obtained from Jinshiyuan Liquor Co., Ltd. Sequencing analysis revealed the 16S rDNA sequence of this strain as shown in SEQ ID No: 1. Comparison of the sequenced sequence with the nucleic acid sequence in GeneBank revealed that the strain is Thermomyces lanuginosus. This strain has excellent acidic protease production and flavor production, including the production of a variety of flavor substances such as esters, alcohols, and ketones. This strain addresses the issues of incomplete or insufficiently active enzyme systems in current Daqu, thereby improving liquor quality and yield. It has broad application prospects and potential value in the production of koji for liquor and related food fermentation processes.

[0034] The lanuginosus thermophilic hyphomycetes involved in the present invention are classified and named as lanuginosus thermophilic hyphomycetes 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 This 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 way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0039] The formula of the PDA culture medium involved in the following examples is: 12 g / L potato extract, 20 g / L glucose, 14 g / L agar powder, and the remainder 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 with an oversize ratio of 20%; the oversize material is evenly mixed with water to obtain a total water content of 35%.

[0043] Example 1

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

[0045] (1) The Daqu sample produced by Jiangsu Jinshiyuan Liquor 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 shaken. The sample was incubated at 150 r / min for 30 min to obtain a bacterial suspension.

[0046] (2) Dilute the bacterial suspension in the triangular flask with sterile water (10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ), 100 μL of each gradient was spread on a PDA medium plate with glass beads, shaken evenly, and the glass beads were poured out after the plate was dry.

[0047] (3) Place the plate in a 50°C constant temperature incubator. After colonies grow, pick single colonies evenly in different areas and separate and purify them on a new sterile PDA medium plate. Streak six times to obtain a single pure strain and store it 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 onto a casein solid medium. After culturing in a 50°C incubator for 48 hours, 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 Figure 2. Figure 1 shown.

[0049] (5) In a clean bench, 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, with each sample repeated 3 times, as shown in Table 1, to obtain a pure strain No. 5 that produces 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 strain No. 5 was performed using the AB solution lysis method. 45 μL of the AB solution 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 capped and marked, and then placed in a PCR instrument for lysis. After lysis, 5 μL of the B solution was added, which served as the DNA template.

[0056] (2) PCR amplification was performed using ITS4 and ITS5 as primers and the DNA obtained in step (1) as a template. The amplification reaction system was 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 were 94°C for 4 min; 35 cycles of 94°C for 40 s, 55°C for 50 s, and 72°C for 40 s; and 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 electrophoresis instrument parameters were adjusted to 130 V and 30 min. After the results were observed using a gel imaging system, a sequencing form was filled out and the cells were 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 by BLAST sequence alignment, 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 as follows: Figure 2 shown.

[0059] (5) The strain was deposited and classified as 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) Thermomyces lanuginosus TL-S5 strain was inoculated on 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, and 100 mL of water was added. The mixture was stirred and ultrasonicated for 30 min to obtain the extract.

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

[0064] Weigh 2 g of sodium chloride, add 5 mL of supernatant extract and 20 μL of 0.2 parts per million of tri-internal standard, 2 rotors, put into a 50°C water bath for preheating for 5 minutes, age the SPME extraction head at 210°C for 30 minutes and then insert it into the sample bottle. After extraction for 45 minutes, desorb the extraction head at 210°C in the GC injection port for 5 minutes and then pull it out; the temperature program of the detection instrument is 50°C for 4 minutes, increase from 4°C / min to 80°C, maintain for 0 minutes, increase to 210°C at 8°C / min, maintain for 15 minutes; the carrier gas (He, 99.999%) has a flow rate of 1.0 mL / min and is injected without splitting; the mass spectrometry conditions are electron bombardment ion source; the electron bombardment energy is 70 eV; the transmission line temperature is 250°C; the ion source temperature is 200°C; the mass scan range m / z is 35-500.

[0065] Chromatogram of flavor substances produced by Thermomyces lanuginosus Figure 3As shown in Table 2, the results of the analysis show that the TL-S5 strain of Thermomyces lanuginosus of the present invention can produce a variety of flavor substances such as esters, alcohols, and ketones. Among them, ethyl hexanoate has complex and unique flavor characteristics such as sweetness, fruitiness, stimulation and astringency. 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 fruity smell, and has a citrus-like fruity aroma like fresh bergamot and white lemon; methyl heptanoate has a sweet and floral berry aroma, which is a kind of aroma characteristic with fruity and sweet aroma; phenylethyl acetate has a floral, rose, honey, and fruity flavor; 2-methyl-3- Methyl hexenoate has a fruity and green aroma similar to that of banana, with a green fruit flavor and a certain spicy taste; phenylethyl alcohol has a sweet rose aroma and is an important aromatic substance in rice-flavored liquor; 3-octanol has earthy, mushroom, moldy, and waxy aromas, with 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 odor of roasted peanuts, chocolate, and cream; this compound is widely used in food, medicine, spices, feed, and other fields; 2-methyl-3-pentanone has a minty odor; acetophenone is considered to have the odor of hawthorn or a sweet aroma similar to that of fruit.

[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 is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure 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, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection 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 appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A Thermomyces lanuginosus that produces acidic protease, characterized in that: The acidic protease-producing Thermomyces lanuginosus is Thermomyces lanuginosus ( Thermomyces lanuginosus )TL-S5 strain, the deposit number is CCTCC NO: M 20241912, and the deposit date is September 4, 2024.

2. The culture of Thermomyces lanuginosus according to claim 1, characterized in that The culture is composed of Thermomyces lanuginosus ( Thermomyces lanuginosus )TL-S5 strain was inoculated into sterile culture medium and cultured at 45-60°C.

3. A microbial preparation producing acidic protease, characterized in that: The strain in the microbial preparation includes the Thermomyces lanuginosus ( Thermomyces lanuginosus )TL-S5 strain.

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 further comprises 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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