Screening and domestication method of high-temperature-resistant mould in Maotai-flavor fermented grains

Abstract

The screening method comprises the following steps: S1, separation and purification of mould: stacking fermented grains of Maotai-flavor liquor, diluting the fermented grains by using sterile normal saline in a gradient manner, inoculating the fermented grains to a PDA culture medium by using a dilution coating plate method, culturing at constant temperature, and purifying by using a plate streaking method to obtain pure mould strains; s2, screening high-temperature-resistant strains: sequentially culturing the pure strains obtained in the step S1 under a temperature gradient, and screening out high-temperature-resistant mould strains with the highest tolerable temperature being greater than or equal to 55 DEG C; and S3, identifying the strain: observing a bacterial colony and a microstructure through morphology, and determining the species of the high-temperature-resistant strain by combining ITS gene PCR amplification, sequencing and phylogenetic tree construction.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganism screening and domestication, and particularly relates to a screening and domestication method of high-temperature-resistant mold in Maotai-flavor fermented grains. BACKGROUND

[0002] Mold, as a kind of fungi with branched mycelium, plays a key role in the production of Maotai-flavor liquor, and its growth and metabolism are directly related to the quality of liquor. On the one hand, mold produces various enzymes such as saccharifying enzyme, protease, cellulase and esterifying enzyme during the growth process, and these enzymes are the main driving force for the degradation of macromolecular substances such as starch, protein and cellulose in fermented grains, which can significantly improve the utilization rate of raw materials and the yield of liquor; on the other hand, the metabolic products of mold include various flavor substances such as higher alcohols, aldehydes, ketones and esters, and the esterifying enzyme produced by mold can promote the esterification reaction of ethanol and organic acids in liquor, further enriching the aroma components of liquor.

[0003] At present, mold found in liquor fermentation includes Aspergillus, Rhizopus, Mucor, Absidia and Geotrichum, and some strains such as Monascus and Clonostachys rosea have been proved to have the function of strengthening flavor generation. However, the activity of mold in the stacking fermentation of Maotai-flavor liquor is significantly affected by environmental factors, and high temperature and low pH value are the main bottlenecks restricting its function. Research data shows that the high-temperature and high-acid characteristics of the stacking fermentation of Maotai-flavor liquor put forward strict requirements for the tolerance of mold, and the temperature of fermented grains in the late stacking fermentation is maintained at 48-52℃, and the pH value is as low as about 3.3. The growth and metabolism of most mold are hindered in this environment, and even die, resulting in a decreasing trend of the number of mold in fermented grains until the end of fermentation. This phenomenon directly leads to insufficient enzyme source, incomplete degradation of macromolecular substances, reduced utilization rate of raw materials, and limited generation efficiency of flavor substances, affecting the stability of liquor quality.

[0004] The existing technology for the screening of mold in fermented grains of liquor mainly focuses on normal-temperature or medium-temperature strains, and lacks a special screening system for the high-temperature and high-acid environment of Maotai-flavor liquor. At the same time, the existing acid-tolerant domestication method mainly uses the one-time reduction of pH value, which is easy to cause strong stress response of the strain and has a high failure rate of domestication. Therefore, it is urgent to develop a high-temperature-resistant mold screening and domestication method with strong pertinence, so as to obtain mold strains with high-temperature tolerance and acid resistance, which has important practical significance and application value for strengthening the fermentation process of Maotai-flavor liquor, stabilizing product quality and improving the technical level of the industry. SUMMARY

[0005] The present application aims to provide a screening and domestication method of high-temperature-resistant mold in Maotai-flavor fermented grains, and obtain mold strains with high-temperature tolerance and acid resistance.

[0006] In order to achieve the above-mentioned purpose, the following technical solutions are provided: A screening method of high-temperature resistant mold in Jiang-flavor liquor fermented grains, comprising the following steps: S1, mold separation and purification: taking Jiang-flavor liquor fermented grains, gradient diluting with sterile normal saline, inoculating on PDA medium by dilution coating plate method, purifying by plate streaking method after constant temperature culture, and obtaining pure mold strains; S2, high-temperature resistant strain screening: sequentially culturing the pure strains obtained in step S1 at temperature gradients, and screening high-temperature resistant mold strains with the highest tolerance temperature of 55 DEG C or above; S3, strain identification: determining the species of the high-temperature resistant strains by observing the colony and microscopic structure, combining with ITS gene PCR amplification, sequencing and phylogenetic tree construction.

[0007] As a preferred, the concentration of gradient dilution in step S1 is 10 -1 ~10 -6 , the culture temperature is 30-33 DEG C, the culture time is 3-5 days, the purification adopts plate streaking by quartering method for not less than 3 times, and the strain is preserved in a Bengal red slant medium at 4 DEG C.

[0008] As a preferred, the temperature gradient in step S2 is 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C and 60 DEG C.

[0009] As a preferred, the culture time at each temperature gradient in step S2 is 3-5 days, and the screening standard is that the strain can grow at 55 DEG C and cannot grow at 60 DEG C.

[0010] A method for acid resistance domestication of a strain, wherein the pH value of PDB liquid medium is adjusted by lactic acid under the condition of 50 DEG C, the minimum growth pH of the strain is determined as a domestication point, the pH value of the medium is gradually reduced through multi-generation subculture domestication, and a domesticated strain with improved acid resistance is obtained.

[0011] As a preferred, the domestication adopts gradient medium with the pH value of 2.0-5.5, the minimum growth pH is determined by determining the mycelium dry weight, the culture time of each generation in the domestication process is 48 h, the medium is domesticated for 3 generations at each pH gradient, the pH value of the medium is gradually reduced by 0.25-0.5, and finally the strain with the acid resistance limit of less than or equal to pH 2.5 is obtained.

[0012] The application of the acid resistance domesticated strain in Jiang-flavor liquor brewing.

[0013] The working principle and beneficial effects of the application are as follows: The present application separates different types of mold strains from fermented grains by gradient dilution plating method, ensuring strain diversity; then gradually selects strains with excellent high-temperature resistance by temperature gradient of 40-60°C, simulating the dynamic temperature rise process in the stacking fermentation process; adjusts the pH value of the culture medium with lactic acid to simulate the gradual increase of acidity in the fermented grains fermentation process, determines the minimum growth pH value of the strain as the acclimation starting point, and gradually adapts the strain to the acidic environment through multiple generations of subculture, so that the acid tolerance of the strain is slightly improved after each acclimation; by controlling the pH gradient and acclimation generation of each acclimation, the stress response of the strain due to environmental mutation is avoided, and the acclimation process is stable and efficient; at the same time, the growth state of the strain is monitored by determining the mycelium dry weight, and finally the strain with significantly improved acid tolerance is obtained. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figures 1-7 Typical colonies and microscopic morphology of B1-B7, respectively; Figure 8 is the microscopic examination result of strain B7 at 50°C; Figure 9 is the gel electrophoresis imaging map of PCR product and DNA of B7 strain; Figure 10 is the ITS sequence gene phylogenetic tree of B7 strain; Figure 11 is the acid tolerance performance of B7 strain at each pH value; Figure 12 is the 72 h growth curve of B7 strain. DETAILED DESCRIPTION

[0015] The following is further described in detail through specific embodiments: Embodiment: A high-temperature resistant mold screening and acclimation method in Maotai-flavor fermented grains, 1. Mold separation and purification 10 g of fermented grain sample was weighed, and 90 mL of sterile normal saline was added under sterile conditions to prepare a 10 -1 mL bacterial suspension, which was gradient diluted to 10 -6 μL; 100 μL of each dilution bacterial suspension was taken and inoculated on PDA plates by dilution plating method, and incubated at 30°C for 3-5 days; single colonies with different morphologies were picked and purified by four-line method plate streaking for more than three times, and the purified strain was transferred to a Bengal red slant medium and preserved at 4°C.

[0016] 2. High-temperature resistant mold screening After the preservation strain is activated, it is sequentially cultured at temperature gradients of 40℃, 45℃, 50℃, 55℃, and 60℃ for 3-5 days (3 parallel groups each time), and the growth is observed (the growth is vigorous, "+" means that the growth is possible, and "-" means that the growth is not possible), and the strain with the highest tolerance temperature is screened out.

[0017] 3. Identification of the target strain Morphological identification: the strain is inoculated on PDA medium by the three-point method, and cultured at 30℃ for 3-5 days, and the characteristics such as the color, texture, and edge of the colony are observed; after lactophenol cottonlan staining, the microscopic structures such as mycelium, conidium, or cleistothecia are observed under a microscope; Molecular biological identification: the mycelium of the strain is collected, and the genomic DNA is extracted; PCR amplification is performed by using ITS1 / ITS4 as primers (the reaction system is 20μL, and the program is 95℃ pre-denaturation for 5min, 95℃ denaturation for 15s, 57℃ annealing for 15s, 72℃ extension for 30s, 35 cycles, and 72℃ final extension for 5min); after the amplification product is sequenced, BLAST comparison is performed in the NCBI database, and a phylogenetic tree is constructed by using MEGA11.0 software to determine the species.

[0018] 4. Acid tolerance domestication of the strain Domestication point determination: PDB medium with pH2.0-5.5 is prepared, the strain is inoculated, and then cultured at 50℃ and 180r / min on a shaking table for 48h, the mycelium dry weight is determined, and the lowest growth pH of the strain is determined as the domestication point; Acid tolerance domestication: starting from the domestication point pH, 100μL of the bacterial solution is inoculated in the same pH PDB medium, and cultured for 48h as one generation, and the domestication is performed for 3 generations for each pH gradient; the medium pH is gradually reduced by 0.25, and the domestication is repeated, and the 72h growth curves before and after the domestication are drawn to verify the effect.

[0019] II. Example results 1. Isolation and purification of molds As Figures 1-7 Seven typical mold strains (named B1-B7) are obtained by isolation and purification from fermented grains, and all of them have mold morphological characteristics: the colony color covers white, orange yellow, green, and brown yellow, the microscopic structure can be observed to have branched mycelium, conidium (spherical / elliptical), or cleistothecia, and no mixed bacteria are confirmed after repeated purification.

[0020] B1 is white and tender yellow at the initial stage, and when it is mature, the texture is felt-like, the edge is white, the center is orange yellow, and there is a protrusion; the reverse side of the colony is red, the conidium stem is short and curved, the small stem is double-layered, the top capsule is hemispherical, and the conidium is spherical; B2 is white at the initial stage of culture, then turns into beige, the central protuberance is green, the edge is white, and the texture is loose; the reverse side is yellow, the conidium stem is long, the small stem of the top capsule surface is double-layered and arranged radially, and the conidium is spherical; B3 texture flocculent or with woolly, grass green, edge white; colony reverse light yellow mycelium with transverse septum, small branches in broom shape, the broom-shaped branches are asymmetric, two rounds of growth; B4 mycelium flocculent, white round, with a convex in the middle; the front and back are the same color, sporangiophore smooth and upright, spore sac pear-shaped, spore sac spore round; B5 loose texture, with a small amount of grooves, initially white, with a large number of black powder particles on it; later, the black powder particles gradually spread to the entire colony; the colony reverse is yellow mycelium, the conidium stalk is swollen at the top, and the double-layer small branches are radially produced from the surface or top of the vesicle; B6 mycelium flocculent, edge white, yellow brown transition from blue to the center; the colony reverse is reddish brown mycelium without transverse septum; the small branches are double-layered; the conidium is single or in a chain, and is round; B7 irregular colony morphology, loose texture, initially white aerial mycelium, gradually yellow, central tight small spots, later mycelium turns brown yellow; the front and back are the same color, mycelium is colorless with branches, septum is clear and complete, spherical cleistothecia are produced, ascospores are oval and arranged in lines.

[0021] 2. Screening of high-temperature resistant mold Among the 7 strains, B7 strain has the best high-temperature resistance: it can grow well at 40℃-50℃ (2 days to cover the plate at 45℃), can survive at 55℃ (mycelium is sparse but can grow), and does not grow at 60℃; the remaining strains have stopped growing at 50℃ or 55℃, so B7 is determined as the target strain.

[0022] 3. Identification results of target strain The microscopic examination results of strain B7 at 50℃ are shown in Figure 8 The mycelium is thick and has branches and septum, and the cleistothecia are single, which is a typical Ascomycotion according to its morphological characteristics.

[0023] The detection results of gel electrophoresis of DNA of strain B7 and PCR amplification product are shown in Figure 9 A single bright band appears at a length of about 5000bp with slight tailing, indicating that the DNA quality is qualified and can meet the requirements of subsequent experiments; the length of the amplified ITS sequence is about 600bp, the band is clear and bright, and no impurity band appears, with slight tailing, indicating that the DNA template of strain B7 is successfully amplified, which meets the sequencing requirements.

[0024] The PCR amplification product is entrusted to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequence results are analyzed and compared for homology on the BLAST website of NCBI website. Take the sequences with similarity of 99% or more to the ITS sequence of B7 strain, use MEGA11.0 for multiple sequence alignment and construct a phylogenetic tree, and the results are as followsFigure 10 .

[0025] 4. Acid-tolerant domestication Reference Figure 11 , domestication point: B7 did not grow below pH 3.0 at 50℃, so the domestication point was determined to be pH 3.0; Reference Figure 12 , domestication effect: after multiple generations of gradient domestication (3 generations per pH gradient, gradually reducing pH by 0.25), B7 can finally grow at pH 2.5, 50℃; the mycelium dry weight at pH 2.5 after domestication was significantly higher than before domestication, the growth curve showed that it could complete the normal growth cycle under this condition, and the acid tolerance was greatly improved.

[0026] This application takes Jiangxiang Baijiu (liquor) piled-up fermented grains as raw material, selects high-temperature resistant mold and domesticates its acid tolerance. The experimental results show that 7 cultivable strains are obtained in the isolation and purification test of mold, which are preserved after observing their colony characteristics and microscopic structure. A mold that can still survive at 55℃ is finally selected by increasing temperature gradient, which is named B7. It is identified by molecular biology as Thermoascus crustaceus. To improve the acid tolerance of the strain, gradient domestication is carried out, and the acid tolerance of the final strain B7 is reduced from 3.0 to 2.5.

[0027] It should be noted that for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can also be made, which should also be considered as the protection scope of the present application, and these will not affect the effect of the present application and the practicality of the patent. The protection scope of the present application should be subject to the content of its claims, and the specific implementation mode and the like in the specification can be used to explain the content of the claims.

Claims

1. A method for screening high-temperature-resistant mold in fermented grains of Maotai-flavor liquor, characterized in that, It comprises the following steps: S1, isolation and purification of mold: take the Jiangxiang Baijiu (liquor) accumulated dregs, gradiently dilute with sterile normal saline, inoculate on PDA medium by dilution coating plate method, purify by plate streaking method after constant temperature culture, and obtain pure mold strain; S2, high temperature resistant strain screening: culture the pure strain obtained in step S1 at temperature gradient, and screen high temperature resistant mold strain with highest tolerance temperature ≥ 55℃; S3, strain identification: determine the species of high temperature resistant strain by morphological observation of colony and microscopic structure, combined with ITS gene PCR amplification, sequencing and phylogenetic tree construction.

2. The screening method according to claim 1, characterized in that, The concentration of gradient dilution in step S1 is 10 -1 ~10 -6 The culture temperature is 30-33℃, the culture time is 3-5 days, the purification adopts the quarter line method plate streaking not less than 3 times, and the purified strain is preserved in a Bengal red slant medium, and the preservation temperature is 4℃.

3. The screening method according to claim 2, characterized in that, The temperature gradient in step S2 is 40℃, 45℃, 50℃, 55℃ and 60℃.

4. The screening method according to claim 3, characterized in that, The culture time at each temperature gradient in step S2 is 3-5 days, and the screening standard is that the strain can grow at 55℃ and cannot grow at 60℃.

5. The method for acid-adaptation of the strain obtained by the screening method according to any one of claims 1 to 4, characterized in that, Under the condition of 50℃, adjust the pH value of PDB liquid medium with lactic acid, determine the minimum growth pH of the strain as the acclimation point, gradually reduce the pH value of the medium by multi-generation passage acclimation, and obtain acclimated strain with improved acid resistance.

6. The method for acclimating the acid tolerance of strains screened according to claim 5, characterized in that, The acclimation adopts gradient medium with pH value of 2.0-5.5, and the minimum growth pH is determined by measuring mycelium dry weight; the culture time of each generation in the acclimation process is 48h, 3 generations are acclimated at each pH gradient, the medium pH value is gradually reduced by 0.25-0.5, and finally the strain with acid resistance limit ≤pH 2.5 is obtained.

7. Application of the strain obtained by the acid resistance acclimation method of claim 6 in Jiangxiang Baijiu (liquor) brewing.

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