Streptomyces cacao for high yield of 2, 3-butanediol and application of streptomyces cacao

Abstract

The invention relates to the technical field of bioengineering, and particularly provides streptomyces cacao for high yield of 2, 3-butanediol and application of the streptomyces cacao, the strain is preserved in China Center for Type Culture Collection on October 29, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) 20242375. According to the streptomyces cacao for high yield of 2, 3-butanediol, the content of 2, 3-butanediol in white spirit can be increased by 211%. The streptomyces cacaoi is separated from high-temperature Daqu liquor yeast, can be used for producing white spirit, and provides a new functional strain for improving the quality of the white spirit.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a high-yield 2,3-butanediol cocoa Streptomyces and applications thereof. Background Art

[0002] In addition to esters, the primary aroma components of Chinese baijiu (white spirits), alcohols also play a crucial role. They are the primary components of the wine's sweetness and aroma, acting as precursors to the formation of flavor compounds. Alcohols react with acids to form various esters, which contribute to baijiu's distinctive aroma. For example, when baijiu contains a small amount of higher alcohols, they impart a unique aroma and enhance the ester aroma, making the aroma more full-bodied. These higher alcohols are both aromatic and flavor components, with most exhibiting an ethanol-like odor, lasting long and having a strong aftertaste, contributing to the flavor of the baijiu. Furthermore, polyols, such as glycerol, 2,3-butanediol, and cyclohexanehexol, are the primary sweeteners in baijiu, contributing to the sweetness, body, and viscosity of the wine. Compared with other polyols, 2,3-butanediol has attracted the attention of researchers because of its relatively low boiling point (183-184°C) and is easier to enter the wine body through the distillation process. Therefore, increasing the content of 2,3-butanediol to enhance the sweetness of liquor is a feasible strategy.

[0003] Numerous bacterial species produce 2,3-butanediol in nature. Bacillus and Paenibacillus polymyxa are primarily biosafe and have promising industrial production potential. Literature reports indicate that some actinomycetes can produce 2,3-butanediol during baijiu fermentation, thereby enhancing the alcoholic sweetness of the liquor. However, in the baijiu industry, research on high-yielding 2,3-butanediol strains is currently lacking. Given the importance of 2,3-butanediol in improving baijiu quality and its promising application prospects, it is necessary to screen for high-yielding actinomycete strains and apply them to production to increase the 2,3-butanediol content in light-flavor baijiu, thereby enhancing the alcoholic sweetness of the liquor. Summary of the Invention

[0004] In view of this, the present invention aims to solve the above technical problems and provides a strain of Streptomyces cacaoi B1624 with high 2,3-butanediol production.

[0005] The technical solution of the present invention is achieved as follows: The present invention provides a strain of Streptomyces cacaoi B1624 with high 2,3-butanediol production, and the Streptomyces cacaoi B1624 has an effect of increasing the 2,3-butanediol content in white wine. The strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 20242375, a deposit date of October 29, 2024, and a deposit address of Wuhan University, Wuhan, China.

[0006] The Streptomyces cacaoi B1624 strain disclosed in the present invention was isolated from high-temperature Daqu. The strain's colony morphology after 5 days of culture on a modified Gaucher plate is characterized by a nearly circular, dry, rough surface with annular patterns, a raised center, and wavy edges. The aerial hyphae are white, while the base hyphae are pale yellow.

[0007] The cocoa Streptomyces (Streptomyces cacaoi) B1624 involved in the present invention is obtained according to the following method:

[0008] High-temperature Daqu was used as the source for strain screening. First, purified actinomycete strains were isolated using a modified Gao's Medium No. 1 supplemented with nalidixic acid and potassium dichromate. The strains were then fermented using sorghum juice medium. After completion, the volatile compound content in the fermentation broth was measured, and strains with relatively high 2,3-butanediol levels were selected. These strains were then inoculated simultaneously with production koji for fermentation and brewing. A control group was inoculated with koji alone for fermentation and distillation. Finally, the strain with the highest 2,3-butanediol content in the base liquor was selected.

[0009] In a second aspect, the present invention further provides a use of the above-mentioned Streptomyces cacaoi B1624 in increasing the 2,3-butanediol content in liquor.

[0010] The high-yield 2,3-butanediol cocoa Streptomyces is cultured to obtain a pure bacterial agent; the pure bacterial agent is then mixed with the production yeast to prepare a microbial compound yeast for fermentation and brewing.

[0011] In some embodiments, the liquor is a light-fragrance liquor.

[0012] In some embodiments, a microbial agent comprising Streptomyces cacaoi B1624 with a deposit number of CCTCC M 20242375 is mixed with distiller's yeast and then fermented and brewed.

[0013] In a third aspect, the present invention further provides a microbial agent comprising Streptomyces cacaoi B1624 with a deposit number of CCTCC M20242375.

[0014] In some embodiments, the microbial agent is a solid agent or a liquid agent.

[0015] In a fourth aspect, the present invention further provides a light-fragrance liquor, which is brewed using the above-mentioned microbial agent.

[0016] The present invention has the following beneficial effects compared to the prior art:

[0017] The high-yield 2,3-butanediol cocoa Streptomyces of the present invention can increase the 2,3-butanediol content in liquor by 211%. The cocoa Streptomyces is isolated from high-temperature Daqu and can be used in liquor production, providing a new functional strain for improving liquor quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a characteristic diagram of the colony morphology of the high-yielding 2,3-butanediol Streptomyces cocoa involved in the present invention. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only 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 making creative efforts are within the scope of protection of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0022] Unless otherwise specified, the methods used in the following examples are conventional methods. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0023] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range. In the present specification and claims, range definitions may be combined and / or interchanged, and if not otherwise stated, such ranges include all subranges contained therein.

[0024] Example 1

[0025] This embodiment provides a method for obtaining a strain of Streptomyces theobrominum, comprising the following steps:

[0026] 1 Screening of 2,3-butanediol-producing actinomycetes

[0027] 1.1 Preparation of culture medium

[0028] Modified Gao's medium No. 1: potassium nitrate 1.0 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 0.01 g / L, sodium chloride 0.5 g / L, soluble starch 20.0 g / L, solid medium supplemented with agar 15.0 g / L, sterilized at 121°C for 20 min;

[0029] Sorghum juice medium: Weigh 1 kg of sorghum, grind it, add 2.5 g each of saccharifying enzyme and amylase, and soak in 5 L of hot water for half an hour. Stir thoroughly, then steam in an autoclave at 105°C for 30 minutes. Remove and cool, then add 5 g each of saccharifying enzyme and amylase, and saccharify in a 60°C oven for 24 hours. Centrifuge the saccharified sorghum juice at 8000 rpm for 15 minutes, filter, and adjust the sugar content to 10.

[0030] 1.2 Isolation and purification of actinomycetes

[0031] Potassium dichromate and nalidixic acid were added to the modified Gao's No. 1 medium as inhibitors to inhibit fungi and non-target bacteria respectively. The koji samples were heat-treated: the samples were placed in a 100°C oven for 1 hour, then added to a conical flask filled with sterile water and shaken at 37°C for 30 minutes. After treatment, the samples were diluted to 10 on a plate.-1 , 10 -2 , 10 -3 , 10 -4 The cells were spread in a gradient manner and cultured at 37°C for one week before observation.

[0032] Select colonies from the screening plate that exhibit the characteristics of actinomycetes: a rough, dry surface that adheres tightly to the culture medium and is difficult to lift; hyphae visible under a microscope, which are significantly thinner than those of molds. Streak these strains onto modified Gao's No. 1 medium until a single colony is obtained.

[0033] 1.3 Sorghum juice fermentation

[0034] Single colonies of the 15 actinomycetes identified in the initial screening were inoculated into Gao's No. 1 liquid medium and cultured at 37°C and 180 rpm for 48 hours to obtain seed liquid. One mL of seed liquid was then added to every 100 mL of sorghum juice medium and cultured at 37°C and 180 rpm for three days. Once a high bacterial concentration was observed, the culture was transferred to an incubator and allowed to ferment for four days. After fermentation, the supernatant was collected and analyzed for the relative content of volatile compounds using gas chromatography-mass spectrometry.

[0035] The results of gas chromatography-mass spectrometry showed that 2,3-butanediol was detected in the fermentation broth of the five actinomycetes strains, with relative contents as follows: 4.14% for B1620, 11.53% for B1622, 19.79% for B1624, 9.87% for B1627, and 16.36% for B1632, indicating that the above strains can produce 2,3-butanediol by fermenting sorghum.

[0036] 1.4 Fermentation of Light-Fragrance Liquor

[0037] Wash the sorghum three times, add enough water to cover the grain, and place it at 65°C for 24 hours. Drain the soaked grain and steam it in an autoclave at 115°C for 10 minutes. Remove it and soak it in hot water for 5 minutes. Drain it and steam it again in an autoclave at 111°C for 10 minutes before cooling it naturally.

[0038] Weigh 1kg of steamed grain onto a clean tray, spread it flat and let it cool. A control group added 1% homemade distiller's yeast (Rhizopus + Saccharomyces cerevisiae), while an experimental group added 1% of the aforementioned actinomycete seed solution. After thorough mixing, the mixture was placed in a sealed bag and opened in a 30°C incubator for saccharification and incubation for 24 hours. Next, the fermented mash from the workshop was sterilized at 121°C for 20 minutes. After cooling, 500g each of the saccharified and sterilized mash was weighed and mixed evenly (two replicates per sample). The mixture was then placed in a gas collection bag and anaerobic fermentation was carried out for 7 days.

[0039] 1.5 Fen-fragrance liquor distillation

[0040] Add approximately 500 mL of water to a distilled wine conical flask, then pour in the fermented mash. Insert the cork attached to the condenser into the conical flask containing the sample and heat it on an electric stove. Once distillation begins, collect 100 mL of the liquid in a graduated cylinder. Analyze the 2,3-butanediol content using liquid chromatography.

[0041] Test results showed that the addition of strains B1622, B1624, and B1632 significantly increased the 2,3-butanediol content in the light-flavor base liquor, reaching 12.10 mg / L, 19.22 mg / L, and 10.55 mg / L, respectively. The addition of strain B1624 had the most significant effect, with the 2,3-butanediol content increasing by 211.88% compared to the existing control group (6.16 mg / L), achieving the purpose of the present invention to increase the 2,3-butanediol content in base liquor.

[0042] 2 Identification of strain B1624

[0043] 2.1 Morphological observation of strain B1624

[0044] The strain B1624 was inoculated on the modified Gao's No. 1 solid medium and cultured in a constant temperature incubator at 37°C for 5 days. The colonies were observed to be nearly circular, with a dry and rough surface with annular patterns, a raised middle, and wavy edges. The aerial hyphae were white and the base hyphae were light yellow. Figure 1 shown.

[0045] 2.2 Molecular identification of strain B1624

[0046] Genomic DNA from the strain was extracted using a soil genomic DNA extraction kit. Using this DNA as a template, the ribosomal RNA subunit encoding gene (16S rDNA) was amplified using the universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification system consisted of 2 μL of template, 0.5 μL of 27F, 0.5 μL of 1492R, 15 μL of PremixTaq, and 12 μL of dd H2O. The PCR amplification procedure was as follows: 95°C for 3 min; 93°C for 60 s, 50°C for 60 s, and 72°C for 70 s; 30 cycles of amplification were performed; and 72°C for 10 min. The amplified product was sent to BGI for sequencing. Sequences obtained were compared with the National Center of Biotechnology Information (NCBI) website, confirming that strain B1624 was Streptomyces cocoae.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A strain of Streptomyces cacaoi B1624 with high yield of 2,3-butanediol, characterized in that: The strain was deposited in the China Center for Type Culture Collection on October 29, 2024, with the deposit number CCTCC M20242375.

2. Use of the Streptomyces cocoa described in claim 1 in increasing the 2,3-butanediol content of liquor.

3. The use according to claim 2, characterized in that The liquor is a light-fragrance liquor.

4. The use according to claim 2 or 3, characterized in that The microbial agent comprising Streptomyces cacaoi B1624 with the deposit number of CCTCC M 20242375 as claimed in claim 1 is mixed with distiller's yeast for fermentation and brewing.

5. A microbial agent, characterized in that: The invention comprises the Streptomyces cacaoi B1624 with the deposit number of CCTCC M 20242375 as described in claim 1.

6. The microbial agent according to claim 5, characterized in that The microbial agent is a solid agent or a liquid agent.

7. A light-fragrance liquor, characterized in that: It is brewed from the microbial agent described in claim 5.

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