Application of acid-resistant lactobacillus paracidophilus in production of Maotai-flavor liquor

Abstract

The invention discloses an application of acid-resistant lactobacillus paracilli FBKL 1.3455 in production of Maotai-flavor liquor. The acid-resistant lactobacillus paracilli has tolerance to a high-acid environment and can still normally grow and metabolize in the middle and later periods of fermentation of the Maotai-flavor liquor, and the acid-resistant lactobacillus paracilli FBKL 1.3455 has the advantages that the acid-resistant lactobacillus paracilli FBKL 1.3455 can be used for preparing acid-resistant lactobacillus paracilli FBKL 1.3455; the strain provides sufficient raw materials for esterification reaction in the long-term aging process, is beneficial for realizing benign acid ester dynamic balance in the aging process of a wine body, and avoids hydrolysis caused by insufficient acidity, so that the long-term flavor stability and high quality of finished wine are guaranteed when the strain is applied to white wine production as a strengthening microbial agent, and the production efficiency is improved. In a high-temperature accumulation or cellar-entering fermentation stage, the acid-resistant capacity of the acid-resistant wine enables the acid-resistant wine to continuously utilize substrates such as saccharides in fermented grains to generate a large amount of lactic acid and other organic acids, so that the total ester content of final base wine is increased, and the wine body is fuller, mellower and more harmonious.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of an acid-resistant Lactobacillus parasiticus in the production of Maotai-flavor liquor. Background Technology

[0002] Maotai-flavor baijiu is a typical representative of traditional Chinese distilled baijiu, renowned for its prominent Maotai aroma, elegant and delicate flavor, mellow body, and long aftertaste. Its complex flavor system originates from a unique "four highs and two longs" production process: high-temperature koji making, high-temperature stacking, high-temperature fermentation, high-temperature distillation, long production cycle, and long storage time. In this process, the microbial community, especially lactic acid bacteria, plays a crucial role. Through activities such as sugar metabolism and protein decomposition, they produce abundant organic acids, esters, and alcohols, making them one of the core contributors to the unique flavor structure of Maotai-flavor baijiu. However, the production environment of Maotai-flavor baijiu, especially the fermentation stage, presents an extremely harsh challenge to microorganisms. During fermentation in the cellar, the acidity of the mash increases significantly as fermentation progresses, accompanied by high concentrations of ethanol and an anaerobic environment. These extreme conditions of high acidity and high alcohol inhibit the growth and metabolic activity of most microorganisms, leading to a sharp decline in microbial community diversity and insufficient fermentation dynamics in the later stages of fermentation. This directly affects the efficiency of the key flavor-forming pathways of acid production and esterification, potentially leading to insufficient flavor richness, lack of body, and even affecting the stability and harmony of the liquor due to an imbalance in the acid-ester ratio. Currently, the industry typically attempts to improve fermentation by optimizing process parameters or adding exogenous agents, but these methods often suffer from a lack of specificity, unstable effects, or the potential introduction of off-flavors. Therefore, the targeted screening, isolation, and utilization of native functional microorganisms with extreme environmental tolerance within the unique micro-ecological environment of the Maotai-flavor liquor brewing system has become an innovative breakthrough for fundamentally improving the quality of Baijiu. Lactic acid bacteria, as the main acid-producing bacteria in Baijiu brewing, depend on their acid tolerance to survive and continue to function in the later stages of fermentation. A lactic acid bacterium capable of maintaining vigorous vitality in a high-acid environment holds promise as a key strain for strengthening the fermentation process of Maotai-flavor Baijiu and optimizing its flavor composition, thus possessing significant value for further exploring the application of lactic acid bacteria resources in Baijiu production. Summary of the Invention

[0003] The purpose of this invention is to provide an acid-resistant *Lactobacillus paralix*. (Lactobacillus acetotolerans) Application of FBKL1.3455 in the production of Maotai-flavor liquor.

[0004] Acid-resistant lactobacillus (Lactobacillus acetotolerans) The separation method for FBKL1.3455 includes the following steps: (a) For acid-resistant lactobacillus paralepticus (Lactobacillus acetotolerans) Separation of FBKL1.3455: 1 kg of production lees was taken from Xiuwen County, a major liquor-producing area in Guizhou Province. 10 g of the lees was added to a 90 ml Erlenmeyer flask containing 2.5 g / L sterile sodium carboxymethyl cellulose and glass beads. The mixture was shaken at 37°C for 30 min to mix. 1 mL of the bacterial suspension was then injected into a test tube containing 9 mL of 2.5 g / L sterile sodium carboxymethyl cellulose for serial dilution. 10 g of the solution was then taken from the flask. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Gradients were poured and spread onto plates, incubated upside down at 37°C for 2 days, and then counted. Single colonies with typical lactic acid bacteria morphology were selected, inoculated, and streaked onto MRS agar medium. This process was repeated 3-4 times until pure cultures were obtained. Each strain was then numbered, and finally, the cultures were streaked on slant plates and stored at 4°C.

[0005] (II) Training: Acid-resistant lactobacillus (Lactobacillus acetotolerans) The culture medium used for FBKL1.3455: MRS liquid culture medium, peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L. Adjust the initial pH to 6.2-6.5, and sterilize at 115℃ for 20 min. MRS solid medium: Based on the above MRS liquid medium, add 15.0-20.0 g / L of agar powder.

[0006] (III) Preservation and subculturing of strains: 1. Culture medium for preserving microbial strains: MRS solid medium: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L, agar powder 20.0 g / L. Adjust the initial pH to 6.2-6.5 and sterilize at 115℃ for 20 min. 2. There are two methods for saving: (1) Subculture method for preserving lactic acid bacteria: Inoculate lactic acid bacteria onto MRS agar slant medium, incubate at 37°C for 2 days, seal the rubber stopper with sterile solid paraffin, and store in a 4°C refrigerator. Generally, lactic acid bacteria can be preserved in a 4°C refrigerator for 3 months, and need to be transplanted periodically. (2) Glycerol tube cryopreservation method: Inoculate lactic acid bacteria onto MRS agar plates and incubate at 37°C for 2 days. Then inoculate onto liquid fermentation medium and incubate at 37°C and 150 r / min for 2 days. Prepare 25% glycerol and autoclave at 121°C for 20 min. When using, mix 25% glycerol and bacterial solution at a ratio of 1:1. Inoculate 0.9 ml of the cultured lactic acid bacteria solution and 0.9 ml of 25% glycerol into a 2 ml cryopreservation tube. Then quickly place the cryopreservation tube into an ultra-low temperature freezer at -80°C for storage.

[0007] (iv) Ecological characteristics of the strain: The colonies formed by this bacterium on MRS solid medium were incubated at 37°C for 2 days. Single colonies were round, small, with a diameter of 2.1 mm–2.4 mm, transparent and flat, with a pubescent outer ring, and easily picked up. Microscopic examination revealed short rod-shaped colonies. Gram staining showed a purple color, indicating it was a Gram-positive bacterium with a diameter of 1.87–2.61 µm. Scanning electron microscopy revealed slender rod-shaped colonies.

[0008] (v) Culture characteristics of the strain (1) The cultivation temperature is 30℃~37℃, and the optimal temperature is 35℃~37℃; (2) The culture pH is 2.8 to 7.5, with the optimal range being 3.0 to 5.2.

[0009] According to Bergey's Manual of Bacterial Identification, the sample was screened, isolated, purified, examined under a microscope, and subjected to physiological and biochemical analysis. ITS sequencing was then performed, and the obtained sequences were reverse-engineered using DNAMAN software, compared with BLAST data in NCBI, and a phylogenetic tree of the isolated strain was constructed using MEGA5 software. This confirmed that the isolated strain was *Pseudomonas aeruginosa*. (Lactobacillus acetotolerans) FBKL1.3455, taxonomically belongs to: Bacteria, Firmicutes, Bacilli, Lactobacillales, Lactobacillaceae, and Paralactobacillus.

[0010] (vi) Verification of the acid resistance characteristics of the strain This invention relates to a type of Lactobacillus parasiticus. (Lactobacillus acetotolerans) Validation of acid resistance of FBKL1.3455. The acid-resistant strain of *Pseudomonas paralepticus* obtained through isolation and screening was used.(Lactobacillus acetotolerans) FBKL1.3455 was inoculated into MRS liquid medium and anaerobically cultured at 37°C for 2 days to serve as a seed culture. Then, an inoculum of 2% v / v seed culture was transferred to MRS liquid medium adjusted to different pH values ​​using lactic acid: pH 2.0, pH 3.0, pH 3.5, pH 4.0, and pH 4.5. These were then anaerobically cultured at 37°C, and the absorbance at 600 nm was periodically measured to monitor bacterial growth. The results demonstrated that this strain could still grow significantly in highly acidic MRS medium at pH 3.5 and below, exhibiting excellent acid tolerance, far exceeding that of ordinary lactic acid bacteria.

[0011] The most significant characteristic of the acid-resistant *Pseudomonas paragallinarum* strain screened in this invention is its exceptional tolerance to high-acid environments. This means that it can still grow and metabolize normally during the mid-to-late stages of fermentation in Maotai-flavor liquor, even when the activity of ordinary microorganisms is inhibited due to acid accumulation. When this strain is used as a fortifying agent in liquor production, especially during high-temperature stacking or cellar fermentation, its strong acid resistance allows it to continuously utilize substrates such as sugars in the mash to produce large amounts of lactic acid and other organic acids. These organic acids are not only important components of the liquor's structure but also precursors for subsequent esterification reactions that produce key flavor esters such as ethyl acetate and ethyl lactate. By increasing acid production in the later stages of fermentation, the generation of esters can be effectively promoted, thereby increasing the total ester content of the final base liquor, resulting in a fuller, more mellow, and harmonious liquor.

[0012] Lactic acid and its derivative, ethyl lactate, are important components of the flavor profile of Maotai-flavor baijiu, directly contributing to its elegant and delicate style. The introduction of the strains in this invention can directionally and stably increase the proportion of lactic acid and ethyl lactate in the baijiu, helping to form a more typical and prominent Maotai-flavor style, making the product's flavor profile clearer and purer. During the storage of baijiu, ester-ester balance is crucial for ensuring the stability of the baijiu.

[0013] This invention increases the organic acid reserves in the base liquor, providing ample raw materials for esterification reactions during long-term aging. This facilitates a healthy dynamic balance of acid and esters in the liquor during aging, avoiding hydrolysis caused by insufficient acidity, such as the risk of ester decomposition, thus ensuring the long-term flavor stability and high quality of the finished liquor. This invention is the first to introduce a type of acid-resistant material derived from the Maotai-flavor liquor brewing system. (Lactobacillus acetotolerans) The strains provide a new technical path and microbial resources for improving the traditional liquor industry through microbial engineering technology, and are of great value in promoting the technological progress and quality upgrading of the liquor industry.

[0014] This invention not only provides a new strain of microorganisms with significant application potential, but also has broad market application prospects. Attached Figure Description

[0015] Appendix Figure 1 The colony morphology of FBKL1.3455 is as follows; Figure 2 Features of an optical microscope at 100x; Figure 3 Features of a 2000x scanning electron microscope. Detailed Implementation

[0016] A type of acid-resistant lactobacillus (Lactobacillus acetotolerans) The separation method for FBKL1.3455 includes the following steps: (a) For acid-resistant lactobacillus paralepticus (Lactobacillus acetotolerans) Separation of FBKL1.3455: 1 kg of production lees was taken from Xiuwen County, a major liquor-producing area in Guizhou Province. 10 g of the lees was added to a 90 ml Erlenmeyer flask containing 2.5 g / L sterile sodium carboxymethyl cellulose and glass beads. The mixture was shaken at 37°C for 30 min to mix. 1 mL of the bacterial suspension was then injected into a test tube containing 9 mL of 2.5 g / L sterile sodium carboxymethyl cellulose for serial dilution. 10 g of the solution was then taken from the flask. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Gradients were poured and spread onto plates, incubated upside down at 37°C for 2 days, and then counted. Single colonies with typical lactic acid bacteria morphology were selected, inoculated, and streaked onto MRS agar medium. This process was repeated 3-4 times until pure cultures were obtained. Each strain was then numbered, and finally, the cultures were streaked on slant plates and stored at 4°C.

[0017] (II) Training: Acid-resistant lactobacillus (Lactobacillus acetotolerans) The culture medium used for FBKL1.3455: MRS liquid culture medium, peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L, adjust the initial pH to 6.2-6.5, and sterilize at 115℃ for 20 min.

[0018] MRS solid medium: Based on the above MRS liquid medium, add 15.0-20.0 g / L of agar powder.

[0019] (III) Preservation and subculturing of strains: 1. Culture medium for preserving microbial strains: MRS solid medium: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L, agar powder 20.0 g / L. Adjust the initial pH to 6.2-6.5 and sterilize at 115℃ for 20 min.

[0020] 2. There are two methods for saving: (1) Subculture method for preserving lactic acid bacteria: Inoculate lactic acid bacteria onto MRS agar slant medium, incubate at 37°C for 2 days, seal the rubber stopper with sterile solid paraffin, and store in a 4°C refrigerator. Generally, lactic acid bacteria can be preserved in a 4°C refrigerator for 3 months and need to be transplanted periodically.

[0021] (2) Glycerol tube cryopreservation method: Inoculate lactic acid bacteria onto MRS agar plates and incubate at 37°C for 2 days. Then inoculate onto liquid fermentation medium and incubate at 37°C and 150 r / min for 2 days. Prepare 25% glycerol and autoclave at 121°C for 20 min. When using, mix 25% glycerol and bacterial solution at a ratio of 1:1. Inoculate 0.9 ml of the cultured lactic acid bacteria solution and 0.9 ml of 25% glycerol into a 2 ml cryopreservation tube. Then quickly place the cryopreservation tube into an ultra-low temperature freezer at -80°C for storage.

[0022] (iv) Ecological characteristics of the strain: The colonies formed by this bacterium were cultured on MRS solid medium at 37°C for 2 days. The single colonies were round, small, with a diameter of 2.1 mm-2.4 mm, transparent and flat, with villi on the outer ring, and easy to pick up. Under a microscope, they appeared as short rods. Gram staining showed purple, indicating that they were Gram-positive bacteria with a diameter of 1.87-2.61 µm. Under a scanning electron microscope, they appeared as slender rods.

[0023] (v) Culture characteristics of the strain (1) The cultivation temperature is 30℃~37℃, and the optimal temperature is 35℃~37℃; (2) The culture pH is 2.8 to 7.5, with the optimal range being 3.0 to 5.2.

[0024] (vi) Verification of the acid resistance characteristics of the strain This invention relates to an acid-resistant *Lactobacillus paragallinarum*. (Lactobacillus acetotolerans) Validation of the acid resistance of FBKL1.3455: The pure strain of acid-resistant *Lactobacillus paragallinarum* obtained through isolation and screening was tested. (Lactobacillus acetotolerans)FBKL1.3455 was inoculated into MRS liquid medium and anaerobically cultured at 37°C for 2 days as a seed culture. Then, an inoculum of 2% v / v seed culture was transferred to MRS liquid medium adjusted to different pH values ​​using lactic acid: pH 2.0, pH 2.5, pH 3.0, pH 3.5, pH 4.0, and pH 4.5. Anaerobic culture was carried out at 37°C, and viable cell counts were periodically measured to monitor bacterial growth. The results demonstrated that this strain could still grow significantly in highly acidic MRS medium at pH 3.5 and below, exhibiting its superior acid tolerance, far exceeding that of ordinary lactic acid bacteria. .

Claims

1. Application of an acid-resistant paraleptic bacterium (Paralactobacillus acetotolerans) FBKL1.3455 in the production of Maotai-flavor liquor.

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