Lactobacillus hilgardii X920 and application thereof

Abstract

The invention relates to lactobacillus hilgardii X920 and application thereof, and belongs to the technical field of beneficial bacterium application, the lactobacillus hilgardii X920 is preserved in China Center for Type Culture Collection on August 21, 2024, the preservation number is CCTCC M 20241828, and the lactobacillus hilgardii X920 is obtained by separation and purification from fermented grains. According to the lactobacillus hilgardii disclosed by the invention, the content of gamma-aminobutyric acid in yellow rice wine is increased to 3.64 g / L The content of ethyl acetate in the fen-flavor white spirit is increased by 95.87%, the content of fusel oil is reduced by 30.26%, the content of acetaldehyde is reduced by 90.91%, and the brewed white spirit has the style characteristics of faint scent, purity, mellowness, fullness, softness, sweetness, cleanness and long aftertaste.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus hilgardii X920 and a use thereof. Background Art

[0002] Lactobacillus hilgardii grows well under anaerobic and facultative conditions. The colonies on MRS agar medium are small, white, round, moist, with a smooth surface, and short rod-shaped cells, either single or in pairs. It is Gram-positive and does not produce spores. Lactobacillus hilgardii was first discovered by Douglas and Cruise in high-alcohol wine in 1936 and named Lactobacillus hilgardii. Lactobacillus hilgardii can be used in the brewing of rice wine and the production of γ-aminobutyric acid in health foods in the food industry. γ-aminobutyric acid (GABA), also known as aminobutyric acid, is a natural amino acid composed of non-proteins. It is widely present in nature and is an important inhibitory neurotransmitter that has been studied in depth. It participates in a variety of metabolic activities and has the effects of lowering blood pressure, regulating arrhythmias, improving sleep, anti-anxiety, improving lipid metabolism, and preventing arteriosclerosis. It has attracted the attention of more and more scientific workers.

[0003] Therefore, Lactobacillus hilgri has become a research hotspot in the food and health products industry and has received increasing attention.

[0004] According to the current literature, different strains of Lactobacillus hilgeni have different probiotic functions. Chinese invention patent application 202310224047.5 reduces the abundance of bacterial species such as Oscillospira, Parasutterella, and Bacteroides, which are significantly increased in the intestines of patients with colitis, by gavaging mice with drinking water containing Lactobacillus hilgeni, and changes the composition of intestinal flora, which can inhibit the infiltration of colonic inflammatory cells and the expression levels of inflammatory factors such as IL-17, IL-23, and TNF-α, and inhibit intestinal inflammatory response. Chinese invention patent application 202010710659.1 produces antimicrobial peptides and organic acids in Lactobacillus hilgeni YH-1, which can be used in feed and breeding fields to promote animal growth and enhance animal immunity and disease resistance. At the same time, the enzyme prepared using the YH-1 strain has good stability. After being stored at room temperature for 6 months, the activity of the enzyme in the product still retains more than 90%. Chinese invention patent application 202310406411.X discloses that when Lactobacillus hilgeni GZ2 is used with glucose as the carbon source, the maximum yield of γ-aminobutyric acid generated is 7.19g / L, but the bacteria does not mention its application in the wine industry. Chinese invention patent application CN 103451134B discloses that by adding a Lactobacillus hilgeni for fermentation in rice wine brewing, the rice wine brewing production process becomes controllable and less affected by seasonality. At the same time, the operation is simple, which can save the rice soaking process and reduce the pollution of rice soaking water. The invented strain can be used in rice wine brewing, but there is no report on improving the function of GABA.

[0005] Although Lactobacillus hilgeni has a wide range of sources and rich genetic and functional diversity. However, the research on the isolation, identification, probiotic properties and metabolic mechanisms of Lactobacillus hilgeni is still relatively insufficient. For example, although Lactobacillus hilgeni is widely used in rice wine brewing, it contributes less to the GABA content in rice wine. It is possible that some strains of Lactobacillus hilgeni cannot metabolize GABA in the rice wine brewing environment; and there is still little research on Lactobacillus hilgeni in the field of liquor koji making and brewing, and there is a lack of Lactobacillus hilgeni with clear functions for use in liquor production. Therefore, it is necessary to further explore more Lactobacillus hilgeni with probiotic functions, and to deeply study its functions according to its different source characteristics, so that it can play an effective role in the brewing field and improve the flavor and active ingredient content in alcoholic products. Summary of the invention

[0006] In order to solve the above defects and shortcomings in the prior art, the present invention separates and purifies a Lactobacillu shilgardii X920 from fermented mash for 21 days, and the strain has multiple functions such as high yield of gamma-aminobutyric acid (GABA), increasing the content of gamma-aminobutyric acid (GABA) in rice wine, increasing ethyl acetate in light-fragrance liquor, reducing fusel oil, reducing acetaldehyde, etc., and has important potential application value.

[0007] To solve the above technical problems:

[0008] The first object of the present invention is to provide a Lactobacillu shilgardii X920 strain, which was deposited in the China Center for Type Culture Collection (CCTCC) on August 21, 2024, with the deposit address being Wuhan University, China, with the deposit number being CCTCC NO: M 20241828, and the classification name being: Lactobacillu shilgardii X920.

[0009] The Lactobacillushilgardii X920 of the present invention is separated from fermented mash for 21 days, and the bacteriological characteristics of the strain are as follows: the bacterial body is rod-shaped, 1.5-4 μm in length, 0.5-1.0 μm in width, without capsule and spore; the colony is white and opaque, the surface is smooth, and the colony edge is diffuse and petal-shaped; the physiological and biochemical characteristics are Gram-positive, peroxidase-negative, VP reaction-negative, gelatin and starch cannot be hydrolyzed, citrate cannot be utilized, glucose is metabolized to produce pyruvic acid, and glucose, sucrose and maltose can be metabolized to produce acid. It has acid resistance pH 3.0, tolerance to 15% ethanol, and tolerance to high temperature of 52°C.

[0010] The second purpose of the present invention is to provide an application of Lactobacillus hilgarnii X920 in increasing the yield of γ-aminobutyric acid in rice wine.

[0011] As a preferred embodiment, in the lower koji stage, 2.5% X920 functional bacterial liquid is added synchronously with the mother koji to carry out rice wine fermentation.

[0012] The third object of the present invention is to provide an application of Lactobacillus hilgari X920 in improving the quality of light-flavor liquor.

[0013] As a preferred embodiment, during the saccharification stage, 10% of X920 functional bacterial liquid is added synchronously with the mother koji to carry out the fermentation of light-fragrance liquor.

[0014] As a preferred embodiment, the fermentation method of the fermentation broth is as follows:

[0015] Lactobacillus hilarii X920 was activated in MRS culture medium, and the activated bacterial solution was inoculated into a triangular flask for fermentation culture for 72-96 hours; wherein, the culture temperature was 37°C, and the culture was statically cultured, and the components of the fermentation culture solution were: MRS culture medium as the basic culture medium, 20 g / L glucose and 50 g / L L-sodium glutamate were added.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention separates and purifies a Lactobacillus hilgardii from white wine mash fermented for 21 days. In yellow wine brewing, the content of functional component gamma-aminobutyric acid (GABA) can be greatly increased, so that the GABA content in the prepared yellow wine liquid reaches 3563.56 mg / L, which is much higher than the GABA content in commercially available yellow wine. For example, the gamma-aminobutyric acid content in 25 batches of commercially available yellow wine is greatly different, and the content range is 7.85-73.0 mg / L.

[0018] In the brewing of liquor, it can effectively increase the key ester substance ethyl acetate in the light-fragrance liquor by 95.93%; reduce the defective components fusel oil by 30.51% and acetaldehyde by 91.26%, and has important potential application value in improving the quality of liquor.

[0019] It can be seen that the newly isolated Lactobacillus hilarii X920 of the present invention can grow and reproduce in the liquor brewing process with a relatively complex fermentation environment (high acid, high alcohol, high temperature), and the tolerance experiment verifies that it has certain acid resistance, alcohol resistance, and high temperature resistance properties (see Figure 3 It provides valuable strain resources for the brewing industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a colony characteristic diagram of the Lactobacillus hilarii X920 strain of the present invention;

[0021] Figure 2 This is a Gram staining effect diagram of the Lactobacillus hilgardii X920 strain of the present invention (microscope 10×100);

[0022] Figure 3 The tolerance of Lactobacillus hilgris X920 of the present invention;

[0023] Figure 4 To study the key chromatographic indicators in the fermentation of fragrant liquor by adding different amounts of functional bacteria liquid. DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with specific embodiments and drawings. It should be understood that the embodiments are only used to illustrate the present invention rather than to limit the present invention, and any modifications, equivalent substitutions, etc. made on the basis of the present invention are within the protection scope of the present invention.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0026] Example 1: Screening of γ-aminobutyric acid-producing lactic acid bacteria

[0027] 1. Screening of lactic acid bacteria in fermented grains

[0028] Ingredients: White wine mash fermented for 21 days in the winery

[0029] Culture medium:

[0030] MRS medium: 10g protein Chen, 8g beef powder, 4g yeast powder, 20g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g diammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, pH 6.5, bottle and place in a 121℃ autoclave for sterilization for 30 minutes, cool and set aside.

[0031] Isolation and purification of lactic acid bacteria: 5 g of fermented grains were added to 95 mL of sterilized distilled water and placed in a shaker at 150 r / min for 30 min. Then, gradient dilution was performed and 200 μL of 10 -3 and 10 -4 The two gradient bacterial suspensions were added to the MRS culture medium for coating, placed in a 37°C incubator for culture observation, the bacterial colonies on the plate were observed and screened, and the colonies were picked for purification and culture for the next step of experimental screening. This time, three morphologically different lactobacilli strains, X920, X925 and X952, were screened.

[0032] 2. Screening of γ-aminobutyric acid-producing lactic acid bacteria

[0033] 2.1 Preliminary screening of γ-aminobutyric acid-producing strains

[0034] Culture medium:

[0035] MRS modified medium: 10g protein Chen, 8g beef powder, 4g yeast powder, 20g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g diammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g sodium L-glutamate, 10g calcium chloride. Bottle and sterilize in a 115℃ autoclave for 20 minutes, cool and set aside.

[0036] Experimental methods:

[0037] Quantitative analysis of γ-aminobutyric acid production activity: The three strains screened were inoculated into MRS modified medium respectively and cultured at 30°C for 3 days. The γ-aminobutyric acid content of lactic acid bacteria in the fermentation broth of MRS modified medium was quantitatively analyzed by liquid chromatography-mass spectrometry, and the strains with high content were selected for yield optimization.

[0038] Experimental results:

[0039] Liquid chromatography-mass spectrometry was used to quantitatively analyze the γ-aminobutyric acid content in the lactic acid bacteria fermentation broth. The results showed that all three lactic acid bacteria produced γ-aminobutyric acid, with the contents of X920, X925 and X952 being 1514.45 mg / L, 69.77 mg / L and 172.47 mg / L, respectively. Among them, the γ-aminobutyric acid content of X920 was relatively the highest.

[0040] 2.2 Optimization of GABA-producing strain conditions

[0041] 2.2.1 Optimization of culture conditions for γ-aminobutyric acid-producing strains

[0042] Experimental method: To explore the optimization experiment of GABA content in fermentation broth with fermentation time, temperature, culture state and other conditions, 4 groups were set up respectively: ①30℃, static culture; ②30℃, 160r / min culture; ③37℃, static culture; ④37℃, 160r / min culture. Samples were taken every day for testing, and samples were taken continuously for 4 days. The cultured fermentation broth was centrifuged to obtain the supernatant of the fermentation broth, and the GABA content in the fermentation broth was detected by liquid chromatography-mass spectrometry. The results are shown in Table 1 below:

[0043] Table 1 γ-aminobutyric acid content in X920 MRS improved fermentation medium

[0044]

[0045] As can be seen from Table 1, with the increase of culture time, the GABA content increases accordingly. The content increases significantly in the first three days, and the increase in the content decreases on the fourth day, so it is more appropriate to culture on the third day. The content in the static culture state is much higher than that in the shaking state, and the content in the 37℃ culture is slightly higher than that in the 30℃ culture. Therefore, under the optimized conditions of 37℃ and static culture for 3d, the GABA content produced by Lactobacillus hilarii X920 metabolism reaches 8700.84mg / L.

[0046] 2.2.2 Optimization of culture medium formula for γ-aminobutyric acid producing strains

[0047] Experimental method: Under the condition of MRS medium as the basic medium, the optimization experiment of adding glucose (0, 10, 20, 30, 40, 50 g / L) and L-glutamate sodium (25, 50, 75 g / L) was explored. The following 8 experimental groups were set up respectively, and the culture was statically cultured at 37°C for 3 days. The cultured fermentation broth was centrifuged to obtain the fermentation broth supernatant, and the GABA content in the fermentation broth was detected by liquid chromatography-mass spectrometry. The results are shown in Table 2 below:

[0048] Table 2 Optimization experiment of medium formula for producing γ-aminobutyric acid by strain X920 (g / L)

[0049]

[0050] From the results of Experiments 1, 2, and 5, it can be seen that at 20 g / L glucose, with the increase of L-glutamate sodium concentration, the γ-aminobutyric acid content first increased and then decreased, and the content was the highest at a concentration of 50 g / L, that is, the 5th group reached 41.88 g / L. From the results of Experiments 3, 4, 5, 6, 7, and 8, it can be seen that at 50 g / L L-glutamate sodium, with the increase of glucose concentration, the γ-aminobutyric acid content first increased and then decreased, and the content was the highest at a concentration of 20 g / L, which was also the 5th group. Based on the above analysis, when MRS medium was used as the basal medium, with the addition of 20 g / L glucose and 50 g / L L-glutamate sodium, the γ-aminobutyric acid content produced by Lactobacillus hilarii X920 was the highest at 41.88 g / L, and it had a good potential for metabolizing GABA.

[0051] Example 2 Identification of lactic acid bacteria strains

[0052] 1. 16S rDNA sequence analysis

[0053] (1) Lactobacillus hilarii X920 DNA extraction

[0054] Collect X920 bacteria and use DNA was extracted using a soil genomic DNA extraction kit. The specific method was referred to its instruction manual.

[0055] (2) Amplification of 16S rDNA fragment of Lactobacillus hilarii X920

[0056] The extracted DNA of Lactobacillus hilarii X920 was used as a template and the bacterial 16S rDNA universal primers 27F and 1492R were used to amplify the 16S rDNA fragment. The amplification reaction system was 30 μL, in which Premix Taq TM (TaKaRa Taq TMVersion2.0 plus dye) 15μL, ddH2O 13μL, primers 27F and 1492R 0.5μL each, DNA template 1μL. Amplification reaction conditions: 94℃5min; 94℃30s; 55℃50s, 72℃30s, 32 cycles; 72℃ extension 10min, 4℃ storage.

[0057] (3) 16S rDNA sequence analysis

[0058] The amplified 16S rDNA fragment was sent to Wuhan BGI for sequencing, and the corresponding 16S rDNA amplification sequence results were obtained (as shown in Table 3). The species information was obtained by comparison through the Blast search program of the National Center for Biotechnology Information (NCBI) (as shown in Table 3).

[0059] Table 3 Phylogenetic information of strains and their 16S rDNA amplification sequence results

[0060]

[0061]

[0062] 2. Identification of morphological and biological characteristics of Lactobacillus

[0063] The bacteria used in the following experiments were inoculated and activated using MRS solid medium and cultured at 37°C for 1-3 days before use in the following experiments:

[0064] (1) Microscopic observation of colonies and bacteria

[0065] Observe the colony morphology of lactobacillus on MRS medium under 37°C culture conditions; pick up a small amount of bacteria with an inoculation needle or a sterilized toothpick and place it on a slide, and observe the bacteria under a microscope (10×100).

[0066] Observation results are shown in Figure 1 :The bacteria are rod-shaped, 1.5-4μm in length, 0.5-1.0μm in width, without capsule and spores; the colonies are white, opaque, with a smooth surface, and the edges of the colonies are diffuse and petal-shaped.

[0067] (2) Gram staining

[0068] Take the newly activated bacteria and fix them on a glass slide for smearing, stain with ammonium oxalate crystal violet for 1 minute, rinse with purified water; add iodine solution to cover the surface and stain for 1 minute; then wash with water and absorb the water with absorbent paper; then add a few drops of 95% alcohol and shake gently to decolorize, wash with water after 30 seconds, absorb the water; finally stain with safranin stain for 10 seconds, rinse with purified water, and examine under a microscope after drying. Figure 2 and Table 4.

[0069] (3) Catalase test

[0070] Directly drip 3% hydrogen peroxide into the lactobacillus MRS liquid culture medium that has been cultured for 3 days, and observe immediately. Those with a large number of bubbles are positive (+), and those without bubbles are negative (-). The results are shown in Table 4.

[0071] (4) Methyl red test

[0072] Pick activated lactobacillus and inoculate it into universal culture medium, and culture it at 36±1℃ or 30℃ (30℃ is better) for 3-5 days. From the second day, take 1mL of culture solution every day, add 1-2 drops of methyl red indicator, positive (+) is bright red, weak positive is light red, and negative is yellow (-). The result can be determined until positive is found or it is still positive on the 5th day. The results are shown in Table 4.

[0073] (5) Starch hydrolysis experiment

[0074] Starch culture medium: soluble starch 10g; peptone 10g; glucose 5g; sodium chloride 5g; beef extract 5g; agar 20g; sterilize at 121℃ for 20min.

[0075] Inoculate the activated lactobacillus on a starch agar plate and culture at 36±1℃ for 24-48h. Then drip the iodine reagent directly on the surface of the culture medium plate. Check the results immediately. A positive reaction is a colorless transparent circle (+) around the colony or culture, and a negative reaction is no transparent circle (-). The results are shown in Table 4.

[0076] (6) Gelatin puncture experiment

[0077] The peptone gelatin medium was dispensed into test tubes and sterilized at 121°C for 20 min. The activated lactobacilli were inoculated and cultured at 25°C for 21 days. The growth of the strain and the liquefaction of gelatin were observed. The liquefaction of gelatin was positive (+), and the absence of liquefaction was negative (-). The results are shown in Table 4.

[0078] (7) Citrate Utilization Experiment

[0079] Culture medium: 1 g sodium citrate; 1 g dipotassium hydrogen phosphate; 0.2 g magnesium sulfate; 5 g sodium chloride; 1 g diammonium hydrogen phosphate; 20 g agar; pH = 6.8, sterilize at 121°C for 20 min, and set aside.

[0080] The activated lactobacillus was inoculated into the culture medium, and then cultured at 37°C for 48 hours. Three drops of bromothymol blue were added. If the culture medium changed from green to blue, it was positive (+); if it did not change color, it was negative (-). The results are shown in Table 4.

[0081] (8) VP experiment

[0082] The activated lactobacillus was inoculated into MRS medium and cultured at 37°C for 48h. 1mL of the culture solution was added with 1mL of O'Meara reagent (40% sodium hydroxide aqueous solution with 0.3% creatine), and after oscillation for 1-2min, it was placed in a 30°C incubator for 15min. The reaction solution was eosin-red, indicating that the experimental result was positive (+), otherwise it was negative (-). The results are shown in Table 4.

[0083] (9) Sugar fermentation experiment

[0084] Lactobacillus culture medium preparation: (NH4)2HPO4: 1g, MgSO4: 0.2g, KCl: 0.2g, yeast extract: 0.2g, sugar: 1% water washing agar: 5-6g, distilled water: 1000mL, bromocresol purple: 0.4% ethanol solution: 2mL, pH 6.8-7.0. Adjust pH first and then add indicator. The above culture medium is divided into test tubes, the culture medium height is about 4-5cm, sterilized at 115℃ for 20min, the color turns yellow for positive (+), and does not turn yellow for negative (-). Four kinds of sugars such as sucrose, glucose, maltose and lactose were determined in total. The results are shown in Table 4.

[0085] Table 4 Physiological and biochemical characteristics of functional bacteria X920

[0086]

[0087]

[0088] Note: “+” indicates positive, “+” indicates negative.

[0089] Example 3: Lactobacillus hilarii X920 tolerance experiment

[0090] Experimental methods:

[0091] Temperature tolerance: The strain was inoculated into MRS liquid culture medium at a 2% inoculation rate, placed in a constant temperature shaker at 30°C, 37°C, 42°C, 47°C, and 52°C, respectively, and cultured at 160 r / min for 24 h. The absorbance was measured at 600 nm.

[0092] pH tolerance: The strain was inoculated at an inoculum size of 2% with initial pH values ​​of 2.5, 3, 3.5, 4.0, and 4.5 in MRS medium at 37°C and shaken at 160 r / min for 24 h. The absorbance was measured at 600 nm.

[0093] Ethanol tolerance: The strain was inoculated at an inoculation dose of 2% into MRS liquid culture medium with ethanol contents of 0% vol, 3% vol, 6% vol, 9% vol, 12% vol, 15% vol, and 18% vol, respectively, and cultured at 37°C and 160 r / min with shaking for 24 h. The absorbance was measured at 600 nm.

[0094] The results are as follows Figure 3 As shown. Lactobacillus hilarii X920 can tolerate high temperatures of around 52°C; can tolerate acidity of around pH 2.5; can tolerate ethanol concentrations below 18%. The above tolerances show that Lactobacillus hilarii X920 has good physiological and biochemical characteristics of temperature resistance, acid resistance and ethanol resistance, and can grow well in a winemaking environment.

[0095] Example 4: Laboratory test of rice wine with γ-aminobutyric acid producing strains

[0096] Experimental methods:

[0097] Soak rice: Wash the glutinous rice and soak it in cold water for 24 hours. Drain the water and divide it into fermentation glass bottles.

[0098] Steamed rice: Place the glass bottle containing rice in a sterilizer and steam at 121℃ for 15 minutes until the glutinous rice is hard on the outside and soft on the inside, without any center, and is loose and not mushy, and cooked evenly.

[0099] Spread out to cool: After the steamed glutinous rice is out of the pot, turn it over and cool it to 20-30℃. Mix the glutinous rice with 1:1.2 boiled water to prevent it from sticking together and affecting fermentation.

[0100] Add koji: Inoculate osmanthus koji at an addition rate of 1%, and inoculate 0, 0.5%, 2.5%, and 5% functional bacteria fermentation liquid respectively, and stir well. Select Lactobacillus hilgeni X925 and functional bacteria X920 screened from the fermented grains for control experiments, and prepare functional bacterial liquids respectively. The preparation method is as follows: activate Lactobacillus hilgeni X920 and X925 in MRS culture medium respectively, and inoculate the activated bacterial liquid into a triangular flask for fermentation and culture for 3 days; wherein, the culture temperature is 37°C, and the culture is statically cultured. The components of the fermentation culture liquid are:

[0101] MRS medium was used as the basal medium, supplemented with 20 g / L glucose and 50 g / L sodium L-glutamate.

[0102] Fermentation: Cover the lid and place in a 30℃ constant temperature incubator for 14 days until the fermentation liquid changes from transparent to off-white and then to light yellow, and from turbid to clear.

[0103] Filtration: After fermentation, squeeze and filter with 300-mesh gauze to obtain filtered fermentation liquid.

[0104] Steaming: Steam the filtered fermentation liquid to precipitate impurities, and then cool it to make rice wine.

[0105] The rice wine sample was sent to the testing center for γ-aminobutyric acid index determination. The results are shown in Table 5. The γ-aminobutyric acid content in the rice wine fermentation sample added with the functional bacteria X920 bacterial solution increased significantly. With the addition of the functional bacterial solution, the γ-aminobutyric acid content first increased and then decreased. At an addition amount of 2.5%, the content reached a maximum of 3563.56 mg / L. It can be seen that the functional bacteria X920 can grow in the rice wine fermentation environment and metabolize to produce γ-aminobutyric acid, and the content is much higher than that of commercially available rice wine. The bacteria X925 was also identified as Lactobacillus hilgardii, and was added to the rice wine brewing process in different proportions, but it had no obvious effect on the GABA content of the rice wine.

[0106] Table 5 γ-aminobutyric acid content in rice wine fermented with different amounts of functional bacteria

[0107]

[0108]

[0109] Example 5: Brewing experiment to improve liquor quality

[0110] Experimental methods:

[0111] (1) Lower koji: Osmanthus koji, a common koji used in brewing, was used as the mother koji, and 0%, 2.5%, 5%, and 10% of Lactobacillus hilgensis X920 and X925 bacterial liquid were added separately for laboratory test effect verification. Preparation method of bacterial liquid: The activated bacterial liquid was inoculated into a triangular flask for fermentation and culture for 3 days; wherein, MRS medium was used, the culture temperature was 37°C, and the culture was static.

[0112] (2) Soaking grains: According to the amount of 500 g of Australian sorghum for each sample, weigh the required grains for the experiment, add 60°C warm water (the water level is one finger higher than the grain surface), and soak the grains in a 60°C oven for 24 hours.

[0113] (3) Steaming the grain: First, put the soaked grain into a sterilizing steamer for initial steaming at 121°C for 30 minutes. After the initial steaming, add 100°C water to steam the grain for 40 minutes. After filtering the water, put the grain into a sterilizing steamer for re-steaming at 115°C for 30 minutes.

[0114] (4) Mixing grain and koji: Cool the re-steamed grain to 30°C, divide it into No. 12 ziplock bags, 1040g in each ziplock bag, and mix it thoroughly with the koji for the experiment.

[0115] (5) Saccharification: Place the mixed koji in an incubator at 30°C for saccharification for about 24 hours. Note that the seal of the ziplock bag must be kept open and the opening must be sealed with a wet towel to ensure the supply of oxygen and water required for the saccharification process.

[0116] (6) Wine lees sterilization: Weigh 500 g of wine lees for the experiment according to the amount of a single sample, put it into a sterilization steamer, and sterilize it at 121°C for 30 min.

[0117] (7) Fermentation in a tank: Mix the saccharified grains with sterilized distiller's grains in a weight ratio of 1:1, put them into a gas sampling bag, seal it, evacuate it, and put it into an incubator at 30°C for fermentation for 7 days.

[0118] (8) Distillation: Add 500 mL of distilled water to the bottom of a special conical flask, put the fermented mash into the special conical flask for open flame distillation, take the first 100 mL of distilled wine sample and send it to the testing center for determination of wine-related gas phase index parameters. The results are shown in Table 6 and Figure 4 shown.

[0119] The gas phase data of the small test samples showed that in the experimental group, the key flavor substance ethyl acetate of the light-flavor liquor was significantly increased, the content of the defective component higher alcohol that causes the liquor to get drunk was significantly reduced, and the defective component acetaldehyde was significantly reduced. Among them, the group adding 10% X920 had the best comprehensive effect, increasing ethyl acetate by 95.87%, reducing fusel oil by 30.26%, and reducing acetaldehyde by 90.91% (see Figure 4 ). It can significantly improve the chromatographic index of light-flavor liquor and improve the liquor quality. However, adding different proportions of Lactobacillus hilgardii X925 has no significant effect on the ester removal and impurity reduction of light-flavor liquor. The sensory evaluation results of the liquor samples of 10% X920+Osmanthus Qu group and Osmanthus Qu group showed that 10% X920+Osmanthus Qu group was a superior liquor, showing the style characteristics of pure fragrance, mellow and full, sweet and refreshing, and long aftertaste.

[0120] Table 6 Key chromatographic indicators of fragrant liquor fermented with different addition amounts of functional bacteria (g / L)

[0121]

[0122]

[0123] Table 7 Sensory evaluation results of fragrant liquor fermented with functional bacteria X920

[0124]

Claims

1. A Lactobacillus hilgardii X920, characterized in that: The Lactobacillus shilgardii X920 was deposited in the China Center for Type Culture Collection on August 21, 2024, with a deposit number of CCTCC M 20241828 and a classification name: Lactobacillus shilgardii X920.

2. Use of the Lactobacillus hilarii described in claim 1 in increasing the content of γ-aminobutyric acid in rice wine.

3. The use according to claim 1, characterized in that: The fermentation method is as follows: In the lower koji stage, 2.5% X920 functional bacterial liquid is added simultaneously with the mother koji to carry out rice wine fermentation.

4. Use of the Lactobacillus hilarii described in claim 1 in increasing the content of ethyl acetate, reducing fusel oil and reducing the content of acetaldehyde in light-flavor liquor.

5. The use according to claim 4, characterized in that: The fermentation method is as follows: During the saccharification stage, 10% of X920 functional bacterial liquid is added simultaneously with the mother koji to ferment the light-fragrance liquor.

6. The use according to claim 3 or 5, characterized in that: The fermentation method of the X920 functional bacterial liquid is as follows: Lactobacillus hilarii X920 was activated in MRS culture medium, and the activated bacterial solution was inoculated into a triangular flask for fermentation culture for 72-96 hours; wherein, the culture temperature was 37°C, and the culture was statically cultured, and the components of the fermentation culture solution were: MRS culture medium as the basic culture medium, 20 g / L glucose and 50 g / L L-sodium glutamate were added.

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