Universal culture medium applicable to proliferation of lactic acid bacteria in wine brewing as well as preparation method and application of universal culture medium
By using lactic acid bacteria culture medium prepared by lees acid water and corn flour, the existing culture medium has been solved, and the effective proliferation of lactic acid bacteria and the protection of liquor flavor is achieved. It is suitable for large-scale Industrialized production.
Patent Information
- Application Number
- CN202411349357.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2024-12-27
AI Technical Summary
Existing lactic acid bacteria culture media such as MRS culture media are poorly separated in a slightly acidic environment, are susceptible to contamination of miscellaneous bacteria, are costly and have an impact on the flavor of liquor, and are difficult to meet the needs of large-scale industrial production.
Using lees acid water as the saccharification solvent and corn flour as the saccharification substrate. By enzymatic saccharification and sterilization, a general culture medium suitable for the proliferation of lactic acid bacteria in winemaking is prepared to ensure its natural and green Harmless and low cost.
The growth needs of lactic acid bacteria in Daquhejiu mash are met, and the culture effect is better than that of MRS culture medium. The number of lactic acid bacteria proliferation can reach 6.55 times that of traditional culture medium, and does not affect the flavor of liquor. It is suitable for large-scale industrial production.
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Figure CN119193385A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of liquor brewing, and particularly relates to a universal culture medium suitable for the proliferation of lactic acid bacteria in wine making, and a preparation method and application thereof. Technical Background
[0002] Lactic acid bacteria are commonly found in the liquor brewing process and are one of the important microorganisms in the liquor brewing system. Lactic acid bacteria produce a large amount of lactic acid during the liquor fermentation process, which can directly affect the acidity environment of the fermented grains, thereby affecting the evolution of the microbial community during the fermentation process, and plays an important role in maintaining the ecological environment of the brewing system. In addition, the lactic acid produced by its metabolism and the ethyl lactate produced after esterification are important flavor components of Luzhou-flavor liquor, which play a key role in improving the product flavor and enriching the product taste.
[0003] With the rise of emerging technologies such as synthetic microbiome and culturomics, it is possible to optimize the fermentation flora and achieve directional regulation of the flora in the production process, thereby improving product stability and quality. Therefore, a culture medium suitable for the cultivation and expansion of liquor microorganisms, with natural, green, harmless ingredients that will not affect liquor fermentation, plays an important role in both the construction of fermentation flora in the future and the application in the current fortified koji.
[0004] At present, the commonly used culture medium for lactic acid bacteria cultivation is MRS culture medium, which has the following problems: the pH is neutral, and the culture effect of lactic acid bacteria isolated from acidic environments such as some fermented grains and Daqu is not good, and it is easily contaminated by miscellaneous bacteria; the culture medium itself has some odor, and its application in liquor brewing will have a certain impact on the flavor of liquor; the cost is high, and it is not suitable for large-scale industrial production. Therefore, it is necessary to explore a culture medium that meets the conditions of natural and green ingredients, suitable for the proliferation of lactic acid bacteria in winemaking, low cost, and little impact on liquor brewing. Summary of the invention
[0005] The purpose of the present invention is to provide a universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking, a preparation method and application thereof, which is used for the proliferation of various lactic acid bacteria in the process of liquor brewing. The raw materials are green and will not bring foreign taste to the liquor brewing. The preparation cost is low and it is suitable for large-scale cultivation of lactic acid bacteria.
[0006] The technical solution of the present invention is: a universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking, which uses lees acid water as a saccharification solvent and corn flour as a saccharification substrate, is prepared by adding sterile inorganic salts in proportion after enzymatic saccharification and sterilization.
[0007] Among them, a method for preparing a universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking comprises the following steps: (1) preparing acid water; (2) preparing saccharification liquid; and (3) adding inorganic salts.
[0008] The method for preparing the acid water is as follows: distiller's grains and water are prepared in a mass ratio of 1:5, extracted with warm water at 40°C for 10 min, the pH range of the extract is 4.0-5.0, filtered, and the resulting aqueous solution is acid water for saccharification.
[0009] Wherein, the preparation of the saccharified liquid comprises the following steps: (1) Ingredients: corn flour and acid water are compounded in a mass ratio of 1:3, and steam is introduced into the mixture to heat it to boiling while stirring; (2) Liquefaction: After boiling, keep the material for 30 minutes, then stop heating, cool it to 95°C, add liquefaction enzyme with an activity unit of 40,000, and the amount of liquefaction enzyme added is 0.1% of the mass of the material after boiling. Stir evenly and keep warm for 10 minutes to liquefy the material; (3) Saccharification: After liquefaction, the material is cooled to 60-65°C, and saccharifying enzyme with an activity unit of 100,000 is added while stirring. The amount of saccharifying enzyme added is 0.2%-0.3% of the mass of the liquefied material. After saccharification for 3-4 hours, the saccharified material is obtained. (4) Inactivation: After saccharification, the supernatant is absorbed and sterilized under high temperature conditions to obtain a saccharification liquid with a sugar content of 13-14 °Bx; the inactivation conditions are: 118 °C, 35 min.
[0010] Wherein: the liquefaction enzyme is the high temperature α-amylase produced by Shandong Longyuan Bioengineering Co., Ltd.; the saccharifying enzyme is the glucoamylase produced by Jiangsu Boli Biological Products Co., Ltd.
[0011] Before strain culture, inorganic salts were added to the sterilized saccharification solution in proportion to obtain a universal culture medium with a sugar content of 11-12°Bx and a pH of 4.0-5.0 suitable for the proliferation of lactic acid bacteria in winemaking; the final concentration of inorganic salts in the culture medium was: K2HPO4, 2.0 g / L; C6H 14 N2O7, 2.0 g / L; CH3COONa, 5.0 g / L; MgSO4, 0.2 g / L; MnSO4·H2O, 0.04 g / L.
[0012] The inorganic salts are prepared by preparing the inorganic salts at a concentration 10 times the final concentration in the culture medium, sterilizing the mixture at 121°C for 20 min, and adding the mixture in proportion after cooling.
[0013] Wherein, the culture medium is used in culturing lactic acid bacteria, the lactic acid bacteria are lactic acid bacteria in the process of liquor brewing, and the lactic acid bacteria inoculation amount is 2% of the mass of the culture medium.
[0014] The beneficial effects of the present invention are as follows: 1. The culture medium provided by the present invention meets the growth requirements of lactic acid bacteria in Daqu and fermented grains, such as high-abundance lactic acid bacteria in the brewing process: Lactobacillus, Weissella, Pediococcus, and Leuconostoc.
[0015] 2. The culture medium provided by the present invention can cultivate lactic acid bacteria isolated from acidic environments such as fermented grains and Daqu, and the culture effect is better than that of conventional MRS culture medium. The number of lactic acid bacteria proliferation can reach up to 6.55 times the number of live bacteria cultured in traditional lactic acid bacteria culture medium MRS.
[0016] 3. The main component of the culture medium provided by the present invention is corn flour, which is natural, green and harmless. The prepared culture medium has a grain aroma and no foreign smell. It is used in liquor brewing without affecting the flavor of the liquor.
[0017] 4. The culture medium provided by the present invention has low production cost and is suitable for large-scale industrial production of lactic acid bacteria. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The difference in live bacteria counts between the culture medium of the present invention and the MRS culture medium after culturing common lactic acid bacteria in winemaking; Figure 2 This is the biological evolutionary tree of lactic acid bacteria in the winemaking process: Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus paracasei, Weissella sinusoids, and Leuconostoc citrinum. DETAILED DESCRIPTION
[0019] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.
[0020] Example 1: Wine lees and water were prepared in a mass ratio of 1:5, and extracted with warm water at 40°C for 10 min, and filtered to obtain an acid water extract with a pH of 4.0; corn flour and acid water extract were compounded in a mass ratio of 1:3, and steam was introduced under stirring to heat to boiling; after boiling, the material was kept for 30 min and then the heating was stopped, and when the temperature was cooled to 95°C, 0.1% of its mass of high-temperature α-amylase with an activity unit of 40,000 was added, and the material was kept warm for 10 min after stirring; when the liquefied material was cooled to 62°C, 0.25% of its mass of glucoamylase with an activity unit of 100,000 was added while stirring, and the saccharified material was obtained after saccharification for 3 h; the supernatant was absorbed from the saccharified material, and sterilized and enzyme-killed under high temperature conditions to obtain a saccharified liquid with a sugar content of 13°Bx; the inactivation conditions were: 118°C, 35 min; sterile water was added to the sterilized saccharification liquid in proportion to prepare a universal culture medium with a sugar content of 12 °Bx and a pH of 4.0 suitable for the proliferation of lactic acid bacteria in winemaking; wherein the sterile water was sterilized at 121 °C for 20 min and added in proportion after cooling.
[0021] Lactobacillus paracasei was inoculated into the prepared culture medium at an inoculum rate of 2% of the culture medium mass, and the viable bacteria were counted after culturing at 37 °C for 24 h.
[0022] Example 2: Wine lees and water were prepared in a mass ratio of 1:5, and extracted with warm water at 40°C for 10 min, and filtered to obtain an acid water extract with a pH of 4.0; corn flour and acid water extract were compounded in a mass ratio of 1:3, and steam was introduced under stirring to heat to boiling; after boiling, the material was kept for 30 min and then the heating was stopped, and when the temperature was cooled to 95°C, 0.1% high-temperature α-amylase with an activity unit of 40,000 was added, and the material was kept warm for 10 min after stirring; when the liquefied material was cooled to 62°C, 0.25% glucoamylase with an activity unit of 100,000 was added while stirring, and the saccharified material was obtained after saccharification for 3 h; the supernatant was absorbed from the saccharified material, and sterilized and enzyme-killed under high temperature conditions to obtain a saccharified liquid with a sugar content of 13°Bx; the inactivation conditions were: 118°C, 35 min; inorganic salts were added to the sterilized saccharification liquid in proportion to obtain a universal medium with a sugar content of 12 °Bx and a pH of 4.0 suitable for the proliferation of lactic acid bacteria in winemaking; wherein the final concentration of inorganic salts in the medium was: K2HPO4, 2.0 g / L; C6H 14 N2O7, 2.0 g / L; CH3COONa, 5.0 g / L; MgSO4, 0.2 g / L; MnSO4·H2O, 0.04 g / L; Among them, the inorganic salts were prepared at a concentration 10 times the final concentration in the culture medium, sterilized at 121 °C for 20 min, and added in proportion after cooling.
[0023] Lactobacillus paracasei was inoculated into the prepared culture medium at an inoculum rate of 2% of the culture medium mass, and the viable bacteria were counted after culturing at 37 °C for 24 h.
[0024] Example 3: Wine lees and water were prepared in a mass ratio of 1:5, and extracted with warm water at 40°C for 10 min, and filtered to obtain an acid water extract with a pH of 4.0; corn flour and acid water extract were compounded in a mass ratio of 1:3, and steam was introduced under stirring to heat to boiling; after boiling, the material was kept for 30 min and then the heating was stopped, and when the temperature was cooled to 95°C, 0.1% high-temperature α-amylase with an activity unit of 40,000 was added, and the material was kept warm for 10 min after stirring; when the liquefied material was cooled to 65°C, 0.2% glucoamylase with an activity unit of 100,000 was added while stirring, and the saccharified material was obtained after saccharification for 3 h; the supernatant was absorbed from the saccharified material, and sterilized and enzyme-killed under high temperature conditions to obtain a saccharified liquid with a sugar content of 12°Bx; the inactivation conditions were: 118°C, 35 min; inorganic salts were added to the sterilized saccharification liquid in proportion to obtain a universal medium with a sugar content of 11 °Bx and a pH of 4.0 suitable for the proliferation of lactic acid bacteria in winemaking; wherein the final concentration of inorganic salts in the medium was: K2HPO4, 2.0 g / L; C6H 14 N2O7, 2.0 g / L; CH3COONa, 5.0 g / L; MgSO4, 0.2 g / L; MnSO4·H2O, 0.04 g / L; Among them, the inorganic salts were prepared at a concentration 10 times the final concentration in the culture medium, sterilized at 121 °C for 20 min, and added in proportion after cooling.
[0025] Lactobacillus paracasei was inoculated into the prepared culture medium at an inoculum rate of 2% of the culture medium mass, and the viable bacteria were counted after culturing at 37 °C for 24 h.
[0026] Example 4: Wine lees and water were prepared in a mass ratio of 1:5, and extracted with warm water at 40°C for 10 min, and filtered to obtain an acid water extract with a pH of 4.0; corn flour and acid water extract were compounded in a mass ratio of 1:3, and steam was introduced under stirring to heat to boiling; after boiling, the material was kept for 30 min and then the heating was stopped, and when the temperature was cooled to 95°C, 0.1% high-temperature α-amylase with an activity unit of 40,000 was added, and the material was kept warm for 10 min after stirring; when the liquefied material was cooled to 60°C, 0.3% glucoamylase with an activity unit of 100,000 was added while stirring, and the saccharified material was obtained after saccharification for 3 h; the supernatant was absorbed from the saccharified material, and sterilized and enzyme-killed under high temperature conditions to obtain a saccharified liquid with a sugar content of 14°Bx; the inactivation conditions were: 118°C, 35 min; inorganic salts were added to the sterilized saccharification liquid in proportion to obtain a universal culture medium with a sugar content of 13 °Bx and a pH of 4.0 suitable for the proliferation of lactic acid bacteria in winemaking; wherein the final concentration of inorganic salts in the culture medium was: K2HPO4, 2.0 g / L; C6H 14 N2O7, 2.0 g / L; CH3COONa, 5.0 g / L; MgSO4, 0.2 g / L; MnSO4·H2O, 0.04 g / L; Among them, the inorganic salts were prepared at a concentration 10 times the final concentration in the culture medium, sterilized at 121 °C for 20 min, and added in proportion after cooling.
[0027] Lactobacillus paracasei was inoculated into the prepared culture medium at an inoculum rate of 2% of the culture medium mass, and the viable bacteria were counted after culturing at 37 °C for 24 h.
[0028] Example 5: Wine lees and water were prepared in a mass ratio of 1:5, and extracted with warm water at 40°C for 10 min, and filtered to obtain an acid water extract with a pH of 4.5; corn flour and acid water extract were compounded in a mass ratio of 1:3, and steam was introduced under stirring to heat to boiling; after boiling, the material was kept for 30 min and then the heating was stopped, and when the temperature was cooled to 95°C, 0.1% high-temperature α-amylase with an activity unit of 40,000 was added, and the material was kept warm for 10 min after stirring; when the liquefied material was cooled to 62°C, 0.25% glucoamylase with an activity unit of 100,000 was added while stirring, and the saccharified material was obtained after saccharification for 3 h; the supernatant was absorbed from the saccharified material, and sterilized and enzyme-killed under high temperature conditions to obtain a saccharified liquid with a sugar content of 13°Bx; the inactivation conditions were: 118°C, 35 min; inorganic salts were added to the sterilized saccharification liquid in proportion to obtain a universal culture medium with a sugar content of 12 °Bx and a pH of 4.5 suitable for the proliferation of lactic acid bacteria in winemaking; wherein the final concentration of inorganic salts in the culture medium was: K2HPO4, 2.0 g / L; C6H 14N2O7, 2.0 g / L; CH3COONa, 5.0 g / L; MgSO4, 0.2 g / L; MnSO4·H2O, 0.04 g / L; Among them, the inorganic salts were prepared at a concentration 10 times the final concentration in the culture medium, sterilized at 121 °C for 20 min, and added in proportion after cooling.
[0029] Lactobacillus paracasei was inoculated into the prepared culture medium at an inoculum rate of 2% of the culture medium mass, and the viable bacteria were counted after culturing at 37 °C for 24 h.
[0030] Example 6: Wine lees and water were prepared in a mass ratio of 1:5, and extracted with warm water at 40°C for 10 min, and filtered to obtain an acid water extract with a pH of 5.0; corn flour and acid water extract were compounded in a mass ratio of 1:3, and steam was introduced under stirring to heat to boiling; after boiling, the material was kept for 30 min and then the heating was stopped, and when the temperature was cooled to 95°C, 0.1% high-temperature α-amylase with an activity unit of 40,000 was added, and the material was kept warm for 10 min after stirring; when the liquefied material was cooled to 62°C, 0.25% glucoamylase with an activity unit of 100,000 was added while stirring, and the saccharified material was obtained after saccharification for 3 h; the supernatant was absorbed from the saccharified material, and sterilized and enzyme-killed under high temperature conditions to obtain a saccharified liquid with a sugar content of 13°Bx; the inactivation conditions were: 118°C, 35 min; inorganic salts were added to the sterilized saccharification liquid in proportion to obtain a universal medium with a sugar content of 12°Bx and a pH of 5.0 suitable for the proliferation of lactic acid bacteria in winemaking; wherein the final concentration of inorganic salts in the medium was: K2HPO4, 2.0 g / L; C6H 14 N2O7, 2.0 g / L; CH3COONa, 5.0 g / L; MgSO4, 0.2 g / L; MnSO4·H2O, 0.04 g / L; Among them, the inorganic salts were prepared at a concentration 10 times the final concentration in the culture medium, sterilized at 121°C for 20 min, and added in proportion after cooling.
[0031] Lactobacillus paracasei was inoculated into the prepared culture medium at an inoculum rate of 2% of the culture medium mass, and the viable bacteria were counted after culturing at 37 °C for 24 h.
[0032] Table 1 Effects of different culture medium components on the number of viable lactic acid bacteria The results show that the initial acid-water sugar solution culture medium after using Example 1 is rich in nutrients and can be used to culture lactic acid bacteria. Compared with the traditional MRS culture medium, the proliferation of lactic acid bacteria is slightly improved, which is 1.36 times that of the traditional MRS culture medium.
[0033] Example 2 Compared with Example 1, the lactic acid bacteria culture effect was better after adding inorganic salts to the initial acid water sugar liquid culture medium, and the viable bacteria count could reach 1.52×10 9 cfu / mL, which is 1.62 times that of Example 1 and 2.2 times that of the traditional MRS medium, indicating that the acid water sugar liquid medium with added inorganic salts has a significant synergistic effect on the proliferation of lactic acid bacteria.
[0034] Compared with Example 2, the main differences of Examples 3 and 4 are the different preparation parameters of saccharification liquid. The saccharification temperature is 60°C~65°C, and the saccharification enzyme dosage is 0.2%~0.3%. The saccharification efficiency is higher when the saccharification process parameters are closer to the optimal reaction conditions of the saccharification enzyme, and the sugar content of the prepared sugar liquid is higher. The sugar content of the culture medium prepared with sugar liquids of different sugar contents is also different, and the growth of lactic acid bacteria is greatly affected by the sugar content. When the sugar content of the culture medium is 12°Bx, the culture effect is better, which is 3.04 times and 1.58 times that of 11°Bx and 13°Bx culture media, respectively.
[0035] Compared with Example 2, Examples 5 and 6 mainly differ in the preparation of acid water. When the pH of the acid water is 4.0-5.0, the liquefaction and saccharification process of the material are realized, ensuring that the sugar content of the saccharifying enzyme meets the requirements. The pH of the acid water can directly affect the pH of the final culture medium. The growth of lactic acid bacteria is greatly affected by the pH of the culture medium. When the pH of the culture medium is 4.5, the culture effect is better, which is 2.97 times and 3.80 times of the culture medium with a pH of 4.0 and 5.0, respectively, and 6.55 times of the traditional MRS culture medium, and the proliferation effect on lactic acid bacteria is most significant.
[0036] Therefore, the culture effect of the lactic acid bacteria proliferation culture medium of the present invention is better than that of the existing culture medium, and the raw materials of the culture medium of the present invention are natural agricultural products, which are natural, green and harmless. The prepared culture medium has a grain aroma and no foreign smell, and is applied to liquor brewing without affecting the flavor of the liquor. At the same time, it has the characteristics of low production cost and can be suitable for large-scale industrial production of lactic acid bacteria.
[0037] The beneficial effects of the present invention are verified by the following experiments: Experiment 1: distiller's grains and water were prepared in a solid-liquid ratio of 1:5, and extracted with warm water at 40°C for 10 minutes. After filtering, an acid water extract with a pH of 4.5 was obtained; corn flour and acid water were compounded in a solid-liquid ratio of 1:3, and steam was introduced under stirring to heat to boiling; after boiling, the material was kept for 30 minutes and then the heating was stopped. When the temperature dropped to 95°C, 0.1% of its mass of high-temperature α-amylase with an activity unit of 40,000 was added, and the material was kept warm for 10 minutes after stirring evenly to liquefy the material; when the liquefied material was cooled to 62°C, 0.25% of its mass of glucoamylase with an activity unit of 100,000 was added while stirring, and the saccharified material was obtained after saccharification for 3-4 hours; the supernatant of the saccharified material was absorbed, and sterilized and enzyme-killed under high temperature conditions to obtain a saccharification liquid with a sugar content of 13°Bx; the inactivation conditions were: 118°C, 35 min; inorganic salts were added to the sterilized saccharification liquid in proportion to obtain a universal culture medium with a sugar content of 12 °Bx and a pH of 4.5 suitable for the proliferation of lactic acid bacteria in winemaking; wherein the final concentration of inorganic salts in the culture medium was: K2HPO4, 2.0 g / L; C6H 14 N2O7, 2.0 g / L; CH3COONa, 5.0 g / L; MgSO4, 0.2 g / L; MnSO4·H2O, 0.04 g / L; Among them, the inorganic salts were prepared at a concentration 10 times the final concentration in the culture medium, sterilized at 121 °C for 20 min, and added in proportion after cooling.
[0038] The prepared culture medium was inoculated with Pediococcus acidilactici at an inoculation rate of 2% of the culture medium mass. After culturing at 37 °C for 24 h, the live bacteria were counted and the number was 2.79 × 10 9 cfu / mL. With the same strain, the same inoculation volume and the same culture conditions, the number of viable bacteria cultured in MRS medium was 2.44×10 9 cfu / mL.
[0039] Experiment 2: The only difference between Experiment 2 and Experiment 1 is that the lactic acid bacteria inoculated is Pediococcus pentosaceus. The rest is the same as Experiment 2. The number of viable bacteria counted is 2.61×10 9 cfu / mL. With the same strain, the same inoculation volume and the same culture conditions, the number of viable bacteria in MRS medium was 2.03×10 9 cfu / mL.
[0040] Experiment 3: The only difference between Experiment 3 and Experiment 1 is that the lactic acid bacteria inoculated is Lactobacillus plantarum. The rest is the same as Experiment 2. The number of viable bacteria count is 7.00×10 9 cfu / mL. With the same strain, the same inoculation volume and the same culture conditions, the number of viable bacteria cultured in MRS medium was 2.77×10 9 cfu / mL.
[0041] Experiment 4: The only difference between Experiment 4 and Experiment 1 is that the lactic acid bacteria inoculated is Lactobacillus paracasei. The rest is the same as Experiment 2. The number of viable bacteria counted is 3.27×10 9 cfu / mL. With the same strain, the same inoculation volume and the same culture conditions, the number of viable bacteria cultured in MRS medium was 1.33×10 9 cfu / mL.
[0042] Experiment 5: The only difference between Experiment 5 and Experiment 1 is that the lactic acid bacteria inoculated is Weissella sinensis, and the rest is the same as Experiment 2. The number of viable bacteria counted is 8.36×10 9 cfu / mL. With the same strain, the same inoculation volume and the same culture conditions, the number of viable bacteria in MRS medium was 3.24×10 9 cfu / mL.
[0043] Experiment 6: The only difference between Experiment 6 and Experiment 1 is that the lactic acid bacteria inoculated is Leuconostoc citrinum. The rest is the same as Experiment 2. The number of viable bacteria counted is 1.96×10 9 cfu / mL. With the same strain, the same inoculation volume and the same culture conditions, the number of viable bacteria cultured in MRS medium was 1.22×10 9 cfu / mL.
[0044] from Figure 1 It can be seen that the culture medium of the present invention can ensure the normal growth of microorganisms when used to cultivate lactic acid bacteria in various winemaking processes, and the number of viable bacteria is greater than that of the common MRS culture medium. The present invention is superior to the currently commonly used culture medium in many aspects such as the source of culture medium components and characteristics, can be widely used in the cultivation of lactic acid bacteria in winemaking, and has the characteristics of low cost and being conducive to large-scale industrialized culture production.
Claims
1. A universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking, which is prepared by using lees acid water as saccharification solvent and corn flour as saccharification substrate, and then adding sterile inorganic salts in proportion after enzymatic saccharification and sterilization.
2. A method for preparing a universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking, characterized in that The preparation method of the universal culture medium comprises the following steps: (1) Preparation of acid water; (2) Preparation of saccharified liquid; (3) Addition of inorganic salts.
3. The method for preparing a universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking according to claim 2, characterized in that The method for preparing the acid water is as follows: distiller's grains and water are prepared in a mass ratio of 1:5, and leaching is performed with warm water at 40°C for 10 minutes, the pH range of the leaching solution is 4.0-5.0, and filtration is performed to obtain the aqueous solution as the acid water for saccharification.
4. The method for preparing a universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking according to claim 3, characterized in that The saccharification liquid preparation The following steps are involved: Ingredients: corn flour and acid water are compounded in a mass ratio of 1:3, and steam is introduced to heat to boiling while stirring; Liquefaction: After boiling, keep the material for 30 minutes and then stop heating. When the temperature drops to 95°C, add liquefaction enzyme with an activity unit of 40,000. The amount of liquefaction enzyme added is 0.1% of the mass of the material after boiling. After stirring evenly, keep warm for 10 minutes to liquefy the material. Saccharification: When the temperature of the liquefied material drops to 60-65°C, add saccharifying enzyme with an activity unit of 100,000 while stirring. The amount of saccharifying enzyme added is 0.2%-0.3% of the mass of the liquefied material. Keep the temperature for 3-4 hours to obtain the saccharified material. Inactivation: After saccharification, the supernatant is absorbed and sterilized and enzyme-killed under high temperature conditions to obtain a saccharification liquid with a sugar content of 13~14 °Bx; the inactivation conditions are: 118 ℃, 35 min.
5. The method for preparing a universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking according to claim 4, characterized in that: The liquefaction enzyme is a high-temperature α-amylase produced by Shandong Longyuan Bioengineering Co., Ltd.; the saccharifying enzyme is a glucoamylase produced by Jiangsu Boli Biological Products Co., Ltd.
6. The method for preparing a universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking according to claim 4, characterized in that: Before strain culture, inorganic salts were added to the sterilized saccharification liquid in proportion to prepare a universal culture medium with a sugar content of 11-12 °Bx and a pH of 4.0-5.0 suitable for the proliferation of lactic acid bacteria in winemaking; wherein the final concentrations of inorganic salts in the culture medium were: K2HPO4, 2.0 g / L; C6H14N2O7, 2.0 g / L; CH3COONa, 5.0 g / L; MgSO4, 0.2 g / L; and MnSO4·H2O, 0.04 g / L.
7. The method for preparing a universal culture medium suitable for the proliferation of lactic acid bacteria in winemaking according to claim 6, characterized in that The inorganic salts are prepared by preparing the inorganic salts at a concentration 10 times the final concentration in the culture medium, sterilizing the inorganic salts at 121° C. for 20 min, and adding the inorganic salts in proportion after cooling.
8. Use of the culture medium according to any one of claims 1 to 7 in culturing lactic acid bacteria, characterized in that: The lactic acid bacteria are lactic acid bacteria used in the liquor brewing process, and the lactic acid bacteria inoculation amount is 2% of the mass of the culture medium.