Lactobacillus fermentation medium and preparation method thereof

Abstract

The invention relates to a lactobacillus fermentation culture medium and a preparation method thereof, and belongs to the technical field of lactobacillus fermentation. The invention provides a lactobacillus fermentation culture medium which comprises the following raw materials in percentage by weight: 95-99.75 wt% of hydrolyzed wheat germ extracting solution and the balance of carbon source, the hydrolyzed wheat germ extracting solution is used for replacing traditional peptone, yeast powder and the like for fermentation, the lactobacillus fermentation culture medium is light in smell and free of peculiar smell, and the fermentation filtrate is high in fermentation efficiency and low in cost. After simple sterilization and clarification, the product can be put into a product for use; in addition, the lactic acid bacteria fermentation medium does not influence the growth of lactic acid bacteria and the expression of protein, the operation is simple and easy to implement, and the cost is lower.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid bacteria fermentation, and relates to a lactic acid bacteria fermentation medium and a preparation method thereof. Background Art

[0002] Lactic acid bacteria are a type of bacteria that can ferment sugars to produce lactic acid, mainly including Lactobacillus, Streptococcus, Enterococcus, etc., and are widely present in fermented foods, the intestine, and the natural environment.

[0003] Since lactic acid bacteria are also probiotics, the filtrate of their fermentation products itself contains components that are very beneficial to the human body, so it is widely used in cosmetics or foods. However, the conventional fermentation media in the prior art mainly use culture medium components with strong odors such as peptone and yeast powder as nitrogen sources, which are likely to produce peculiar smells or the odors of peptone and yeast powder will remain after fermentation. This will not only affect the smell of the product, but also requires removing the peculiar smell generated by the fermentation filtrate and purifying it before it can be put into use in the product, undoubtedly increasing the cost. Summary of the Invention

[0004] The purpose of the present invention is to provide a lactic acid bacteria fermentation medium and a preparation method thereof, which use hydrolyzed wheat germ extract instead of traditional peptone, yeast powder, etc. for fermentation. It has a light smell, no peculiar smell, and the fermentation filtrate can be put into the product after simple sterilization and clarification; in addition, the lactic acid bacteria fermentation medium of the present invention does not affect the growth of lactic acid bacteria and the expression of proteins, and is simple and easy to implement, with lower costs.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] A lactic acid bacteria fermentation medium, the raw materials of the fermentation medium are calculated by weight percentage, including 95 - 99.75 wt% of hydrolyzed wheat germ extract and the balance of carbon source.

[0007] Further, the preparation method of the hydrolyzed wheat germ extract comprises the following steps:

[0008] Step X1: Mix wheat germ and deionized water evenly, then add protease for treatment and inactivate to obtain hydrolyzed wheat germ crude liquid;

[0009] Step X2: Triple-filter the hydrolyzed wheat germ crude liquid to obtain hydrolyzed wheat germ extract.

[0010] Further, the raw materials of the hydrolyzed wheat germ crude liquid in Step X1 are calculated by weight percentage, including 4 - 9 wt% of wheat germ, 0.1 - 2 wt% of protease, and the balance of deionized water; the protease is selected from one or more of papain and bromelain.

[0011] Further, the treatment described in step X1 refers to stirring at 50 - 65 °C at a rotation speed of 25 - 200 rpm for 50 - 100 min.

[0012] Further, the inactivation described in step X1 refers to heat preservation at 90 - 100 °C and a rotation speed of 25 - 200 rpm for 50 - 100 min.

[0013] Further, the triple filtration described in step X2 refers to first filtering through a 40 - 100 mesh filter membrane, then through a 200 - 400 mesh filter membrane, and finally filtering using a 0.1 - 0.65 μm hollow fiber membrane.

[0014] Further, the carbon source is selected from one or more of glucose, lactose, and lactic acid bacteria.

[0015] Further, the preparation method includes the following steps:

[0016] Step Y1: Mix the hydrolyzed wheat germ extract and the carbon source evenly, and sterilize at 115 °C for 15 - 20 min to obtain the product.

[0017] Advantages of the present invention:

[0018] The present invention provides a lactic acid bacteria fermentation medium, which uses a hydrolyzed wheat germ extract to replace traditional peptone, yeast powder, etc. for fermentation. It has a light smell and no peculiar smell, and the fermentation filtrate can be put into the product for use after simple sterilization and clarification; in addition, the lactic acid bacteria fermentation medium of the present invention does not affect the growth of lactic acid bacteria and the expression of proteins, and is simple to operate and easy to implement, with lower costs. Description of the Drawings

[0019] For the convenience of those skilled in the art to understand, the present invention will be further described below with reference to the drawings.

[0020] Figure 1 It is the protein electrophoresis diagrams of the supernatant samples and disrupted samples obtained by culturing Lactococcus lactis with the media obtained in Example 2 and Comparative Example 3. Among them, the first on the left is the disrupted sample of Example 2, the second on the left is the supernatant sample of Example 2, the third on the left is the disrupted sample of Comparative Example 3, the fourth on the left is the supernatant sample of Comparative Example 3, and the fifth on the left is the control Marker. Detailed Embodiments

[0021] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following will, in combination with the drawings and preferred embodiments, describe in detail the specific embodiments, structures, features, and their effects according to the present invention.

[0022] In all the examples and comparative examples of the present invention, the fresh wheat germ is from Suzhou, Anhui; the papain is papain with an activity of 400,000 u / g, purchased from Hanzhong Hansuyuan Biotechnology Co., Ltd. (270 yuan / kg); the bromelain is bromelain with an activity of 1,200,000 u / g, purchased from Hanzhong Hansuyuan Biotechnology Co., Ltd. (1270 yuan / kg); the lactose is α-lactose (monohydrate), purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the M17 broth medium is purchased from Beijing Sanyao Technology Co., Ltd.

[0023] Example 1

[0024] A lactic acid bacteria fermentation medium. The raw materials of the fermentation medium in this example are calculated by weight percentage and include 99.5 wt% of the hydrolyzed wheat germ extract and the balance of lactose.

[0025] The preparation method of the hydrolyzed wheat germ extract in this example includes the following steps:

[0026] Step X1: Mix wheat germ and deionized water evenly, then add papain and bromelain, stir at 55 °C at a speed of 50 rpm for 60 min, and then keep warm at 95 °C and 50 rpm for 60 min to obtain the crude hydrolyzed wheat germ liquid;

[0027] Step X2: Triple-filter the crude hydrolyzed wheat germ liquid, that is, first filter through an 80-mesh filter membrane, then through a 300-mesh filter membrane, and finally filter using a 0.22-μm hollow fiber membrane to obtain the hydrolyzed wheat germ extract.

[0028] The raw materials of the crude hydrolyzed wheat germ liquid in step X1 of this example are calculated by weight percentage and include 5 wt% of wheat germ, 0.25 wt% of papain, 0.25 wt% of bromelain, and the balance of deionized water.

[0029] The preparation method of this example includes the following steps:

[0030] Step Y1: Mix the hydrolyzed wheat germ extract and lactose evenly, and sterilize at 115 °C for 20 min to obtain the product.

[0031] Example 2

[0032] A lactic acid bacteria fermentation medium. The raw materials of the fermentation medium in this example are calculated by weight percentage and include 99.5 wt% of the hydrolyzed wheat germ extract and the balance of lactose.

[0033] The preparation method of the hydrolyzed wheat germ extract in this example includes the following steps:

[0034] Step X1: After mixing wheat germ and deionized water evenly, add papain and bromelain, stir at 50 rpm for 60 min at 55°C, and then keep warm at 95°C and 50 rpm for 60 min to obtain the crude hydrolyzed wheat germ liquid.

[0035] Step X2: Triple-filter the crude hydrolyzed wheat germ liquid, that is, first filter through an 80-mesh filter membrane, then through a 300-mesh filter membrane, and finally filter using a 0.22-μm hollow fiber membrane to obtain the hydrolyzed wheat germ extract.

[0036] In this Example, for the raw materials of the crude hydrolyzed wheat germ liquid in Step X1, by weight percentage, it includes 5 wt% of wheat germ, 0.5 wt% of papain, 0.5 wt% of bromelain, and the balance of deionized water.

[0037] The preparation method of this Example includes the following steps:

[0038] Step Y1: After mixing the hydrolyzed wheat germ extract and lactose evenly, sterilize at 115°C for 20 min to obtain the product.

[0039] Example 3

[0040] For a lactic acid bacteria fermentation medium, for the raw materials of the fermentation medium in this Example, by weight percentage, it includes 99.5 wt% of the hydrolyzed wheat germ extract and the balance of lactose.

[0041] The preparation method of the hydrolyzed wheat germ extract in this Example includes the following steps:

[0042] Step X1: After mixing wheat germ and deionized water evenly, add papain, stir at 50 rpm for 60 min at 55°C, and then keep warm at 95°C and 50 rpm for 60 min to obtain the crude hydrolyzed wheat germ liquid.

[0043] Step X2: Triple-filter the crude hydrolyzed wheat germ liquid, that is, first filter through an 80-mesh filter membrane, then through a 300-mesh filter membrane, and finally filter using a 0.22-μm hollow fiber membrane to obtain the hydrolyzed wheat germ extract.

[0044] In this Example, for the raw materials of the crude hydrolyzed wheat germ liquid in Step X1, by weight percentage, it includes 5 wt% of wheat germ, 1 wt% of papain, and the balance of deionized water.

[0045] The preparation method of this Example includes the following steps:

[0046] Step Y1: After mixing the hydrolyzed wheat germ extract and lactose evenly, sterilize at 115°C for 20 min to obtain the product.

[0047] Comparative Example 1

[0048] A lactic acid bacteria fermentation medium. In this example, the raw materials of the fermentation medium are calculated by weight percentage and include 99.5 wt% of hydrolyzed wheat germ extract and the balance of lactose.

[0049] The preparation method of the hydrolyzed wheat germ extract in this example includes the following steps:

[0050] Step X1: Mix wheat germ and deionized water evenly, then add protease, stir at 50 rpm for 60 min at 55 °C, and then keep warm at 95 °C and 50 rpm for 60 min to obtain a crude hydrolyzed wheat germ solution.

[0051] Step X2: Triple-filter the crude hydrolyzed wheat germ solution, that is, first filter through an 80-mesh filter membrane, then through a 300-mesh filter membrane, and finally filter using a 0.22-μm hollow fiber membrane to obtain the hydrolyzed wheat germ extract.

[0052] In this example, the raw materials of the crude hydrolyzed wheat germ solution in Step X1 are calculated by weight percentage and include 5 wt% of wheat germ and the balance of deionized water.

[0053] The preparation method in this example includes the following steps:

[0054] Step Y1: Mix the hydrolyzed wheat germ extract and lactose evenly, and sterilize at 115 °C for 20 min to obtain the product.

[0055] Comparative Example 2

[0056] A lactic acid bacteria fermentation medium. In this example, the raw materials of the fermentation medium are calculated by weight percentage and include 99.5 wt% of hydrolyzed wheat germ extract and the balance of lactose.

[0057] The preparation method of the hydrolyzed wheat germ extract in this example includes the following steps:

[0058] Step X1: Mix wheat germ and deionized water evenly, then add papain and bromelain and mix evenly, and keep warm at 95 °C and 50 rpm for 60 min to obtain a crude hydrolyzed wheat germ solution.

[0059] Step X2: Triple-filter the crude hydrolyzed wheat germ solution, that is, first filter through an 80-mesh filter membrane, then through a 300-mesh filter membrane, and finally filter using a 0.22-μm hollow fiber membrane to obtain the hydrolyzed wheat germ extract.

[0060] In this example, the raw materials of the crude hydrolyzed wheat germ solution in Step X1 are calculated by weight percentage and include 5 wt% of wheat germ, 0.25 wt% of papain, 0.25 wt% of bromelain and the balance of deionized water.

[0061] The preparation method of this embodiment comprises the following steps:

[0062] Step Y1: Mix the hydrolyzed wheat germ extract and lactose evenly, and sterilize at 115°C for 20 min to obtain the product.

[0063] Comparative Example 3

[0064] Take M17 broth medium. According to the usage requirements of M17, weigh 42.25 grams of M17 broth medium in 1 L of distilled water or deionized water, and sterilize at 115°C for 20 min, which can be used for the cultivation of lactic acid bacteria.

[0065] Among them, the components of 42.25 grams of M17 broth medium include: 5.0 grams of tryptone, 5.0 grams of soy peptone, 5.0 grams of beef extract powder, 2.5 grams of yeast extract powder, 0.5 grams of ascorbic acid, 0.25 grams of magnesium sulfate, 19.0 grams of sodium glycerophosphate, 5.0 grams of lactose, and the pH value is 6.9 ± 0.2 (25°C); at this time, the lactose ratio is 0.5%.

[0066] 1. Take the wheat germ extracts prepared in Examples 1-3 and Comparative Examples 1-2 as samples, and measure the free total amino acids, soluble proteins, total sugar content and extract pH in the samples. Evaluate the degree of hydrolysis through the comparison of the free total amino acids, soluble proteins and total sugar content, and evaluate whether it is conducive to the growth of microorganisms through the pH. The measurement results are recorded in Table 1 below.

[0067] Table 1

[0068]

[0069] As can be seen from Table 1, the total amino acids and total sugars in the wheat germ extracts prepared by the processes of Examples 1, 2, and 3 are higher than those in Comparative Examples 1 and 2. This indicates that normal hydrolysis with protease is beneficial for increasing the total amino acids and total sugars, resulting in a higher degree of hydrolysis and more beneficial substances. Although protease was added in Comparative Example 2, it was directly inactivated at high temperature, so the protease could not take effect. However, the total amino acids and total sugars are still higher than those in Comparative Example 1, probably because the enzyme was not immediately inactivated during the heating process, thus producing a small amount of hydrolysis. Comparative Example 2 is used for comparison with the wheat germ extract hydrolyzed normally with protease. At the same time, it can be seen that the higher the degree of hydrolysis, the lower the soluble protein content, indicating that proteins are converted into amino acids, polypeptides and other substances during hydrolysis. The order of total amino acids and total sugars from large to small is Example 2 > Example 3 > Example 1 > Comparative Example 2 > Comparative Example 1. The order of total protein from small to large is Example 2 > Example 3 > Example 1 > Comparative Example 2 > Comparative Example 1. This shows that at the same protease concentration, the composite protease has a better hydrolysis degree than single papain, and in the case of different concentrations, the higher the concentration of the composite protease, the better the hydrolysis degree. However, generally speaking, Examples 1, 2, and 3 do not differ much. From the perspective of pH value, the extracts all meet the requirements for microorganism culture.

[0070] 2. Using the lactic acid bacteria media prepared in Examples 1 - 3 and Comparative Examples 1 - 3 as samples, Lactococcus lactis (CICC 20408), Lactobacillus plantarum (CICC 25034), and Lactobacillus reuteri (CICC 6124) were cultured for 24 hours respectively, and then their OD 600 and the pH comparison data of the bacterial liquid at this time were measured, as shown in Table 2 below.

[0071] Table 2

[0072]

[0073] As can be seen from Table 2, the OD 600 of Examples 1, 2, and 3 are all higher than those of Comparative Examples 1 and 2, indicating that after the hydrolyzed wheat germ extract is made into a medium, it is more conducive to the growth of lactic acid bacteria. Among them, the OD 600 of Example 2 is higher, indicating that the medium prepared in this example is more conducive to the growth of lactic acid bacteria. However, generally speaking, the differences among Examples 1, 2, and 3 are not very significant, and the OD 600 of Examples 1, 2, and 3 all reach the level of Comparative Example 3 and exceed it, indicating that after adding a carbon source to the hydrolyzed wheat germ extract, it does not affect the growth of lactic acid bacteria compared with the conventional medium. The higher pH of Comparative Example 1 and the lower pH of Example 2 also indicate from the side that the growth of lactic acid bacteria in Comparative Example 1 is poor, while the growth of lactic acid bacteria in Example 2 is better.

[0074] 3. Using the lysate of Lactococcus lactis, observe the protein expression and make a comparison.

[0075] The specific method is as follows: Using the fermentation media prepared in Example 2 and Comparative Example 3, after culturing Lactococcus lactis for 24 hours, first take 1.5 mL of the Lactococcus lactis culture solution, centrifuge at 12,000 rpm for 3 min, then take 500 μL of the centrifuged supernatant and mix it evenly with 1000 μL of absolute ethanol, refrigerate and let it stand for 24 h, then centrifuge at 12,000 rpm for 3 min, take the precipitate of the centrifuged supernatant, and perform electrophoresis. This sample is the Lactococcus lactis supernatant sample. Take another 50 mL of the Lactococcus lactis culture solution, select an ultrasonic power of 1 KW, keep the temperature constant at 25 °C, and ultrasonicate for 20 min. Take 40 μL of the lysate. This sample is the Lactococcus lactis lysate sample. Add 40 μL of protein loading buffer (SDS-PAGE Sample Loading Buffer, 2X) to the above-mentioned Lactococcus lactis supernatant sample and lysate sample respectively, mix evenly, then place in a water bath at 95 - 100 °C for 5 min, centrifuge at 12,000 rpm for 3 min, and then take 10 μL of the supernatant for unified loading. After loading, perform SDS protein gel electrophoresis (separating gel with a concentration of 12%). First, run at 60 V for 30 min, then run at 150 V for 1.5 h, and after staining overnight, perform decolorization. The protein electrophoresis pattern is as Figure 1 shown (Note: The secretion ability of Lactococcus lactis is very poor, so there are basically no protein bands in the supernatant).

[0076] According to Figure 1 It can be seen that the proteins of Lactococcus lactis cultured with the medium prepared in Example 2 and the conventional M17 medium are the same, indicating that their growth conditions are not very different, and Example 2 does not affect growth.

[0077] 4. Compare the prices of 1 kg of fermentation media

[0078] Compare the prices of Example 3 and Comparative Example 3 as shown in Table 3 below.

[0079] Table 3

[0080] Serial number Item Feeding amount for 1 kg fermentation culture Input price (excluding water) 1 Example 3 995 g of single-enzyme hydrolyzed wheat germ extract and 5 g of lactose 10.55 yuan 2 Comparative Example 3 42.25 g of M17 broth medium, with the remainder being water 185.9 yuan

[0081] The price calculation method is as follows:

[0082] (1) In Example 3, the price of wheat germ is: 15.52 yuan / kg, the price of papain is: 270 yuan / kg, and the price of α-lactose (monohydrate) is: 234.76 yuan / kg.

[0083] (2) In the wheat germ extract, the extraction rate of the extract is calculated at 80%. That is, in Example 3, after 1 kg of wheat germ hydrolyzate reacts and is filtered and microfiltered, 800 g of single-enzyme hydrolyzed wheat germ extract can be obtained (because 5% of the wheat germ has certain filter residues). That is, to prepare 1 kg of the fermentation medium in Example 3, the amount of wheat germ consumed is 995 / 0.8 * 5% = 62.1875 g, approximately 0.97 yuan. The amount of protease consumed is 995 / 0.8 * 1% = 12.4375 g, approximately 3.36 yuan; the amount of lactose consumed is 5 g, approximately 1.17 yuan; that is, the cost of pure raw materials is: 5.5 yuan.

[0084] (3) Labor: Calculated based on production employees in Guangzhou, at 8000 yuan per month and working 24.5 days per month, that is, the labor cost per day is approximately 326.53 yuan; the operation in Example 3 is simple. With 1 person for operation and 1 person for review, testing, etc., on average, 2 people can carry out hydrolysis of approximately 500 kg in Example 3 per day, that is, 400 kg of single-enzyme hydrolyzed wheat germ extract is obtained. That is, the labor cost per kilogram of wheat germ extract is approximately 1.62 yuan.

[0085] (4) Hydrolysis consumables: 1 80-mesh filter bag and 1 300-mesh filter bag are needed, with an average cost of 100 yuan each. Each can be used for 10 batches, so the consumable cost of the filter bag is: that is, the filter bag consumption per kilogram of wheat germ extract is approximately 0.05 yuan; a 0.22-μm hollow fiber microfiltration column is needed for microfiltration. Approximately each 5 m2 microfiltration column can filter 20 kg of clear liquid at a time and can be used for 50 batches, and the cost of each is 1000 yuan. Then the filter bag consumption per kilogram of wheat germ extract is approximately 1 yuan; then the filter consumable cost is 1.05 yuan / kg.

[0086] (5) Equipment: A 500L cooking pot with stirring costs approximately 50,000 yuan, and a 50kW steam generator (Tian Shen brand) costs 15,000 yuan; a pump (flow rate: 200 L / h) costs 5000 yuan; the equipment can be used continuously for 5 years, with 200 batches produced each year. That is, the usage cost of the equipment is: 0.175 yuan / kg.

[0087] (6) Factory building rent: For a factory building of 100 m 2 with a rent of 80 yuan per square meter per month in the near suburbs of Huangpu District, Guangzhou, and 200 batches produced each year, the cost of the factory building rent is: 1.2 yuan / kg.

[0088] (7) Other costs: Electricity, water, and reagent consumable costs are calculated at 1.0 yuan / kg.

[0089] Therefore, the other prices for producing 995 g of single-enzyme hydrolyzed wheat germ extract are: 1.62 + 1.05 + 0.175 + 1.2 + 1.0 = 5.045 yuan. That is, for every 1 kg of the fermentation medium of Example 3, 5.5 + 5.045 = 10.545 yuan needs to be invested, approximately 10.55 yuan; while for the M17 broth medium in Comparative Example 3, the price is 4,400 yuan / kg, the input amount is 42.25 g, and the total price is 185.9 yuan.

[0090] Therefore, the usage price of the culture medium of the present invention is much lower than that of M17. Even if using the cheapest Zhongshan Baiwei brand (480 yuan / kg) and putting in 42.25 g of M17, 20.28 yuan still needs to be consumed.

[0091] The above are only the preferred embodiments of the present invention and do not impose any formal restrictions on the present invention. Although the present invention has been disclosed as above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the disclosed technical content without departing from the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any indirect modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A lactic acid bacteria fermentation medium, characterized in that: The raw materials of the fermentation medium, by weight percentage, include 95 - 99.75 wt% of hydrolyzed wheat germ extract and the balance carbon source.

2. The lactic acid bacteria fermentation medium according to claim 1, characterized in that: The preparation method of the hydrolyzed wheat germ extract comprises the following steps: Step X1: Mix wheat germ and deionized water evenly, add protease for treatment, and inactivate to obtain crude hydrolyzed wheat germ liquid; Step X2: Triple-filter the crude hydrolyzed wheat germ liquid to obtain hydrolyzed wheat germ extract.

3. A lactic acid bacteria fermentation medium according to claim 1, characterized in that: The raw materials of the crude hydrolyzed wheat germ liquid in Step X1, by weight percentage, include 4 - 9 wt% of wheat germ, 0.1 - 2 wt% of protease, and the balance deionized water; the protease is selected from one or more of papain and bromelain.

4. A lactic acid bacteria fermentation medium according to claim 1, characterized in that: The treatment in Step X1 refers to stirring at 50 - 65 °C at a rotation speed of 25 - 200 rpm for 50 - 100 min.

5. A lactic acid bacteria fermentation medium according to claim 1, characterized in that: The inactivation in Step X1 refers to heat preservation at 90 - 100 °C and a rotation speed of 25 - 200 rpm for 50 - 100 min.

6. The lactic acid bacteria fermentation medium according to claim 1, characterized in that: The triple filtration in Step X2 refers to first filtering through a 40 - 100 mesh filter membrane, then through a 200 - 400 mesh filter membrane, and finally filtering using a 0.1 - 0.65 μm hollow fiber membrane.

7. A lactic acid bacteria fermentation medium according to claim 1, characterized in that: The carbon source is selected from one or more of glucose, lactose, and lactic acid bacteria.

8. A method for preparing a lactic acid bacteria fermentation medium according to any one of claims 1-7, characterized in that: The preparation method comprises the following steps: Step Y1: Mix the hydrolyzed wheat germ extract and the carbon source evenly, and sterilize at 115 °C for 15 - 20 min to obtain the product.

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