Method for screening lactobacillus paracasei flora from Kefir grains and application of lactobacillus paracasei flora

Abstract

The invention provides a method for screening lactobacillus paracasei flora from Kefir granules and application of the lactobacillus paracasei flora, and belongs to the technical field of microorganisms. The method disclosed by the invention comprises the following steps: (1) mixing soybeans and water according to a mass-volume ratio of 1g: (5-10) mL, grinding into thick liquid, and sterilizing to obtain soybean milk; (2) inoculating Kefir granules into the soybean milk obtained in the step (1), continuously culturing for 2-5 months, and collecting fungus granules; and (3) separating and purifying the bacteria particles obtained in the step (2) to obtain the lactobacillus paracasei flora. According to the method disclosed by the invention, the soybean milk is used as a culture medium to continuously culture the Kefir grains, so that the flora in the Kefir grains is successfully and remarkably changed. The screened lactobacillus paracasei flora can be used for fermenting the soybean milk, and a foundation is laid for developing a new fermented soybean milk product.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a method for screening Lactobacillus paracasei flora from Kefir grains and its application. Background Art

[0002] Kefir grains are a microbial consortium rich in lactic acid bacteria, acetic acid bacteria, and yeast microbial resources, and are commonly used in yogurt fermentation in the prior art. Soy milk is mainly composed of stachyose, raffinose, and sucrose, and Lactobacillus kefiranofaciens, the dominant bacterium in Kefir grains, is not suitable for fermenting soy milk. Therefore, it is particularly important to obtain strains suitable for soy milk fermentation from Kefir grains, apply them to soy milk fermentation, and develop new fermented soy milk products. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for screening Lactobacillus paracasei flora from Kefir grains and its application. By continuously culturing Kefir grains with soy milk as the culture medium, the present invention successfully causes significant changes in the flora in Kefir grains, and successfully screens out Lactobacillus paracasei through pure culture. The screened Lactobacillus paracasei can be used to ferment soy milk.

[0004] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a method for screening Lactobacillus paracasei flora from Kefir grains, comprising the following steps:

[0006] (1) Mix soybeans and water at a mass-to-volume ratio of 1 g:5 - 10 mL, grind to obtain soy milk, and sterilize it.

[0007] (2) Inoculate Kefir grains into the soy milk obtained in step (1), continuously culture for 2 - 5 months, and collect the bacterial grains.

[0008] (3) Isolate and purify the bacterial grains obtained in step (2) to obtain Lactobacillus paracasei flora.

[0009] Preferably, before mixing soybeans and water in step (1), there is also a step of soaking the soybeans, and the soaking time is 6 - 10 h.

[0010] Preferably, the sterilization temperature is 100 - 130 °C, and the sterilization time is 10 - 30 min.

[0011] Preferably, the inoculation amount of the Kefir grains is 0.5% - 2% of the mass of the soy milk.

[0012] Preferably, the method of continuous culture is to subculture the Kefir grains, and the subculture time is 45 - 50 h / time.

[0013] Preferably, the operation method of subculture is to collect the precipitate generated during the culture using a sterile filter screen, wash the precipitate with sterile water, and transfer the washed precipitate into fresh soymilk, thus completing one subculture.

[0014] Preferably, the mesh number of the sterile filter screen is 30 - 70 meshes, and the number of washing times is 1 - 5 times.

[0015] Preferably, the culture temperature is 20 - 30 °C.

[0016] Preferably, the medium used for purification is MRS solid medium.

[0017] The present invention also provides the application of the Lactobacillus paracasei flora obtained by the above method in fermenting soymilk.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for screening the Lactobacillus paracasei flora from Kefir grains and its application. By continuously culturing Kefir grains using soymilk as the medium, the flora in Kefir grains has changed significantly. The dominant bacteria have changed from the original Lactobacillus kefiranofaciens to Lactobacillus paracasei. And through a simple purification and culture process, Lactobacillus paracasei can be successfully screened out. Using the screened Lactobacillus paracasei flora, the fermentation of soymilk can be achieved, laying a foundation for the development of new fermented soymilk products. Description of the Drawings

[0019] Figure 1 It is a succession diagram of the flora in Kefir grains with the subculture time in soymilk;

[0020] Figure 2 It is a metabolic pathway diagram of the main soybean oligosaccharides (stachyose, raffinose, and sucrose) in soymilk, involving the enzymatic catalysis of α-galactosidase (EC 3.2.1.22), β-fructofuranosidase (EC 3.2.1.26), and α-glucosidase (EC 3.2.1.20);

[0021] Figure 3 It is a time dynamic change diagram of the microbial groups related to the MAP00052 lactose metabolic pathway during continuous subculture (from m0 to m4);

[0022] Figure 4 It is a succession diagram of the microbial source of α-galactosidase (EC 3.2.1.22) during subculture;

[0023] Figure 5For the microbial source succession diagram of β-fructofuranosidase (EC 3.2.1.26) during subculture;

[0024] Figure 6 For the microbial source succession diagram of α-glucosidase (EC 3.2.1.20) during subculture;

[0025] Figure 7 For the microbial source succession diagram of oligo-α-1,6-glucosidase (EC 3.2.1.10) during subculture. Detailed implementation manners

[0026] The present invention provides a method for screening Lactobacillus paracasei flora from Kefir grains, comprising the following steps:

[0027] (1) Mix soybeans and water at a mass-to-volume ratio of 1 g: 5 - 10 mL, grind to obtain soymilk, and sterilize it;

[0028] (2) Inoculate Kefir grains into the soymilk obtained in step (1), continuously culture for 2 - 5 months, and collect the bacterial grains;

[0029] (3) Separate and purify the bacterial grains obtained in step (2) to obtain the Lactobacillus paracasei flora.

[0030] In the present invention, soybeans and water are mixed at a mass-to-volume ratio of 1 g: 5 - 10 mL, ground to obtain soymilk. In the present invention, before mixing soybeans and water, there is also a step of soaking the soybeans; the soaking time is 6 - 10 h, preferably 7 - 9 h, more preferably 8 h. The mass of the soybeans is the mass before soaking, the mass-to-volume ratio of the soybeans and water is preferably 1 g: 6 - 8 mL, more preferably 1 g: 7 mL, the sterilization temperature is 100 - 130 °C, preferably 115 - 125 °C, more preferably 121 °C, and the sterilization time is 10 - 30 min, preferably 15 - 25 min, more preferably 20 min. The present invention has no special limitation on the grinding method, and a conventional grinding method can be used.

[0031] In the present invention, Kefir grains are inoculated into the soy milk obtained in step (1), continuously cultured for 2 to 5 months, and the bacterial grains are collected. The Kefir grains are inoculated into the sterilized soy milk at 0.5% to 2% of the mass of the soy milk, and subcultured during the continuous culture process. The time for subculture is once every 45 to 50 h. The operation method for each subculture is as follows: Use a sterile filter screen to collect the precipitate generated during the culture process, wash the precipitate with sterile water, and transfer the washed precipitate into fresh soy milk, thus completing one subculture. The inoculation amount of the Kefir grains is preferably 0.7% to 1.5% of the mass of the soy milk, more preferably 1%; the time for each subculture is preferably 47 to 49 h, more preferably 48 h; the temperature for the culture is 20 to 30 °C, preferably 23 to 28 °C, more preferably 25 °C; the mesh number of the sterile filter screen is 30 to 70 meshes, preferably 40 to 60 meshes, more preferably 50 meshes; the number of washing times is 1 to 5 times, preferably 2 to 4 times, more preferably 3 times; the time for continuous culture is preferably 3 to 4.5 months, more preferably 4 months.

[0032] In the present invention, the bacterial grains obtained in step (2) are separated and purified to obtain a Lactobacillus paracasei flora. The collected bacterial grains are ground with a sterile grinder and dissolved in 0.85% physiological saline for gradient dilution. The diluted solution is spread on an MRS solid medium, and the culture dish is incubated at 37 °C for 48 h. Colonies are picked and transferred to a fresh MRS solid culture for streak separation, and this process is repeated 3 times to obtain the Lactobacillus paracasei flora.

[0033] The present invention also provides the application of the Lactobacillus paracasei flora obtained by the method in fermenting soy milk.

[0034] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Embodiment

[0036] 100 g of disease-free and plump soybeans are selected, rinsed, soaked in cold water for 8 h and then fished out, 700 g of water is added, and grinding is carried out. It is dispensed into 250 mL conical flasks at a rate of 100 mL / bottle, sterilized at 121 °C for 20 min, and then cooled to obtain sterile soy milk for standby.

[0037] The Kefir grains are inoculated into the sterile soy milk at an inoculation amount of 1% of the mass of the soy milk, activated at 25 °C for 48 h, then the bacterial grains are collected using a 50-mesh sterile filter screen, the collected bacterial grains are repeatedly washed three times with sterile water, the washed bacterial grains are inoculated into fresh sterile soy milk, continuously cultured at 25 °C for 4 months, and the soy milk is updated every 48 h until the culture ends, and the bacterial grains are collected.

[0038] The collected kefir grains were ground with a sterile grinder and dissolved in 0.85% physiological saline for gradient dilution. The diluted solution was spread on MRS solid medium, and the culture dishes were incubated at 37 °C for 48 h. Colonies were picked and transferred to fresh MRS solid medium for streak isolation. This process was repeated three times to obtain the Lactobacillus paracasei population.

[0039] Experimental Example

[0040] Kefir grains were obtained in the manner of the examples. At 0 months (m0), 1 month (m1), 2 months (m2), and 4 months (m4) respectively, 1 g of kefir grain samples were collected for metagenomic analysis to observe the succession of the microbial community in kefir grains over time during the subculture of soy milk (the results are shown in Figure 1 ), the metabolic pathways of the main soy oligosaccharides (stachyose, raffinose, and sucrose) in soy milk (the results are shown in Figures 2 - 3 ), and the succession of the microbial sources of key carbohydrate-active enzymes during the subculture process (the results are shown in Figures 4 - 7 ).

[0041] As Figure 1 can be seen, after the kefir grains were transferred to soy milk and subcultured continuously for 2 months, the microbial community changed significantly. The relative abundance of the absolute dominant bacterium Lactobacillus kefiranofaciens (L. kefiranofaciens) decreased from 95.00% at the initial stage (m0) to 10.88%, while the relative abundance of Lactobacillus paracasei (Lacticaseibacillus paracasei, L. paracasei), which is beneficial to the fermentation of soy milk, increased from 0.32% to 51.82%; after continuous culture for 4 months, the relative abundance of L. paracasei could be increased to 76.94% and become the absolute dominant bacterium.

[0042] As Figures 2 - 3 can be seen, compared with cow's milk, the unique sugar composition and concentration in soy milk may be the key driving factors regulating the dynamics of the microbial community in kefir grains. Soy milk is mainly composed of stachyose, raffinose, and sucrose, and its key metabolic processes are as Figure 2 shown. These metabolic processes are mainly related to the lactose metabolic pathway (MAP00052) and involve key enzymes such as EC3.2.1.22 (α-galactosidase), EC 3.2.1.26 (β-fructofuranosidase), EC 3.2.1.20 (α-glucosidase), and EC 3.2.1.10 (oligo-α-1,6-glucosidase). Figure 3Shows the changes in the microbial species related to the MAP00052 pathway during continuous subculture in soymilk. During the continuous subculture in soymilk, the microbial composition related to the MAP00052 pathway changed significantly. At the initial stage (m0), L. kefiranofaciens was the main dominant bacterial species related to this pathway. However, as the subculture time increased, the microbial community became more diverse, the relative abundance of L. kefiranofaciens decreased, while the relative abundances of L. paracasei group and L. paracasei increased synchronously. This trend became more obvious over time. By subculture m4, the L. paracasei group and L. paracasei had become the main dominant bacterial species.

[0043] It can be seen from Figures 4 - 7 that further analysis of the microbial sources of key enzymes showed that during continuous subculture, the main microbial sources of EC3.2.1.22 and EC 3.2.1.26 changed from L. kefiranofaciens to L. paracasei group and L. paracasei. Notably, EC3.2.1.20 and EC 3.2.1.10 were not detected at the initial stage (m0), but one month after subculture (m1), these enzymes were mainly derived from L. paracasei group and L. paracasei, and their relative abundances continued to increase with further subculture. These results indicate that L. paracasei has the key enzymes for metabolizing raffinose, stachyose and sucrose, showing a strong ability to metabolize soybean oligosaccharides. Therefore, during continuous subculture, L. paracasei gradually replaced L. kefiranofaciens and finally became the dominant bacterial species.

[0044] Therefore, the L. paracasei flora obtained in the examples can be used for soymilk fermentation, laying a foundation for the development of new fermented soymilk products.

[0045] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for screening Lactobacillus paracasei flora from Kefir grains, characterized in that, It includes the following steps: (1) Mix soybeans with water at a mass-to-volume ratio of 1 g: 5-10 mL, grind into a slurry, and sterilize to obtain soy milk; (2) Inoculate Kefir grains into the soy milk obtained in step (1), continuously culture for 2-5 months, and collect the grains; (3) Separate and purify the grains obtained in step (2) to obtain a Lactobacillus paracasei flora.

2. The method according to claim 1, wherein Before mixing soybeans with water in step (1), it also includes a step of soaking the soybeans, and the soaking time is 6-10 h.

3. The method according to claim 1, characterized in that, The sterilization temperature is 100-130 °C, and the sterilization time is 10-30 min.

4. The method according to claim 1, wherein The inoculation amount of the Kefir grains is 0.5%-2% of the mass of the soy milk.

5. The method according to claim 1, wherein The method of continuous culture is to subculture the Kefir grains, and the subculture time is 45-50 h / time.

6. The method according to claim 5, wherein The operation method of subculture is to use a sterile filter screen to collect the precipitate generated during the culture process, wash the precipitate with sterile water, and transfer the washed precipitate into fresh soy milk, thus completing one subculture.

7. The method according to claim 6, wherein The mesh number of the sterile filter screen is 30-70 meshes, and the number of washing times is 1-5 times.

8. The method according to claim 5, wherein The culture temperature is 20-30 °C.

9. The method according to claim 1, wherein The medium used for purification is MRS solid medium.

10. Application of the Lactobacillus paracasei flora obtained by the method according to claims 1-9 in fermenting soy milk.

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