Application of lactobacillus brevis CCFM1267 capable of improving content of phytosphingosine in fermented milk

Abstract

The invention discloses an application of lactobacillus brevis CCFM1267 capable of increasing the content of phytosphingosine in fermented milk, and belongs to the technical field of microbial fermentation. The invention provides a bacterial strain composition. The bacterial strain composition comprises lactobacillus brevis CCFM1267, streptococcus thermophilus CCFM1094 and lactobacillus delbrueckii subsp. Bulgaricus 2038. The strain composition is used for fermenting dairy products, and the content of phytosphingosine in the dairy products can be remarkably increased. Therefore, the sake lactobacillus brevis CCFM1267 or the strain composition has a huge application prospect in preparation of fermented dairy products.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and particularly to the application of Latilactobacillus sakei CCFM1267 capable of increasing the content of phytosphingosine in fermented milk. Background Art

[0002] Phytosphingosine is a kind of sphingosine and is a precursor of ceramides. Phytosphingosine usually exists in mammals, plants and fungi. Phytosphingosine has a hydrophilic repair barrier effect on skin tissues and has anti-inflammatory activity both in vitro and in vivo. Phytosphingosine is widely used in cosmetics and pharmaceuticals and has potential applications in dairy products. The content of sphingosines in foods is relatively low, but they have a profound impact on the developmental fates of many cell types. As intracellular messengers, they participate in cell cycle regulation and apoptosis induction. Therefore, developing food products with high phytosphingosine content is of positive significance for maintaining good health.

[0003] Current research shows that phytosphingosine can be produced by microorganisms. For example, Saccharomyces cerevisiae (CN116103176A) has a phytosphingosine production of about 150.54 mg / g dry weight, but Saccharomyces cerevisiae cannot be applied to the fermented milk system, and there is no report on the production of phytosphingosine by microbial fermentation and Latilactobacillus sakei fermentation. Providing a strain capable of increasing the sphingosine content in fermented milk is of great value for expanding the application scope of fermented dairy products. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain composition and a new use of Latilactobacillus sakei CCFM1267.

[0005] The present invention provides a strain composition, which comprises Latilactobacillus sakei CCFM1267, Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038;

[0006] In one embodiment, the Latilactobacillus sakei CCFM1267 is Latilactobacillus sakei CCFM1267, which has been disclosed in patent CN115029270A and has a deposit number of GDMCC No: 62426; the Streptococcus thermophilus CCFM1094 has been disclosed in patent CN111187738A and has a deposit number of GDMCC No: 60809.

[0007] In one embodiment, in the strain composition, the viable cell count ratio of Lactobacillus pantarum CCFM1267, Streptococcus thermophilus CCFM1094, and Lactobacillus delbrueckii subsp. bulgaricus 2038 is (1000 - 20000):(100 - 2000):(1 - 10).

[0008] The present invention provides a food containing the strain composition, and the food includes health products.

[0009] The present invention provides a medicine containing the strain composition.

[0010] The present invention provides a daily chemical product or feed containing the strain composition.

[0011] The present invention discovers that adding Lactobacillus pantarum CCFM1267 to existing commercial starters can increase the content of phytosphingosine in fermented milk.

[0012] The present invention provides a method for fermentatively synthesizing phytosphingosine. The method is to ferment in dairy products using a starter containing Lactobacillus pantarum CCFM1267 to obtain a fermentation product containing phytosphingosine.

[0013] In one embodiment, the commercial starter contains Lactobacillus pantarum CCFM1267, Streptococcus thermophilus CCFM1094, and Lactobacillus delbrueckii subsp. bulgaricus 2038; the viable cell count ratio of Lactobacillus pantarum CCFM1267, Streptococcus thermophilus CCFM1094, and Lactobacillus delbrueckii subsp. bulgaricus 2038 is (1000 - 20000):(100 - 2000):(1 - 10).

[0014] In one embodiment, the dairy product is raw milk, reconstituted milk, or cream.

[0015] In one embodiment, the milk includes whole milk, skim milk, or reduced-fat milk.

[0016] In one embodiment, the fermentation conditions are: 35°C - 45°C, and ferment for at least 16 h.

[0017] In one embodiment, the fermentation includes post-fermentation at a low temperature.

[0018] In one embodiment, the temperature of the post-fermentation is 2 - 6°C, and the time is at least 1 day.

[0019] The present invention provides the use of Lactobacillus pantarum CCFM1267 or the strain composition in the preparation of a food rich in phytosphingosine.

[0020] In one embodiment, the application is to ferment a food containing phytosphingosine in an emulsion using Lactobacillus sakei CCFM1267 or the strain composition.

[0021] In one embodiment, the food includes health products.

[0022] In one embodiment, the food includes fermented milk, milk beverages, fermented cream, milk powder or solid beverages.

[0023] The present invention provides the application of Lactobacillus sakei CCFM1267 or the strain composition in the preparation of drugs rich in phytosphingosine.

[0024] The present invention provides the application of Lactobacillus sakei CCFM1267 or the strain composition in the preparation of daily chemical products or feeds rich in phytosphingosine.

[0025] Beneficial effects:

[0026] The present invention discovers that Lactobacillus sakei CCFM1267 ferments milk matrix and has good ability to synthesize phytosphingosine. The Lactobacillus sakei CCFM1267 strain can be used in the production applications such as fermented milk, milk beverages and daily chemical products of phytosphingosine. The present invention uses Lactobacillus sakei CCFM1267 with high yield of phytosphingosine combined with Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038 as a compound starter to ferment milk, and develops a compound fermented milk product rich in phytosphingosine. The content of phytosphingosine in the obtained product reaches 10.75 nmol / mL. Description of the drawings

[0027] Figure 1 Growth curve of Lactobacillus sakei CCFM1267;

[0028] Figure 2 Changes in pH, colony number of Lactobacillus sakei CCFM1267 and total viable count during the co-fermentation of Lactobacillus sakei CCFM1267. Specific embodiments

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the drawings. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the examples, all the original reagent materials can be obtained commercially. The experimental methods without specific conditions are the conventional methods and conventional conditions well-known in the art, or the conditions recommended by the instrument manufacturers.

[0030] The culture media involved in the following examples are as follows:

[0031] MRS solid medium (g / L): Peptone 10 g / L, Beef extract 10 g / L, Glucose 20 g / L, Sodium acetate 2 g / L, Yeast extract 5 g / L, Diammonium hydrogen citrate 2 g / L, K 2 HPO 4 ·3H 2 O 2.6 g / L, MgSO 4 ·7H 2 O 0.1 g / L, MnSO 4 ·H 2 O 0.05 g / L, Tween 80 1 mL / L, Agar 20 g / L.

[0032] MRS liquid medium (g / L): Peptone 10 g / L, Beef extract 10 g / L, Glucose 20 g / L, Sodium acetate 2 g / L, Yeast extract 5 g / L, Diammonium hydrogen citrate 2 g / L, K 2 HPO 4 ·3H 2 O 2.6 g / L, MgSO 4 ·7H 2 O 0.1 g / L, MnSO 4 ·H 2 O 0.05 g / L, Tween 80 1 mL / L.

[0033] Lactobacillus sakei CCFM1267 involved in the following examples is Lactobacillus sakei CCFM1267, which has been disclosed in Patent CN115029270A and has a deposit number of GDMCC No: 62426; Streptococcus thermophilus CCFM1094 has been disclosed in Patent CN111187738A and has a deposit number of GDMCC No: 60809; Lactobacillus delbrueckii subsp. bulgaricus 2038 was isolated from Meiji yogurt LB81 (Meiji Dairies Corporation, Japan); Lactobacillus sakei CCFM1199 has been disclosed in Patent CN114317334A and has a deposit number of GDMCC No: 61953; Lactobacillus sakei HD, Lactobacillus sakei Z1, and Lactobacillus sakei Z4 were self-screened.

[0034] Example 1 Growth curve of Lactobacillus sakei CCFM1267

[0035] 1. Activation and preparation of the strain

[0036] (1) Strain activation

[0037] Lactobacillus sakei CCFM1267 in the glycerol preservation tube was spread on an MRS solid plate and cultured at 37 °C for 48 h; a single colony was picked and inoculated into 5 mL of MRS medium, cultured at 37 °C for 18 h, and the strain in the logarithmic growth phase was taken and stored in a glycerol tube for later use.

[0038] (2) Growth curve

[0039] During the growth process, the OD values of Lactobacillus sakei CCFM1267 group were measured at 0 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, and 18 h, and the viable cell count was measured by plate counting method. The growth curve is shown in Figure 1 , and the end of the logarithmic growth phase was around 12 h.

[0040] Example 2 Lactobacillus sakei CCFM1267 complex starter increases the content of phytosphingosine in fermented milk

[0041] 1. Experimental strains

[0042] The strains used in the technical solution of the present invention include Lactobacillus sakei CCFM1267, Lactobacillus sakei CCFM1199, Lactobacillus sakei HD, Lactobacillus sakei Z1, Lactobacillus sakei Z4, Streptococcus thermophilus CCFM1094, and Lactobacillus delbrueckii subsp. bulgaricus 2038 (Lactobacillus sakei CCFM1267 has been disclosed in patent CN115029270A, and the preservation number is GDMCC No: 62426; Streptococcus thermophilus CCFM1094 has been disclosed in patent CN111187738A, and the preservation number is GDMCC No: 60809; Lactobacillus delbrueckii subsp. bulgaricus 2038 was isolated from Meiji yogurt LB81 (Meiji Dairies Corporation, Japan); Lactobacillus sakei CCFM1199 has been disclosed in patent CN114317334A, and the preservation number is GDMCC No: 61953; Lactobacillus sakei HD, Lactobacillus sakei Z1, and Lactobacillus sakei Z4 were self-screened).

[0043] 2. Activation and preparation of strains

[0044] Lactobacillus sakei CCFM1267, Lactobacillus sakei CCFM1199, Lactobacillus sakei HD, Lactobacillus sakei Z1, Lactobacillus sakei Z4, Streptococcus thermophilus CCFM1094, and Lactobacillus delbrueckii subsp. bulgaricus 2038: The glycerol preservation tubes of Lactobacillus sakei CCFM1267, Lactobacillus sakei CCFM1199, Lactobacillus sakei HD, Lactobacillus sakei Z1, Lactobacillus sakei Z4, Streptococcus thermophilus CCFM1094, and Lactobacillus delbrueckii subsp. bulgaricus 2038 were respectively spread on MRS solid plates and cultured at 37 °C for 48 h; single colonies were respectively picked and inoculated into 5 mL MRS medium, cultured at 37 °C for 18 h, and the strains in the logarithmic phase of growth were taken and stored in glycerol tubes for later use.

[0045] 3. Sphingosine synthesis by Lactobacillus sakei CCFM1267-fermented milk

[0046] Fermentation groups: control group, inoculating the seed liquid of Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038 into sterilized skim milk; CCFM1267 group, inoculating the seed liquid of Lactobacillus sakei CCFM1267, Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038 into sterilized skim milk simultaneously; CCFM1199 group, inoculating Lactobacillus sakei CCFM1199, Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038 into sterilized skim milk simultaneously; HD group, inoculating Lactobacillus sakei HD, Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038 into sterilized skim milk simultaneously; Z1 group, inoculating Lactobacillus sakei Z1, Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038 into sterilized skim milk simultaneously; Z4 group, inoculating Lactobacillus sakei Z4, Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038 into sterilized skim milk simultaneously.

[0047] Preparation of fermented milk: 12% skim milk powder was dissolved in water at room temperature, heated to 95 °C for 10 min for sterilization, cooled to 37 °C, and the activated strains were added according to the groups. The inoculation concentration ratio was Lactobacillus sakei: Streptococcus thermophilus CCFM1094: Lactobacillus delbrueckii subsp. bulgaricus 2038 = 10000:1000:1 (the inoculation volume ratio was 2%), and the initial inoculation concentration of Lactobacillus sakei was 10 8 CFU / mL, and the inoculation concentrations of Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038 in the control group were the same as those in other groups. Each sample was fermented at 37 °C for 16 h, then transferred to 4 °C for cold storage for 1 d, and the post-fermentation process was continued. Samples were collected at 0, 4, 8, 12, 16 h (1 day), 2, 7, and 14 days of storage for analysis. All samples were stored at -80 °C waiting for further analysis.

[0048] During the fermentation process, the pH values of the control group and the CCFM1267 group were measured at 0 h, 4 h, 8 h, 12 h, and 16 h, the total viable count was measured by the plate counting method, and the total colony count of CCFM1267 at different time points was measured by qPCR.

[0049] During the post-fermentation process, the fermented milk was centrifuged at 8000 r / min for 15 min at different time points, the fermentation supernatant was collected, passed through a 0.22 μm filter membrane, and the filtrate was used for the determination of sphingosine content. The content of specific sphingosine was determined using a sphingosine assay kit.

[0050] Table 1 Content of phytosphingosine in the fermentation supernatant (nmol / mL)

[0051] Sample Name 1d 2d 14d control 6.28±0.20 7.02±0.95 6.35±0.55 CCFM1267 9.54±0.08 10.66±0.71 10.75±0.20 CCFM1199 6.13±0.09 6.14±0.03 6.39±0.11 HD 6.58±0.15 7.07±0.62 6.84±0.91 Z1 5.98±0.03 7.14±0.14 7.41±1.16 Z4 7.08±0.13 7.77±0.81 5.90±0.90

[0052] From Figure 2 it can be seen that in the CCFM1267 group, the pH reached 4.5 at 8 hours of fermentation, while in the control group, the pH did not drop below 4.5 until 12 hours. Further evaluate the growth of CCFM1267 in fermented milk. Figure 2 A shows that CCFM1267 showed rapid growth between 4 - 8 hours and reached a plateau at 12 hours, which was consistent with the observed pH drop in the CCFM1267 group. On the second day of storage, after the ripening period was completed, the viable bacteria count in the CCFM1267 group was 5×10 8 CFU / mL, significantly higher than 10 8 CFU / mL in the control group (p<0.01)( Figure 2 B). Compared with the control group, the CCFM1267 group maintained a higher viable bacteria count throughout the storage period (p<0.01).

[0053] It can be seen from the results in Table 1 that compared with the CCFM1267 group, the control group, HD group, Z1 group, and Z4 group produced less phytosphingosine during fermentation, almost maintaining at about 6 - 7 nmol / mL; when Lactobacillus sakei CCFM1267, Streptococcus thermophilus CCFM1094, and Lactobacillus delbrueckii subsp. bulgaricus 2038 were used together to prepare fermented milk, the content of phytosphingosine increased significantly compared with other groups, reaching 9 - 10 nmol / ml.

[0054] Example 3 Preparation of a phytosphingosine - rich milk beverage by fermenting Lactobacillus sakei CCFM1267

[0055] Heat 12% skim milk to 95°C for 10 min for sterilization, cool to 37°C, and add Lactobacillus sakei CCFM1267, Streptococcus thermophilus CCFM1094, and Lactobacillus delbrueckii subsp. bulgaricus 2038 (10000:1000:1) at an inoculation amount of 2% (v / v), and ferment at 35°C - 45°C for at least 16 h. Dilute the fermentation product 2 - 4 times with a mixture of sterilized and cooled water, syrup, emulsifying stabilizer, etc. to make a directly drinkable lactic acid bacteria beverage. This lactic acid bacteria beverage not only has a sour - sweet taste and good flavor, but also contains a relatively high content of phytosphingosine.

[0056] Although the present invention has been disclosed above in its preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A strain composition, characterized in that: The strain composition comprises Latilactobacillus sakei CCFM1267, Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subsp. bulgaricus 2038; The preservation number of the sake lactobacillus CCFM1267 is GDMCC No: 62426; the preservation number of the thermophilic streptococcus CCFM1094 is GDMCC No: 60809.

2. The strain composition according to claim 1, characterized in that: In the strain composition, the ratio of the live bacterial counts of Lactobacillus sakei CCFM1267, Streptococcus thermophilus CCFM1094 and Lactobacillus delbrueckii subspecies bulgaricus 2038 is (1000-20000):(100-2000):(1-10).

3. A product containing the strain composition according to claim 1 or 2, characterized in that: The products include food, daily chemicals, feed or medicine; the food includes health products.

4. A method for synthesizing phytosphingosine, characterized in that: The strain composition according to claim 1 or 2 is inoculated into dairy products for fermentation.

5. The method according to claim 4, characterized in that The dairy product includes raw milk, reconstituted milk or cream.

6. The method according to claim 5, characterized in that Ferment at 35℃~45℃ for at least 16h.

7. Use of Latilactobacillus sakei CCFM1267 or the strain composition according to claim 1 or 2 in preparing food rich in phytosphingosine.

8. The use according to claim 7, characterized in that: The food includes health products; or the food includes fermented milk, milk drinks, fermented cream, milk powder or solid beverages.

9. Use of Latilactobacillus sakei CCFM1267 or the strain composition according to claim 1 or 2 in the preparation of a medicine rich in phytosphingosine.

10. Use of Latilactobacillus sakei CCFM1267 or the strain composition according to claim 1 or 2 in the preparation of daily chemical products or feeds rich in phytosphingosine.

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