A lactobacillus salivarius ccfm1266 capable of increasing the content of niacin in fermented milk

Abstract

The application discloses lactobacillus salivarius CCFM1266 capable of improving the content of nicotinic acid in fermented milk, and belongs to the technical field of microbial fermentation. The application discloses lactobacillus salivarius CCFM1266 capable of increasing the content of nicotinic acid in fermented milk, and a composite starter is prepared based on lactobacillus salivarius CCFM1266. The lactobacillus salivarius CCFM1266 provided by the application can utilize fructooligosaccharide and mannooligosaccharide as fermentation substrates to prepare fermented milk rich in nicotinic acid. In the fermentation system containing fructooligosaccharide and / or mannooligosaccharide: (1) the number of bacteria of lactobacillus salivarius is significantly increased; and (2) the content of nicotinic acid is significantly increased. Therefore, the lactobacillus salivarius CCFM1266 has great application prospect in the preparation of fermented milk products.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus salivarius CCFM1266 that can increase the niacin content in fermented milk. Specifically, it relates to a method and related applications of using a strain of Lactobacillus salivarius CCFM1266 as a starter culture and fructooligosaccharide and mannose as substrates to co-ferment and produce niacin, belonging to the field of microbial fermentation technology. Background Technology

[0002] Niacin participates in energy metabolism, maintains the health of the skin, nervous system, and digestive system, and helps lower cholesterol and prevent cardiovascular disease. The "Dietary Guidelines for Chinese Residents" recommends a daily niacin intake of 13-19 mg for adults, 20 mg for pregnant women, and 22 mg for breastfeeding mothers. Niacin is widely found in animal and plant tissues, especially in meat, fish, nuts, and grains. Some niacin can also be synthesized in the human intestine, but usually in small amounts. Therefore, developing food products with high niacin content is of positive significance for increasing people's niacin intake and maintaining good health.

[0003] Current research indicates that niacin can be produced through lactic acid bacteria fermentation, such as *Bifidobacterium lactis* BL-99 (CN113355376A), *Lactobacillus paracasei* ET-22 (CN113355374B), and *Lactobacillus paracasei* K56 (CN113355375B). However, there are no reports on the production of niacin by *Lactobacillus salivarius* fermentation. The niacin content in currently commercially available fermented milk is approximately 0.2 mg / 100g.

[0004] In addition, Lactobacillus salivarius has demonstrated antibacterial activity, immune effects, and the ability to regulate the gut microbiota in both in vivo and in vitro studies (Appl Microbiol Biotechnol 106, 57–80, 2022). The addition of Lactobacillus salivarius can provide more active functions for fermented dairy products. Summary of the Invention

[0005] One object of the present invention is to provide a new use for Lactobacillus salivarius CCFM1266.

[0006] Lactobacillus salivarius CCFM1266, with accession number GDMCC No: 62418, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 17, 2022.

[0007] This invention discovers that adding Lactobacillus salivarius CCFM1266 to existing commercial starter cultures can increase the niacin content in fermented milk.

[0008] This invention provides a method for the fermentation synthesis of niacin, wherein the method involves using Lactobacillus salivarius CCFM1266 to ferment dairy products containing fructooligosaccharides and / or mannose to prepare a fermentation product containing niacin.

[0009] In one embodiment, the amount of fructooligosaccharides added is 1-5%.

[0010] In one embodiment, the amount of oligomannose added is 1-5%.

[0011] In one implementation, a commercial starter culture is added during the fermentation process.

[0012] In one embodiment, the commercial fermenting agent contains Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0013] In one embodiment, the dairy product is raw milk, reconstituted milk, or cream.

[0014] In one embodiment, the milk includes whole milk, skim milk, or reduced-fat milk.

[0015] In one embodiment, the fermentation conditions are: 35℃~45℃, fermentation for 1 day~14 days.

[0016] In one embodiment, the fermentation includes post-fermentation at low temperature.

[0017] This invention provides the application of Lactobacillus salivarius CCFM1266 in the fermentation preparation of products rich in nicotinic acid.

[0018] In one embodiment, the application is to prepare a product containing nicotinic acid by fermentation with Lactobacillus salivarius CCFM1266 in an emulsion containing fructooligosaccharides and / or mannose.

[0019] In one embodiment, the product includes food, daily chemical products, feed, or pharmaceuticals.

[0020] In one embodiment, the food product includes health supplements.

[0021] In one embodiment, the food includes fermented milk, dairy drinks, fermented cream, milk powder, and solid beverages.

[0022] Beneficial effects:

[0023] This invention discovers that *Lactobacillus salivarius* CCFM1266, a fermented milk substrate, possesses excellent niacin synthesis capabilities. This *Lactobacillus salivarius* CCFM1266 strain can be used in the production of fermented milk, dairy beverages, and daily chemical products containing niacin. This invention utilizes the high-niacin-producing *Lactobacillus salivarius* CCFM1266 as a starter culture for fermenting milk, while simultaneously adding fructooligosaccharides and mannose to promote the growth of *Lactobacillus salivarius* CCFM1266, thus developing a niacin-rich compound fermented milk product. Fermentation for one day (24 hours) can achieve a niacin content of over 60 μg / mL in the product, meeting consumer demand for functional foods.

[0024] Preservation of biological materials

[0025] Lactobacillus salivarius CCFM1266 was deposited on May 17, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNo: 62418. Attached Figure Description

[0026] Figure 1 Changes in viable cell count during fermentation of Lactobacillus salivarius CCFM1266;

[0027] Figure 2 Changes in pH and titratable acidity during fermentation of Lactobacillus salivarius CCFM1266;

[0028] Figure 3 Changes in metabolites during the fermentation process of Lactobacillus salivarius CCFM1266. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. In the embodiments, all original reagent materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

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

[0031] MRS solid medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, K2HPO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4·H2O 0.05g / L, Tween 80 1mL / L, agar 20g / L.

[0032] MRS liquid culture medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, K2HPO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4·H2O 0.05g / L, Tween 80 1mL / L.

[0033] Example 1: Lactobacillus salivarius CCFM1266 compound starter culture increases niacin content in fermented milk.

[0034] 1. Experimental strains

[0035] The technical solution of this invention uses Lactobacillus salivarius CCFM1266 and commercial starter YO-MIX300 (Danisco yogurt starter YO-MIX300 components: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus).

[0036] 2. Activation and preparation of bacterial strains

[0037] (1) Activation of strains

[0038] Lactobacillus salivarius CCFM1266: Spread Lactobacillus salivarius CCFM1266 from glycerol storage tubes onto MRS solid plates and incubate at 37°C for 48 h; pick a single colony and inoculate it into 5 mL of MRS medium and incubate at 37°C for 36 h; collect the strain that has grown to the logarithmic phase and store it in glycerol tubes for later use.

[0039] YO-MIX300: Inoculate YO-MIX300 bacterial powder into 5 mL of MRS medium and incubate at 37°C for 36 h. Collect the strain that has grown to the logarithmic phase and store it in glycerol tubes for later use.

[0040] (2) Preparation of seed liquid

[0041] Lactobacillus salivarius CCFM1266: Dissolve 10% whole milk powder in water at room temperature, sterilize at 95℃ for 5 min, cool to 36℃±1℃, inoculate the strain, ferment at 36℃±1℃ for 48h±2h, which can be extended to 72h±2h, and culture to the logarithmic growth phase to use as seed culture.

[0042] YO-MIX300: Dissolve 10% whole milk powder in water at room temperature, sterilize at 95℃ for 5 min, cool to 36℃±1℃, inoculate with the strain, ferment at 36℃±1℃ for 48h±2h, which can be extended to 72h±2h, and culture to the logarithmic growth phase to serve as seed culture.

[0043] 3. Lactobacillus salivarius CCFM1266 synthesizes niacin in fermented milk.

[0044] Fermentation groups: YO-MIX300, seed culture of sterilized raw milk inoculated with starter culture YO-MIX300; LAS, seed culture of sterilized raw milk inoculated with Lactobacillus salivarius CCFM1266; LASMIX, seed culture of sterilized raw milk inoculated with both Lactobacillus salivarius CCFM1266 and starter culture YO-MIX300.

[0045] Fermented milk preparation: Raw milk was heated to 95℃ for 5 minutes to sterilize, then cooled to 37℃. Seed culture of each strain was added according to the group (inoculation volume ratio of 3%). Each sample was fermented at 37℃ for 0, 1, 3, 6, 10, 12, 24 h, 2 d, 7 d, and 14 d. After the pH value was less than 4.5, it was transferred to 4℃ for refrigeration to continue the post-fermentation process.

[0046] The fermentation emulsion was centrifuged at 8000 r / min for 15 min, and the fermentation supernatant was collected and filtered through a 0.22 μm filter membrane. The filtrate was then used to determine the nicotinic acid content. The specific determination of nicotinic acid content was performed in accordance with the national standard method GB5009.89-2016 "Determination of Nicotinic Acid and Nicotinamide in Food".

[0047] The sample from hour 0 was used as a control. Since coagulation did not occur during the same fermentation period, the supernatant of the blank control could not be obtained by centrifugation. Therefore, the following data are all nicotinic acid contents directly measured in the fermentation broth supernatant, without deducting the nicotinic acid content in the blank milk matrix.

[0048] Table 1. Nicotinic acid content (μg / mL) in fermentation supernatant

[0049] Sample Name 0h 12h 1d 2d 7d 14d YO-MIX300 0.56±0.023 5±2.36 3.57±1.27 2.54±0.59.27 2.03±0.97 2.63±3.97 LAS 0.50±0.084 7.19±0.56 14.09±0.96 15.75±1.36 14.05±1.66 9.15±0.55 LASMIX 0.49±0.057 11.6±1.02 19.32±1.22 22.6±2.39 22.04±1.57 15.77±0.96

[0050] As can be seen from the results in Table 1, compared with Lactobacillus salivarius CCFM1266, the nicotinic acid content produced by the starter culture YO-MIX300 during fermentation was lower, remaining at around 2-3 μg / mL. When Lactobacillus salivarius CCFM1266 and starter culture YO-MIX300 were used together to prepare fermented milk, the nicotinic acid content was significantly higher than that of the YO-MIX300 group and the LAS group.

[0051] Example 2: Lactobacillus salivarius CCFM1266 compound starter culture increases niacin content in fermented milk

[0052] 1. Experimental strains

[0053] The Lactobacillus salivarius CCFM1266 used in the technical solution of this invention

[0054] 2. Activation and preparation of bacterial strains

[0055] Lactobacillus salivarius CCFM1266: Spread Lactobacillus salivarius CCFM1266 from glycerol storage tubes onto MRS solid plates and incubate at 37°C for 48 h; pick a single colony and inoculate it into 5 mL of MRS medium and incubate at 37°C for 36 h; collect the strain that has grown to the logarithmic phase and store it in glycerol tubes for later use.

[0056] YO-MIX300: Inoculate YO-MIX300 bacterial powder into 5 mL of MRS medium and incubate at 37°C for 36 h. Collect the strain that has grown to the logarithmic phase and store it in glycerol tubes for later use.

[0057] (2) Preparation of seed liquid

[0058] Lactobacillus salivarius CCFM1266: Dissolve 10% whole milk powder in water at room temperature, sterilize at 95℃ for 5 min, cool to 36℃±1℃, inoculate the strain, ferment at 36℃±1℃ for 48h±2h, which can be extended to 72h±2h, and culture to the logarithmic growth phase to use as seed culture.

[0059] YO-MIX300: Dissolve 10% whole milk powder in water at room temperature, sterilize at 95℃ for 5 min, cool to 36℃±1℃, inoculate with the strain, ferment at 36℃±1℃ for 48h±2h, which can be extended to 72h±2h, and culture to the logarithmic growth phase to serve as seed culture.

[0060] 3. Lactobacillus salivarius CCFM1266 synthesizes niacin in fermented milk.

[0061] Fermentation groups: LAS, sterilized raw milk inoculated with Lactobacillus salivarius CCFM1266; LASF, sterilized raw milk containing 2% fructooligosaccharides (m / v) inoculated with Lactobacillus salivarius CCFM1266; LASM, sterilized raw milk containing 2% mannose oligosaccharides (m / v) inoculated with Lactobacillus salivarius CCFM1266; LASFM, sterilized raw milk containing 2% fructooligosaccharides and 2% mannose oligosaccharides inoculated with Lactobacillus salivarius CCFM1266.

[0062] Fermented milk preparation: Raw milk containing or without 2% fructooligosaccharides and 2% mannose was heated to 95℃ for 5 minutes for sterilization, cooled to 37℃, and seed liquid was added at an inoculation rate of 3% (v / v). Each sample was fermented at 37℃ for 0, 1, 3, 6, 10, 12, 24 h, 2 d, 7 d, and 14 d. After the pH value was less than 4.5, it was transferred to 4℃ for refrigeration to continue the post-fermentation process.

[0063] The fermented milk broth was centrifuged at 8000 r / min for 15 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The filtrate was then used to determine the nicotinic acid content. Colony counts were performed at each time point using plate counting. Changes in metabolites in the fermented milk supernatant were determined using liquid chromatography-mass spectrometry (LC-MS), with specific operating conditions following those described in DanYang Li, Yan Zheng, Lai-yu Kwok, WenYiZhang, TianSong Sun, "Metabolic footprinting revealed key biochemical changes in a brown fermented milk product using Streptococcus thermophilus," Journal of Dairy Science, Volume 103, Issue 3, 2020, 2128-2138. The nicotinic acid content was determined according to the national standard GB5009.89-2016, "Determination of Nicotinic Acid and Nicotinamide in Food."

[0064] The sample from hour 0 was used as a control. Since coagulation did not occur during the same fermentation period, the supernatant of the blank control could not be obtained by centrifugation. Therefore, the following data are all nicotinic acid contents directly measured in the fermentation broth supernatant, without deducting the nicotinic acid content in the blank milk matrix.

[0065] Table 2. Nicotinic acid content in fermentation supernatant (μg / mL)

[0066] Sample Name 0h 12h 1d 2d 7d 14d LAS 0 6.15±0.89 15.89±1.36 16.77±0.47 16.05±1.76 10.15±1.25 LASF 0 12.39±1.07 22.2±1.30 21.44±1.48 21.45±1.87 16.77±1.77 LASM 0 15.16±0.89 22.12±0.19 24.89±1.27 24.01±0.97 18.87±2.01 LASFM 0 40.69±1.28 65.89±1.57 63.37±1.98 60.33±1.46 55.46±1.46

[0067] from Figure 1 The results showed that Lactobacillus salivarius CCFM1266 could proliferate in dairy products, reaching a plateau phase at approximately 12 hours, and remained above 10 during subsequent fermentation and storage. 7 CFU / mL. The simultaneous addition of fructooligosaccharides and mannose significantly increased the viable count of *Lactobacillus salivarius* CCFM1266. After approximately 12 hours of fermentation, the pH of the fermented milk reached 4.5. Figure 2 ).

[0068] The metabolite profile of fermented milk from Lactobacillus salivarius CCFM1266 starter culture was analyzed using partial least squares discriminant analysis (PLS-DA). Figure 3As shown in Figure A, compared to the baseline at 0 h, the metabolites of all groups underwent significant changes after 12 h of fermentation. At 12 h, the composition of the LASF, LASM, and LASFM groups was clearly separated, indicating that the composition induced by *Lactobacillus salivarius* CCFM1266 fermentation underwent significant changes. The addition of fructooligosaccharides or mannooligosaccharides alone, as well as the simultaneous addition of fructooligosaccharides and mannooligosaccharides, had a significant impact on the composition of the fermented milk. Furthermore, on day 14 of storage, the changes in LAS and LASF were smaller compared to the changes at 12 h, while the changes in LASM and LASFM were larger. This suggests that the post-fermentation maturation process of the fermented milk has a relatively smaller impact on the LAS and LASF groups, but a larger impact on the LASM and LASFM groups.

[0069] Nicotinamide and niacin are two forms of vitamin B3. Nicotinamide levels significantly increased in the LASM and LASFM groups after 12 hours of fermentation, but significantly decreased after 14 days of storage. Niacin levels significantly increased in all four groups after 12 hours of fermentation. The addition of fructooligosaccharides, mannooligosaccharides, or both significantly increased niacin content. Furthermore, the niacin level in the LASFM group was higher than in the other groups at both 12 hours of fermentation and 14 days of storage. The addition of fructooligosaccharides and mannooligosaccharides increased niacin levels in fermented milk containing *Lactobacillus salivarius* CCFM1266. Figure 3 BE).

[0070] The niacin content was determined as shown in Table 2. Adding 2% fructooligosaccharides and / or 2% mannosaccharides to raw milk can significantly increase the niacin content in fermented milk prepared using Lactobacillus salivarius CCFM1266.

[0071] Example 3: Preparation of a niacin-rich dairy beverage by fermentation of Lactobacillus salivarius CCFM1266

[0072] Raw milk containing 2% fructooligosaccharides and 2% mannose oligosaccharides is heated to 95°C for 5 minutes to sterilize, then cooled to 37°C. Seed culture of *Lactobacillus salivarius* CCFM1266 and / or starter culture YO-MIX300 is added at a 3% (v / v) inoculation rate, and fermented at 37°C for 24 hours. The fermentation product is diluted 2-4 times with a mixture of sterilized and cooled water, syrup, emulsifying stabilizer, etc., to produce a ready-to-drink lactic acid bacteria beverage. This beverage not only has a sweet and sour taste and good flavor, but also contains a high level of niacin.

[0073] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for fermentative synthesis of nicotinic acid, characterized in that, The method is to use Lactobacillus salivarius CCFM1266 to ferment to obtain a fermentation product containing nicotinic acid in a dairy product containing fructooligosaccharides and mannan oligosaccharides; the Lactobacillus salivarius CCFM1266 has been preserved in the Guangdong Microbial Culture Collection Center on May 17, 2022, and the preservation number is GDMCC No: 62418; The addition amount of the fructooligosaccharides is 2%, and the addition amount of the mannan oligosaccharides is 2%. The fermentation conditions are 35-45 DEG C, and the fermentation time is 1-7 days.

2. The method of claim 1, wherein, Commercial leavening agents are added during the fermentation process.

3. The method of claim 2, wherein, The commercial leavening agents contain Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

4. The method of claim 1, wherein, The dairy product is raw milk, reconstituted milk or cream.

5. Application of Lactobacillus salivarius CCFM1266 in the fermentation preparation of a product rich in nicotinic acid; the Lactobacillus salivarius CCFM1266 has been preserved in the Guangdong Microbial Culture Collection Center on May 17, 2022, and the preservation number is GDMCC No: 62418; The application is to use Lactobacillus salivarius CCFM1266 to ferment to obtain a product containing nicotinic acid in a dairy product containing fructooligosaccharides and mannan oligosaccharides; the addition amount of the fructooligosaccharides is 2%, and the addition amount of the mannan oligosaccharides is 2%.

6. Use according to claim 5, characterized in that, The product includes food, daily chemical product, feed or medicine.

7. Use according to claim 6, characterized in that, The food includes health products.

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