A probiotic formula for increasing vitamin B in fermented milk and its application

Abstract

The present invention belongs to the field of microbial fermentation, and specifically relates to a probiotic formula for increasing the B vitamins in fermented milk and its application. The present invention provides a method for increasing the folic acid level in fermented milk. The present invention finds that the combined use of bifidobacteria and lactobacilli can significantly increase the folic acid content in fermented milk. The folic acid level of single bacteria fermentation is in the range of 0.5 to 1 μg / g, while the folic acid content can be increased to 18 to 30 μg / g after compound use, which is significantly increased by nearly 18 to 60 times. The present invention further verifies that the combined use of bifidobacteria and lactobacilli does not necessarily achieve better results. Among other B vitamins, the vitamin level content is not effectively improved after compounding. It can be seen that the microbial composition has an unexpected technical effect in increasing the folic acid content of fermented milk, and can be widely used in the preparation of products with higher folic acid content.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and in particular relates to a probiotic formula for improving B vitamins in fermented milk and an application thereof. Background Art

[0002] Vitamin B9, also known as folic acid, is abundant in dark green leafy vegetables, nuts, beans, dairy products, meat, poultry and grains. The World Health Organization WHO / FAO recommends that adults consume 400 μg of vitamin B9 per day. Vitamin B9 is involved in DNA synthesis, amino acid synthesis and erythropoiesis. At the same time, vitamin B9 is involved in the conversion of homocysteine ​​to methionine, and plays a certain role in preventing megaloblastic anemia. In addition, vitamin B9 has a regulatory effect on the homeostasis of immune function. Studies have shown that regulatory T cells highly express folic acid receptors on their surface. In in vitro culture experiments, culturing regulatory T cells under conditions of insufficient folic acid will cause their cell growth to be impaired, and folic acid deficiency will block the cell cycle of CD8+T cells, inhibit the maturation of dendritic cells, and lead to immune dysfunction. Folic acid deficiency can lead to megaloblastic anemia, weakness, intestinal diseases, neurological diseases, etc.

[0003] Studies have confirmed that Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus delbrueckii subspecies bulgaricus, and Streptococcus thermophilus can all synthesize folic acid. [1,2] Most human-derived Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum and Bifidobacterium adolescentis have the ability to synthesize folic acid, but the synthesis capacity of different strains varies. [3]

[0004] Studies have shown that Lactobacillus sakei LZ217 isolated from raw milk can synthesize folic acid up to 239.70±0.03ng / mL, and the active form of folic acid, 5-methyltetrahydrofolate, was detected in its fermentation broth; [4] Lactobacillus plantarum P2R3FA screened out from traditional grain fermented foods has been proven to be able to synthesize folic acid and can significantly alleviate folic acid deficiency in a rat model of folic acid deficiency. [5]

[0005] In addition, the folic acid content in the fermented milk obtained by fermenting cow milk with folic acid-producing Lactobacillus plantarum ATCC 14917, Lactobacillus casei ATCC 15008, and Lactobacillus acidophilus ATCC 4356 increased from 10.80 μg / mL to 63.23 μg / mL, 45.41 μg / mL, and 42.78 μg / mL, respectively. [6] Adding probiotics Lactobacillus paracasei SE160, Lactobacillus casei VC199 and Lactobacillus plantarum VS513 that can synthesize folic acid during cheese making can increase the folic acid content in cheese from 56.39±3.19μg / 100g to 83.28±0.26μg / 100g, 86.68±4.74μg / 100g and 80.20±1.80μg / 100g respectively after 30 days of ripening, and from 58.92±3.84μg / 100g to 122.58±6.03μg / 100g, 104.52±2.24μg / 100g and 102.25±2.32μg / 100g respectively after 60 days of ripening. [7] Fermentation of cereals with Lactobacillus plantarum P2R3FA, which can synthesize folic acid, can significantly increase the folic acid content in the fermented product. [8] Therefore, lactic acid bacteria have the ability to synthesize folic acid, but the effect of combining probiotics with prebiotics in different proportions on the synthesis level of folic acid in fermented milk has not been reported.

[0006] [1] Albano C, Silvetti T, Brasca M. Screening of lactic acid bacteria producing folate and their potential use as adjunct cultures for cheese bio-enrichment[J]. Fems Microbiology Letters, 2020, 367(9).

[0007] [2]Hossain KS,Amarasena S,Mayengbam SB Vitamins and Their Roles in Gut Health[J].Microorganisms,2022,10(6).

[0008] [3]Sugahara H,Odamaki T,Hashikura N,et al.Differences in folateproduction by bifidobacteria of different origins[J].Biosci Microbiota FoodHealth,2015,34(4):87-93.

[0009] [4]Liu M,Chen Q,Sun Y,et al.Probiotic Potential of a Folate-ProducingStrain Latilactobacillus sakei LZ217 and Its Modulation Effects on Human GutMicrobiota[J].Foods,2022,11(2).

[0010] [5]Tamene A,Baye K,Kariluoto S,et al.Lactobacillus plantarum P2R3FAIsolated from Traditional Cereal-Based Fermented Food Increase Folate Status in Deficient Rats[J].Nutrients,2019,11(11).

[0011] [6]Wu Z,Wu J,Cao P,et al.Characterization of probiotic bacteriainvolved in fermented milk processing enriched with folic acid[J].J DairySci,2017,100(6):4223-4229.

[0012] [7] Albano C, Silvetti T, Brasca M. Screening of lactic acid bacteria producing folate and their potential use as adjunct cultures for cheese bio-enrichment[J]. FEMS Microbiol Lett, 2020, 367(9). [8] Tamene A, Baye K, Humblot C. Folate content of a staple food increased by fermentation of a cereal using selected folate-producing microorganisms[J].Heliyon,2022,8(5):e09526. Summary of the invention

[0013] The first aspect of the present invention aims to provide a method.

[0014] The second aspect of the present invention aims to provide a fermented milk.

[0015] The third aspect of the present invention aims to provide a food.

[0016] The purpose of the fourth aspect of the present invention is to provide an application of the method of the first aspect of the present invention.

[0017] The fifth aspect of the present invention aims to provide the use of the fermented milk according to the second aspect of the present invention.

[0018] The sixth aspect of the present invention aims to provide a microbial composition.

[0019] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0020] The first aspect of the present invention provides a method for increasing the content of vitamin B9 in fermented milk, comprising fermenting a fermentation raw material using a microbial composition.

[0021] The microbial composition comprises bifidobacteria and lactobacilli.

[0022] Preferably, the ratio of the number of live bifidobacteria to that of lactobacilli is (1:10) to (10:1).

[0023] Preferably, the ratio of the number of live bifidobacteria to that of lactobacilli is (1:5) to (5:1).

[0024] Preferably, the bifidobacterium comprises at least one of Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. lactis and Bifidobacterium bifidum.

[0025] Preferably, the animal Bifidobacterium lactis subspecies includes at least one of animal Bifidobacterium lactis subspecies BB-12, animal Bifidobacterium lactis subspecies BL-04, animal Bifidobacterium lactis subspecies B420, animal Bifidobacterium lactis subspecies HN019, animal Bifidobacterium lactis subspecies Y6 and animal Bifidobacterium lactis subspecies CECT8145.

[0026] Preferably, the Bifidobacterium longum subsp. longum includes Bifidobacterium longum subsp. longum W68.

[0027] Preferably, the Bifidobacterium bifidum comprises Bifidobacterium bifidum Y22.

[0028] Preferably, the lactobacillus comprises at least one of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus gasseri and Lactobacillus reuteri;

[0029] Preferably, the Lactobacillus plantarum includes at least one of Lactobacillus plantarum FEED8, Lactobacillus plantarum LP45 and Lactobacillus plantarum CN2018;

[0030] Preferably, the Lactobacillus acidophilus includes at least one of Lactobacillus acidophilus K43, Lactobacillus acidophilus NCFM and Lactobacillus acidophilus LA-5;

[0031] Preferably, the Lactobacillus rhamnosus includes at least one of Lactobacillus rhamnosus YGG and Lactobacillus rhamnosus HN001;

[0032] Preferably, the Lactobacillus paracasei comprises at least one of Lactobacillus paracasei K9 and Lactobacillus paracasei Lpc-37;

[0033] Preferably, the Lactobacillus casei includes Lactobacillus casei K35;

[0034] Preferably, the Lactobacillus gasseri comprises Lactobacillus gasseri BDUP;

[0035] Preferably, the Lactobacillus reuteri includes Lactobacillus reuteri K07.

[0036] In some embodiments of the present invention, the bifidobacterium used is Bifidobacterium animalis lactis subspecies BB-12 or Bifidobacterium animalis lactis subspecies BL-04; and the lactobacillus used is Lactobacillus rhamnosus HN001.

[0037] Preferably, the fermentation raw materials include raw milk and prebiotics.

[0038] Preferably, the raw milk comprises animal milk and plant milk.

[0039] Preferably, the animal milk includes at least one of cow's milk, buffalo's milk, yak's milk, goat's milk and camel's milk.

[0040] Preferably, the plant milk comprises at least one of soy milk, peanut milk, oat milk, walnut milk, coconut milk and almond milk.

[0041] Preferably, the prebiotics include at least one of fructooligosaccharides, maltooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides and 2'-fucosyllactose.

[0042] The second aspect of the present invention provides a fermented milk, wherein the fermented milk is prepared by the method according to the first aspect of the present invention.

[0043] The third aspect of the present invention provides a food, comprising the fermented milk according to the second aspect of the present invention.

[0044] Preferably, the food comprises at least one of dairy products, cereal products, bean products, condiments, ice products and beverages.

[0045] More preferably, the dairy product is selected from at least one of sterilized milk, sterilized milk, reconstituted milk, yogurt, yogurt, milk powder, formula milk powder, condensed milk, cheese, casein, whey powder and milk fat.

[0046] The fourth aspect of the present invention provides application of the method of the first aspect of the present invention in promoting the content of vitamin B9 in fermented milk.

[0047] The fifth aspect of the present invention provides the use of the method of the first aspect of the present invention and / or the fermented milk of the second aspect of the present invention in any one of (1) to (3):

[0048] (1) Preparation of food;

[0049] (2) Preparation of health products for improving immunity;

[0050] (3) Preparation of drugs for preventing or treating anemia.

[0051] A sixth aspect of the present invention provides a microbial composition comprising bifidobacteria and lactobacilli.

[0052] Preferably, the ratio of the number of live bifidobacteria to that of lactobacilli is (1:10) to (10:1).

[0053] Preferably, the ratio of the number of live bifidobacteria to that of lactobacilli is (1:5) to (5:1).

[0054] Preferably, the bifidobacterium comprises at least one of Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. lactis and Bifidobacterium bifidum.

[0055] Preferably, the animal Bifidobacterium lactis subspecies includes at least one of animal Bifidobacterium lactis subspecies BB-12, animal Bifidobacterium lactis subspecies BL-04, animal Bifidobacterium lactis subspecies B420, animal Bifidobacterium lactis subspecies HN019, animal Bifidobacterium lactis subspecies Y6 and animal Bifidobacterium lactis subspecies CECT8145.

[0056] Preferably, the Bifidobacterium longum subsp. longum includes Bifidobacterium longum subsp. longum W68.

[0057] Preferably, the Bifidobacterium bifidum comprises Bifidobacterium bifidum Y22.

[0058] Preferably, the lactobacillus comprises at least one of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus gasseri and Lactobacillus reuteri;

[0059] Preferably, the Lactobacillus plantarum includes at least one of Lactobacillus plantarum FEED8, Lactobacillus plantarum LP45 and Lactobacillus plantarum CN2018;

[0060] Preferably, the Lactobacillus acidophilus includes at least one of Lactobacillus acidophilus K43, Lactobacillus acidophilus NCFM and Lactobacillus acidophilus LA-5;

[0061] Preferably, the Lactobacillus rhamnosus includes at least one of Lactobacillus rhamnosus YGG and Lactobacillus rhamnosus HN001;

[0062] Preferably, the Lactobacillus paracasei comprises at least one of Lactobacillus paracasei K9 and Lactobacillus paracasei Lpc-37;

[0063] Preferably, the Lactobacillus casei includes Lactobacillus casei K35;

[0064] Preferably, the Lactobacillus gasseri comprises Lactobacillus gasseri BDUP;

[0065] Preferably, the Lactobacillus reuteri includes Lactobacillus reuteri K07.

[0066] In some embodiments of the present invention, the bifidobacterium used is Bifidobacterium animalis lactis subspecies BB-12 or Bifidobacterium animalis lactis subspecies BL-04; and the lactobacillus used is Lactobacillus rhamnosus HN001.

[0067] The beneficial effects of the present invention are:

[0068] The present invention provides a method for increasing the folic acid level of fermented milk. The present invention finds that the combined use of bifidobacteria and lactobacilli can significantly increase the folic acid content in fermented milk. The folic acid level of single bacteria fermentation is in the range of 0.5 to 1 μg / g, while the folic acid content can be increased to 18 to 30 μg / g after compound use, which is significantly increased by nearly 18 to 60 times. The present invention further verifies that the combined use of bifidobacteria and lactobacilli does not necessarily achieve better results. Among other B vitamins, the vitamin level content is not effectively improved after compounding. It can be seen that the microbial composition has an unexpected technical effect in increasing the folic acid content of fermented milk, and can be widely used in the preparation of products with higher folic acid content. DETAILED DESCRIPTION

[0069] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0070] Example 1 Recovery and cultivation of strains

[0071] Table 1 shows the sources of microorganisms in the examples

[0072] Table 1 Microbial sources and numbers

[0073]

[0074] (1) Take a small amount of freeze-dried bacterial powder in a clean bench and inoculate it into MRSc liquid culture medium. Incubate it in a 37°C incubator for 24 h. Mix the cultured bacterial solution with an equal volume of 50% glycerol and store it at -80°C.

[0075] (2) Streak the previously preserved strain on the MRSc plate, culture it at 37°C for 48 h, pick a single colony and culture it in MRSc liquid culture medium for 24 h.

[0076] (3) Transfer the cells to fresh MRSc liquid culture medium at a 1% inoculum volume, culture at 37°C for 24 h, centrifuge at 4°C, 6000 rpm, discard the supernatant, wash the cells 2-3 times with sterile PBS, and resuspend in sterile PBS for later use.

[0077] Example 2 Effects of different formulations on folic acid levels

[0078] 1. Fermentation

[0079] Weigh 485g of raw milk and 15g of oligofructose (Yuanye Biotechnology S11133), fully dissolve them at 65℃, sterilize them at 95℃ for 5-10min, and adjust the number of viable probiotic strains to 1×10 9 CFU / mL, and then inoculated into sterilized milk at different ratios, fermented at 37°C for 24 hours, demulsified and placed at 4°C for post-maturation, and then samples were taken for processing (previously stored at -80°C before the experiment) to determine the changes in the level of folic acid in fermented milk after different probiotics were compounded at different ratios. The preparation of probiotic fermented milk is shown in Table 2.

[0080] Table 2 Grouping of probiotic fermented milk

[0081]

[0082]

[0083] 2. Fermented milk sample processing

[0084] (1) Take out the sample from -80℃ and freeze-dry 1 mL of the sample;

[0085] (2) Add 500 μL of pre-cooled methanol / acetonitrile / water (2:2:1, v / v) solution containing 0.3% formic acid to the freeze-dried sample, vortex mix, sonicate on ice for 20 min, and let stand at -20°C for 2 h;

[0086] (3) The samples were centrifuged at 16,000 g for 20 min at 4°C, freeze-dried, and stored at -80°C until use;

[0087] (4) Before detection, 100 μL of pre-cooled 50% methanol solution (containing 0.1% formic acid) was added to the sample to reconstitute the freeze-dried sample. After centrifugation at 20,000 g for 15 min at 4°C, 50 μL of the supernatant was added to the injection bottle for injection analysis.

[0088] 3. Folic acid determination

[0089] (1) Liquid phase conditions

[0090] The samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system. The samples were placed in an autosampler at 4°C, the injection volume was 5 μL, the chromatographic column was Waters UPLC HSS T3 column (1.8 μm 2.1×100 mm Column), and the column temperature was 45°C. The mobile phase A was 5 mM ammonium formate and 0.3% formic acid in water, and the mobile phase B was pure methanol, with a flow rate of 300 μL / min.

[0091] Liquid elution gradient: 0-2min, liquid B is 0%, 2-3min linearly changes from 0% to 60%; 3-4.5min, liquid B changes linearly from 60% to 100%; 4.5-6min, liquid B changes linearly from 100% to 5%, 6-11min, liquid B is 5%.

[0092] (2) Mass spectrometry conditions

[0093] Mass spectrometry analysis was performed using a 5500QTRAP mass spectrometer (AB SCIEX) in positive ion mode, with multiple reaction monitoring (MRM) as the detection method. Ion source temperature: 550°C; nebulizer gas (Gas1): 55 psi; auxiliary gas (Gas2): 55 psi; curtain gas (CUR): 40 psi; electrospray gas pressure (ISVF): +4500 V. After the determination results were calculated, the data were analyzed for intergroup significance.

[0094] 4. Experimental results

[0095] According to Table 2, probiotic fermented milk with different proportions was prepared, and the folic acid in the fermented milk samples was determined by high performance liquid chromatography-mass spectrometry. The results showed that:

[0096] After animal Bifidobacterium lactis subspecies BB12, BB04 and Lactobacillus rhamnosus LGG were compounded, there were differences in the level of folic acid in probiotic fermented milk containing 3% oligofructose. Among them, the level of vitamin B9 (folic acid) in fermented milk after animal Bifidobacterium lactis subspecies BB04 and Lactobacillus rhamnosus LGG were higher than that of animal Bifidobacterium lactis subspecies BB12 and Lactobacillus rhamnosus LGG. After animal Bifidobacterium lactis subspecies BB04 and Lactobacillus rhamnosus LGG were compounded in a ratio of 3:2, the level of folic acid in fermented milk containing 3% oligofructose reached 28.5372±1.6134μg / g. The results are shown in Table 3.

[0097] The folic acid levels of different single-bacteria fermentations were only in the range of 0.5 to 1 μg / g, while the lowest folic acid content after compound use was 18 μg / g and the highest was close to 30 μg / g. Compared with single-bacteria fermentation, compound use significantly increased the folic acid content by nearly 18 to 60 times.

[0098] Table 3 Folic acid levels in fermented milk prepared with different proportions of probiotics (μg / g)

[0099]

[0100]

[0101] Note: The same letters indicate no significant difference, and different letters indicate significant difference (p<0.05).

[0102] Comparative Example 1 Effect of different formulas on vitamin B1 levels

[0103] Weigh 485g of raw milk and 15g of oligofructose, fully dissolve them at 65℃, sterilize them at 95℃ for 5-10min, and adjust the number of viable probiotic strains to 1×10 9 CFU / mL, and then inoculated into sterilized bovine milk in different proportions. After fermentation at 37°C for 24 hours, the milk was broken and placed at 4°C for post-ripening. Samples were then taken for processing to determine the changes in thiamine (vitamin B1) levels in fermented milk after different probiotics were compounded in different proportions.

[0104] The grouping and experimental method were the same as those in Example 2. The results are shown in Table 4.

[0105] Table 4 Thiamine levels in fermented milk prepared with different ratios of probiotics (μg / g)

[0106]

[0107] Note: One-way ANOVA (Tukey's post hoc test) was used; the same letters represent no significant difference, and different letters represent significant difference (p<0.05).

[0108] From the above results, it can be seen that the level of thiamine in the compound probiotic fermented milk is significantly lower than that in the single-strain probiotic fermented milk, and among the single-strain probiotic fermented milk, the level of thiamine in Lactobacillus rhamnosus LGG fermented milk is significantly lower than that in the animal Bifidobacterium lactis subspecies BB12 or BB04 fermented milk, and the level of thiamine in animal Bifidobacterium lactis subspecies BB12 fermented milk is significantly lower than that in animal Bifidobacterium lactis subspecies BB04 fermented milk.

[0109] Comparative Example 2 Effects of different formulas on vitamin B2 levels

[0110] Weigh 485g of raw milk and 15g of oligofructose, fully dissolve them at 65℃, sterilize them at 95℃ for 5-10min, and adjust the number of viable probiotic strains to 1×10 9 CFU / mL, and then inoculated into sterilized bovine milk in different proportions. After fermentation at 37°C for 24 hours, the milk was broken and placed at 4°C for post-ripening. Samples were then taken for processing to determine the changes in riboflavin (vitamin B2) levels in fermented milk after different probiotics were compounded in different proportions.

[0111] The grouping and experimental method were the same as those in Example 2. The results are shown in Table 5.

[0112] Table 5 Riboflavin levels in fermented milk prepared with different ratios of probiotics (μg / g)

[0113]

[0114] Note: One-way ANOVA (Tukey's post hoc test) was used; the same letters represent no significant difference, and different letters represent significant difference (p<0.05).

[0115] From the above results, it can be seen that the level of riboflavin in the compound probiotic fermented milk is significantly lower than that in the single-strain probiotic fermented milk, and among the single-strain probiotic fermented milk, the level of riboflavin in Lactobacillus rhamnosus LGG fermented milk is significantly lower than that in the animal Bifidobacterium lactis subspecies BB12 or BB04 fermented milk.

[0116] Comparative Example 3 Effects of different formulas on vitamin B3 levels

[0117] Weigh 485g of raw milk and 15g of oligofructose, fully dissolve them at 65℃, sterilize them at 95℃ for 5-10min, and adjust the number of viable probiotic strains to 1×10 9 CFU / mL, and then inoculated into sterilized bovine milk in different proportions. After fermentation at 37°C for 24 hours, the milk was broken and placed at 4°C for post-ripening. Samples were then taken for processing to determine the changes in niacin (vitamin B3) levels in fermented milk after different probiotics were compounded in different proportions.

[0118] The grouping and experimental method were the same as those in Example 2. The results are shown in Table 6.

[0119] Table 6 Niacin levels in fermented milk prepared with different ratios of probiotics (μg / g)

[0120]

[0121] Note: One-way ANOVA (Tukey's post hoc test) was used; the same letters represent no significant difference, and different letters represent significant difference (p<0.05).

[0122] The results are shown in Table 6. After Bifidobacterium animalis subspecies lactis BB12 or BB04 was compounded with Lactobacillus rhamnosus LGG in different proportions, there were differences in the niacin levels in the fermented milk, and as the inoculation amount of Lactobacillus rhamnosus increased, the niacin level in the fermented milk decreased.

[0123] After animal Bifidobacterium lactis subspecies BB12 or BB04 and Lactobacillus rhamnosus LGG were compounded in a ratio of 4:1, the level of niacin in the fermented milk after fermentation with 3% oligofructose was high, 98.7596±2.6948μg / g and 87.3926±2.5279μg / g, respectively. It can be seen that for niacin (vitamin B3), the niacin level of Lactobacillus rhamnosus was the highest in the case of single bacteria fermentation, but in the case of compounding, the niacin level decreased as the proportion of Lactobacillus rhamnosus increased. Lactobacillus rhamnosus played a technical effect that was completely opposite to that of the single bacteria case in the case of compounding.

[0124] Comparative Example 4 Effects of different formulations on the levels of vitamin B3 derivatives

[0125] Weigh 485g of raw milk and 15g of oligofructose, fully dissolve them at 65℃, sterilize them at 95℃ for 5-10min, and adjust the number of viable probiotic strains to 1×10 9CFU / mL, and then inoculated into sterilized bovine milk in different proportions. After fermentation at 37°C for 24 hours, the milk was broken and placed at 4°C for post-ripening. Samples were then taken for processing to determine the changes in the level of nicotinamide (a vitamin B3 derivative) in fermented milk after different probiotics were compounded in different proportions.

[0126] The grouping and experimental method were the same as those in Example 2. The results are shown in Table 7.

[0127] Table 7 Nicotinamide levels in fermented milk prepared with different ratios of probiotics (μg / g)

[0128]

[0129] Note: One-way ANOVA (Tukey's post hoc test) was used; the same letters represent no significant difference, and different letters represent significant difference (p<0.05).

[0130] From the above results, it can be seen that the level of nicotinamide in the fermented milk of Lactobacillus rhamnosus LGG and the fermented milk of compound probiotics is significantly lower than the level of nicotinamide in the fermented milk of Bifidobacterium lactis subspecies. And the level of nicotinamide in the fermented milk prepared by fermentation of Bifidobacterium lactis subspecies BB12 is significantly higher than the level of nicotinamide in the fermented milk of Bifidobacterium lactis subspecies BB04. Comparative Example 5 Effect of different formulas on vitamin B5 level

[0131] Weigh 485g of raw milk and 15g of oligofructose, fully dissolve them at 65℃, sterilize them at 95℃ for 5-10min, and adjust the number of viable probiotic strains to 1×10 9 CFU / mL, and then inoculated into sterilized bovine milk in different proportions. After fermentation at 37°C for 24 hours, the milk was broken and placed at 4°C for post-ripening. Samples were then taken for processing to determine the changes in the level of pantothenic acid (vitamin B5) in the fermented milk after different probiotics were compounded in different proportions.

[0132] The grouping and experimental method were the same as those in Example 2. The results are shown in Table 8.

[0133] Table 8 Levels of pantothenic acid in fermented milk prepared with different ratios of probiotics (μg / g)

[0134]

[0135]

[0136] Note: One-way ANOVA (Tukey's post hoc test) was used. The same letters represent no significant difference, and different letters represent significant difference (p<0.05).

[0137] From the above results, it can be seen that the level of pantothenic acid in milk fermented with Lactobacillus rhamnosus LGG and fermented milk fermented with compound probiotics is significantly lower than that in milk fermented with single strain Bifidobacterium lactis subspecies, while the level of pantothenic acid in the fermented milk samples prepared by compounding Bifidobacterium lactis subspecies BB12 or BB04 with Lactobacillus rhamnosus LGG at a ratio of 1:4 is higher than that in other ratios. And among the single strain probiotic fermented milk, the level of pantothenic acid in the fermented milk prepared by fermentation with single strain Bifidobacterium lactis subspecies BB12 is significantly higher than that in the fermented milk fermented with Bifidobacterium lactis subspecies BB04.

[0138] Comparative Example 6 Effects of different formulations on the levels of pyridoxine, pyridoxal and pyridoxal phosphate

[0139] Weigh 485g of raw milk and 15g of oligofructose, fully dissolve them at 65℃, sterilize them at 95℃ for 5-10min, and adjust the number of viable probiotic strains to 1×10 9 CFU / mL, and then inoculated into sterilized bovine milk in different proportions. After fermentation at 37°C for 24 hours, the milk was broken and placed at 4°C for post-ripening. Samples were then taken for processing to determine the changes in the levels of pyridoxine, pyridoxal and pyridoxal phosphate (vitamin B6 and its derivatives) in the fermented milk after different probiotics were compounded in different proportions.

[0140] The grouping and experimental method were the same as those in Example 2. The results are shown in Table 9.

[0141] Table 9 Levels of pyridoxine, pyridoxal and pyridoxal phosphate in fermented milk prepared with different ratios of probiotics (μg / g)

[0142]

[0143]

[0144] Note: One-way ANOVA (Tukey's post hoc test) was used; the same letters represent no significant difference, and different letters represent significant difference (p<0.05).

[0145] From the above results, it can be seen that the levels of pyridoxine, pyridoxal and pyridoxal phosphate in the compound probiotic fermented milk are significantly lower than those in the single strain Bifidobacterium lactis fermented milk, while the levels of pyridoxal and pyridoxal phosphate in the fermented milk samples prepared by compounding Bifidobacterium lactis BB12 or BB04 with Lactobacillus rhamnosus LGG at a ratio of 4:1 are higher than those in other ratios. And among the single strain probiotic fermented milk, the levels of pyridoxine, pyridoxal and pyridoxal phosphate in the fermented milk prepared by fermentation of a single strain of Bifidobacterium lactis strain are significantly higher than those in the Lactobacillus rhamnosus LGG fermented milk.

[0146] Comparative Example 7 Effects of different formulations on biotin levels

[0147] Weigh 485g of raw milk and 15g of oligofructose, fully dissolve them at 65℃, sterilize them at 95℃ for 5-10min, and adjust the number of viable probiotic strains to 1×10 9 CFU / mL, and then inoculated into sterilized bovine milk in different proportions. After fermentation at 37°C for 24 hours, the milk was broken and placed at 4°C for post-ripening. Samples were then taken for processing to determine the changes in biotin (vitamin B7) levels in fermented milk after different probiotics were compounded in different proportions.

[0148] The grouping and experimental method were the same as those in Example 2. The results are shown in Table 10.

[0149] Table 10 Biotin levels in fermented milk prepared with different ratios of probiotics (μg / g)

[0150]

[0151] Note: One-way ANOVA (Tukey's post hoc test) was used; the same letters represent no significant difference, and different letters represent significant difference (p<0.05).

[0152] From the above results, it can be seen that the level of biotin in the compound probiotic fermented milk is significantly lower than the level of pantothenic acid in the single probiotic fermented milk. And the level of biotin in the single animal Bifidobacterium lactis fermented milk is significantly higher than the level of biotin in the Lactobacillus rhamnosus fermented milk.

[0153] Comparative Example 8 Effect of different formulas on vitamin B12 levels

[0154] Weigh 485g of raw milk and 15g of oligofructose, fully dissolve them at 65℃, sterilize them at 95℃ for 5-10min, and adjust the number of viable probiotic strains to 1×10 9 CFU / mL, and then inoculated into sterilized bovine milk in different proportions, fermented at 37°C for 24 hours, demulsified and placed at 4°C for post-ripening, and then samples were taken for processing to determine the changes in the level of cobalamin (vitamin B12) in fermented milk after different probiotics were compounded in different proportions.

[0155] The grouping and experimental method were the same as those in Example 2. The results are shown in Table 11.

[0156] Table 11 Cobalamin levels in fermented milk prepared with different ratios of probiotics (μg / g)

[0157]

[0158]

[0159] Note: One-way ANOVA (Tukey's post hoc test) was used; the same letters represent no significant difference, and different letters represent significant difference (p<0.05).

[0160] After the animal Bifidobacterium lactis subspecies BB12, BB04 and Lactobacillus rhamnosus LGG were compounded, there were differences in the level of cobalamin in probiotic fermented milk containing 3% oligofructose. Among them, the level of vitamin B12 (cobalamin) in the fermented milk after the animal Bifidobacterium lactis subspecies BB04 and Lactobacillus rhamnosus LGG were compounded more effectively than the animal Bifidobacterium lactis subspecies BB04 and Lactobacillus rhamnosus LGG. After the animal Bifidobacterium lactis subspecies BB04 and Lactobacillus rhamnosus LGG were compounded in a ratio of 3:2, the level of cobalamin in the fermented milk containing 3% oligofructose reached 527.1965±31.3172ng / g.

[0161] In summary, among the numerous B vitamins, only the content of folic acid was effectively increased after compounding with Bifidobacterium and Lactobacillus, while the other B vitamins achieved the opposite technical effects after compounding.

Claims

1. A method for increasing the content of vitamin B9 in fermented milk, characterized in that: fermenting a fermentation feedstock using a microbial composition; The microbial composition includes bifidobacteria and lactobacilli; The ratio of the number of live bifidobacteria to that of lactobacilli is (1:10) to (10:1); The bifidobacterium is Bifidobacterium animalis subspecies lactis BB-12 and / or Bifidobacterium animalis subspecies lactis BL-04; The lactobacillus is Lactobacillus rhamnosus HN001.

2. The method according to claim 1, characterized in that: The fermentation raw materials include raw milk and prebiotics.

3. The method according to claim 2, characterized in that: The raw milk includes animal milk and plant milk; The animal milk includes at least one of cow's milk, goat's milk and camel's milk; The plant milk comprises at least one of soybean milk, peanut milk, oat milk, walnut milk, coconut milk and almond milk; The prebiotics include at least one of fructooligosaccharides, maltooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides and 2'-fucosyllactose.

4. The method according to claim 2, characterized in that: The raw milk includes animal milk and plant milk; The animal milk includes at least one of buffalo milk and yak milk; The plant milk comprises at least one of soybean milk, peanut milk, oat milk, walnut milk, coconut milk and almond milk; The prebiotics include at least one of fructooligosaccharides, maltooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides and 2'-fucosyllactose.

5. Use of the method according to any one of claims 1 to 4 in increasing the content of vitamin B9 in fermented milk.

6. Use of the method according to any one of claims 1 to 4 in any one of (1) to (3): (1) Preparation of food; (2) Preparation of health products for improving immunity; (3) Preparation of drugs for preventing or treating anemia.

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