A synbiotic combination capable of fermenting and synthesizing vitamin B1, and a fermentation method and application thereof

Abstract

The present invention discloses a synbiotic combination capable of fermenting and synthesizing vitamin B1, and a fermentation method and application thereof, and relates to the field of biotechnology. The synbiotic combination of the present invention comprises: bifidobacteria and / or lactobacilli, and glucose or prebiotics; wherein the prebiotics are selected from at least one of oligofructose, oligogalactose, oligoisomaltose, and 2'-fucosyllactose; the bifidobacteria or lactobacilli are inoculated in a fermentation substrate in the form of seed liquid, the inoculation amount is 1% to 5%, and the inoculation concentration is 1×10 7 cfu / mL~1×10 11 cfu / mL; the concentration of the prebiotic is 10g / L to 50g / L. The synbiotic combination of the present invention can effectively increase the content of vitamin B1 in the fermentation broth, and can promote the growth of related probiotics in the fermentation broth, improve the ability of probiotics to form biofilm, and has wide application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a synbiotic combination capable of fermenting and synthesizing vitamin B1, and a fermentation method and application thereof. Background Art

[0002] Vitamin B1, also known as thiamine, is abundant in cereals, pork, fish, beans and nuts, but is less abundant in wheat flour, refined rice, vegetables and fruits. The World Health Organization (WHO) / Food and Agriculture Organization (FAO) of the United Nations recommends that adults consume 1.1-1.2 mg of vitamin B1 per day. Thiamine is usually absorbed in the duodenum and then converted into its active form, thiamine pyrophosphate (TPP), which can act as a coenzyme in the tricarboxylic acid cycle and the pentose phosphate pathway, such as the oxidative decarboxylation reaction of α-keto acids in the tricarboxylic acid cycle. Vitamin B1 deficiency can lead to abnormal energy metabolism, impaired oxidation reactions, and blocked neurotransmitter synthesis, ultimately leading to impaired health.

[0003] Related studies have shown that some probiotics such as Lactobacillus can effectively synthesize vitamin B1. Lactobacillus casei VKPM B-2873 and Lactobacillus rhamnosus Lactobacillus rhamnosus VKPM B-8238 can synthesize vitamin B1. Lactobacillus fermentum screened from fermented foods Lactobacillus fermentum MT903311 and MT903312 can produce a variety of B vitamins, including vitamin B1, B2, B3, B9 and B 12 . However, its synthesis efficiency is low and it is difficult to meet actual needs.

[0004] Therefore, there is an urgent need to find a composition that can significantly increase the content of vitamin B1 in the fermentation broth. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a synbiotic combination that can ferment and synthesize vitamin B1, which can effectively increase the content of vitamin B1 in the fermentation broth, promote the growth of related probiotics in the fermentation broth, and improve the ability of probiotics to form biofilms.

[0006] The present invention also provides a fermentation medium having the synbiotic combination.

[0007] The invention also provides a fermentation method.

[0008] The present invention also proposes an application of the above synbiotic combination, fermentation medium or fermentation method in the preparation of a product containing vitamin B1.

[0009] The first aspect of the present invention provides a synbiotic combination capable of fermenting and synthesizing vitamin B1, the synbiotic combination comprising:

[0010] A1), Bifidobacterium and / or Lactobacillus, and

[0011] A2), glucose or prebiotics;

[0012] The prebiotic is selected from at least one of oligofructose, oligogalactose, oligoisomaltose and 2'-fucosyllactose; the inoculation amount of bifidobacterium or lactobacillus is 1% to 5% and the inoculation concentration is 1×10 7 cfu / mL~1×10 11 cfu / mL; the concentration of the prebiotic is 10 g / L~50 g / L.

[0013] The synbiotic combination according to the embodiments of the present invention has at least the following beneficial effects: the synbiotic combination of the present invention that can be fermented to synthesize vitamin B1 can effectively increase the content of vitamin B1 in the fermentation broth, and can promote the growth of related probiotics in the fermentation broth, and improve the ability of probiotics to form biofilms.

[0014] Those skilled in the art will appreciate that the inoculation amount of the bifidobacterium or lactobacillus of 1% to 5% means that the volume in the fermentation broth or culture broth is 1% to 5%, and the concentration of the prebiotic of 10 g / L to 50 g / L means that the initial concentration of the prebiotic in the fermentation broth or culture broth after addition is 10 g / L to 50 g / L.

[0015] In some preferred embodiments of the present invention, the bifidobacterium is selected from at least one of Bifidobacterium animalis subsp. lactis, Bifidobacterium longum subsp. longum, and Bifidobacterium bifidum.

[0016] In some embodiments of the present invention, the bifidobacterium is selected from at least one of Bifidobacterium animalis subspecies lactis strain BB-12, Bifidobacterium animalis subspecies lactis strain BL-04, Bifidobacterium animalis subspecies lactis strain B420, Bifidobacterium animalis subspecies lactis strain HN019, Bifidobacterium animalis subspecies lactis strain Y6, Bifidobacterium animalis subspecies lactis strain CECT8145, Bifidobacterium longum subspecies longum W68, and Bifidobacterium bifidum Y22.

[0017] In some preferred embodiments of the present invention, the bifidobacterium is selected from at least one of Bifidobacterium animalis subsp. lactis strain BL-04 and Bifidobacterium longum subsp. longum W68.

[0018] When the bifidobacterium is animal Bifidobacterium lactis subspecies BL-04, it can significantly promote the synthesis of vitamin B1 by adding it in combination with oligofructose to the fermentation broth, which is 6036.1% higher than when glucose is used as a prebiotic. When the bifidobacterium is or Bifidobacterium longum subspecies longum W68, it can significantly promote the synthesis of vitamin B1 by adding it in combination with oligomaltose to the fermentation broth, which is 1750.0% higher than when glucose is used as a prebiotic.

[0019] In some embodiments of the present invention, the lactobacillus is selected from at least one of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus gasseri, and Lactobacillus reuteri.

[0020] In some embodiments of the present invention, the lactobacillus is selected from at least one of Lactobacillus plantarum FEED8, Lactobacillus plantarum LP45, Lactobacillus plantarum CN2018, Lactobacillus acidophilus K43, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus LA-5, Lactobacillus rhamnosus YGG, Lactobacillus rhamnosus HN001, Lactobacillus paracasei K9, Lactobacillus paracasei Lpc-37, Lactobacillus casei K35, Lactobacillus gasseri BDUP, and Lactobacillus reuteri K07.

[0021] In some preferred embodiments of the present invention, the lactobacillus is selected from at least one of Lactobacillus acidophilus K43, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus LA-5, Lactobacillus rhamnosus YGG, Lactobacillus rhamnosus HN001, Lactobacillus paracasei K9, Lactobacillus paracasei Lpc-37, and Lactobacillus casei K35.

[0022] In some embodiments of the present invention, the prebiotic is selected from at least one of fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, and 2'-fucosyllactose.

[0023] In some embodiments of the present invention, when glucose is used as the carbon source, the bifidobacterium is selected from Bifidobacterium animalis subsp. lactis B420 and / or Bifidobacterium longum subsp. longum W68.

[0024] In some embodiments of the present invention, when glucose is used as the carbon source, the lactobacillus is selected from at least one of Lactobacillus plantarum LP45, Lactobacillus acidophilus K43, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus LA-5, and Lactobacillus rhamnosus HN001.

[0025] In some preferred embodiments of the present invention, when glucose is used as the carbon source, the lactobacillus is Lactobacillus acidophilus LA-5.

[0026] In some preferred embodiments of the present invention, when glucose is used as the carbon source, the content of vitamin B1 in the fermentation supernatant of Lactobacillus acidophilus LA-5 is increased by 877.1% relative to the combination of glucose and Lactobacillus rhamnosus YGG.

[0027] In some embodiments of the present invention, the prebiotic is fructooligosaccharide, and the bifidobacterium is selected from Bifidobacterium animalis subsp. lactis strain BL-04 and / or Bifidobacterium longum subsp. longum W68.

[0028] When the synbiotic is a combination of fructooligosaccharide and Bifidobacterium animalis subsp. lactis strain BL-04 or Bifidobacterium longum subsp. longum W68, the content of vitamin B1 in the fermentation supernatant is increased by 14451.4% and 9531.4% respectively compared with the combination of fructooligosaccharide and Bifidobacterium animalis subsp. lactis Y6.

[0029] In some embodiments of the present invention, the prebiotic is oligofructose, and the lactobacillus is selected from at least one of Lactobacillus acidophilus K43, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus LA-5, Lactobacillus rhamnosus YGG, Lactobacillus rhamnosus HN001, Lactobacillus paracasei K9, and Lactobacillus casei K35.

[0030] In some preferred embodiments of the present invention, the prebiotic is fructooligosaccharide, and the lactobacillus is selected from at least one of Lactobacillus rhamnosus YGG and Lactobacillus rhamnosus HN001.

[0031] When the synbiotic is a combination of fructooligosaccharide and Lactobacillus rhamnosus YGG or Lactobacillus rhamnosus HN001, the content of vitamin B1 in the fermentation supernatant is increased by 21062.2% and 22414.0% respectively compared with the combination of fructooligosaccharide and Lactobacillus plantarum FEED8.

[0032] In some embodiments of the present invention, the prebiotic is galacto-oligosaccharide, and the bifidobacterium is selected from Bifidobacterium animalis subsp. lactis strain BL-04 and / or Bifidobacterium longum subsp. longum W68.

[0033] When the synbiotic is a combination of galacto-oligosaccharide and Bifidobacterium animalis subsp. lactis strain BL-04 or Bifidobacterium longum subsp. longum W68, the content of vitamin B1 in the fermentation supernatant is increased by 3093.5% and 15416.1% respectively compared with the combination of galacto-oligosaccharide and Bifidobacterium animalis subsp. lactis Y6.

[0034] In some embodiments of the present invention, the prebiotic is galacto-oligosaccharide, and the lactobacillus is selected from at least one of Lactobacillus acidophilus K43, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus LA-5, and Lactobacillus rhamnosus HN001.

[0035] In some preferred embodiments of the present invention, the prebiotic is galacto-oligosaccharide, and the lactobacillus is selected from at least one of Lactobacillus acidophilus NCFM and Lactobacillus acidophilus LA-5.

[0036] When the synbiotic is a combination of galacto-oligosaccharide and Lactobacillus acidophilus NCFM or Lactobacillus acidophilus LA-5, the content of vitamin B1 in the fermentation supernatant is increased by 6802.3% and 8624.1% respectively compared with the combination of galacto-oligosaccharide and Lactobacillus plantarum CN2018.

[0037] In some embodiments of the present invention, the prebiotic is isomaltooligosaccharide, and the bifidobacterium is selected from Bifidobacterium animalis subsp. lactis strain BL-04 and / or Bifidobacterium longum subsp. longum W68.

[0038] When the synbiotic is a combination of isomaltooligosaccharide and Bifidobacterium animalis subsp. lactis strain BL-04 or Bifidobacterium longum subsp. longum W68, the content of vitamin B1 in the fermentation supernatant is increased by 4030.3% and 1187.9% respectively compared with the combination of isomaltooligosaccharide and Bifidobacterium animalis subsp. lactis Y6.

[0039] In some embodiments of the present invention, the prebiotic is isomaltooligosaccharide, and the lactobacillus is selected from at least one of Lactobacillus acidophilus NCFM, Lactobacillus acidophilus LA-5, Lactobacillus rhamnosus YGG, Lactobacillus paracasei K9, Lactobacillus paracasei Lpc-37, and Lactobacillus casei K35.

[0040] In some preferred embodiments of the present invention, the prebiotic is isomaltooligosaccharide, and the lactobacillus is Lactobacillus casei K35.

[0041] When the synbiotic is a combination of isomaltooligosaccharide and Lactobacillus casei K35, the content of vitamin B1 in the fermentation supernatant is increased by 80600.0% compared with the combination of isomaltooligosaccharide and Lactobacillus reuteri strain LR.

[0042] In some embodiments of the present invention, the prebiotic is 2'-fucosyllactose, and the lactobacillus is selected from at least one of Lactobacillus rhamnosus YGG, Lactobacillus rhamnosus HN001, Lactobacillus paracasei K9, Lactobacillus paracasei Lpc-37, and Lactobacillus casei K35.

[0043] According to the detection results of the present invention, it can be seen that Lactobacillus rhamnosus YGG, Lactobacillus rhamnosus HN001, Lactobacillus paracasei K9, Lactobacillus paracasei Lpc-37 and Lactobacillus casei K35 have a higher utilization rate of 2'-fucosyllactose (2'-FL) than that of Lactobacillus acidophilus strains, and the combination of 2'-fucosyllactose and 2'-fucosyllactose can not only effectively increase the content of vitamin B1 in the fermentation supernatant, but also promote the biofilm formation ability of the strain.

[0044] In some preferred embodiments of the present invention, the prebiotic is 2'-fucosyllactose, and the lactobacillus is Lactobacillus paracasei Lpc-37.

[0045] When the synbiotic is a combination of 2'-fucosyllactose and Lactobacillus paracasei Lpc-37, the content of vitamin B1 in the fermentation supernatant is increased by 530.3%, 3488.3%, 499.8% and 149.4% respectively compared with the combination of glucose, fructooligosaccharide, galacto-oligosaccharide or isomaltooligosaccharide and Lactobacillus paracasei Lpc-37.

[0046] The second aspect of the present invention provides a fermentation culture medium, wherein the raw materials for preparing the culture medium include the synbiotic combination capable of fermenting and synthesizing vitamin B1 as described in the first aspect above, and a basal culture medium.

[0047] In some embodiments of the present invention, the basal culture medium includes but is not limited to MRSc culture medium.

[0048] In some preferred embodiments of the present invention, the raw materials for preparing the MRSc culture medium include beef extract powder, tryptone, yeast extract powder, dipotassium hydrogen phosphate, triammonium citrate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, sodium acetate, Tween-80 and cysteine ​​hydrochloride.

[0049] In some embodiments of the present invention, the concentration of the beef extract powder in the MRSc medium is 8-12 g / L, the concentration of tryptone is 8-12 g / L, the concentration of yeast extract powder is 4-6 g / L, the concentration of dipotassium hydrogen phosphate is 1-3 g / L, the concentration of triammonium citrate is 1-3 g / L, the concentration of magnesium sulfate heptahydrate is 0.4-0.6 g / L, the concentration of manganese sulfate tetrahydrate is 0.2-0.3 g / L, the concentration of sodium acetate is 4-6 g / L, the concentration of cysteine ​​hydrochloride is 0.4-0.6 g / L, and the concentration of Tween-80 is 0.05-0.2% (v / v).

[0050] In some embodiments of the present invention, the inoculation amount of the bifidobacterium or lactobacillus in the fermentation medium is 1% to 5%, and the inoculation concentration is 1×10 7 cfu / mL~1×10 11 cfu / mL.

[0051] In some embodiments of the present invention, the final concentration of the prebiotics in the fermentation medium is 10 g / L to 50 g / L.

[0052] The third aspect of the present invention provides a fermentation method, comprising adding the synbiotic combination capable of fermenting and synthesizing vitamin B1 described in the first aspect to a fermentation broth for fermentation.

[0053] In some embodiments of the present invention, the inoculation amount of the bifidobacterium or lactobacillus is 1% to 5%, and the inoculation concentration is 1×10 7 cfu / mL~1×10 11 cfu / mL.

[0054] In some embodiments of the present invention, the inoculation concentration of the bifidobacterium or lactobacillus is about 1×10 9 cfu / mL.

[0055] In some embodiments of the present invention, the final concentration of the prebiotic is 10 g / L to 50 g / L.

[0056] In some embodiments of the present invention, the final concentration of the prebiotic is 15 g / L to 25 g / L.

[0057] In some preferred embodiments of the present invention, the final concentration of the prebiotics is 20 g / L.

[0058] In some embodiments of the present invention, the fermentation temperature is 37±2°C.

[0059] In some embodiments of the present invention, the fermentation time is 20 to 28 hours.

[0060] In some preferred embodiments of the present invention, the fermentation time is 24 h.

[0061] The fourth aspect of the present invention provides the use of the synbiotic combination capable of fermenting and synthesizing vitamin B1 described in the first aspect, the fermentation medium described in the second aspect, or the fermentation method described in the third aspect in the preparation of a product containing vitamin B1.

[0062] In some embodiments of the present invention, the vitamin B1-containing products include but are not limited to dairy products.

[0063] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0065] Figure 1 The test results of the effects of different prebiotics on the growth of bifidobacteria in Example 7 of the present invention, wherein A is Bifidobacterium animalis subsp. lactis, B is Bifidobacterium longum subsp. longum, and C is Bifidobacterium bifidum;

[0066] Figure 2The test results of the effects of different prebiotics on the growth of lactobacilli in Example 8 of the present invention are shown in Figure 1, wherein A is Lactobacillus plantarum, B is Lactobacillus acidophilus, C is Lactobacillus gasseri, D is Lactobacillus reuteri, E is Lactobacillus rhamnosus, F is Lactobacillus paracasei, and G is Lactobacillus casei;

[0067] Figure 3 The test results of the effects of different prebiotics on the biofilm formation ability of bifidobacteria in Example 9 of the present invention, wherein A is Bifidobacterium animalis subsp. lactis, B is Bifidobacterium longum subsp. longum, and C is Bifidobacterium bifidum;

[0068] Figure 4 The test results of the effects of different prebiotics on the biofilm forming ability of lactobacilli in Example 10 of the present invention are shown, wherein A is Lactobacillus plantarum, B is Lactobacillus acidophilus, C is Lactobacillus gasseri, D is Lactobacillus reuteri, E is Lactobacillus rhamnosus, F is Lactobacillus paracasei, and G is Lactobacillus casei. 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] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0071] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0072] The preparation method of the MRSc medium used in this example includes: weighing 10 g of beef extract, 10 g of trypsin, 5 g of yeast extract powder, 2 g of dipotassium hydrogen phosphate, 2 g of triammonium citrate, 0.5 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate tetrahydrate, 5 g of sodium acetate, 1 mL of Tween-80 and 0.5 g of cysteine ​​hydrochloride, dissolving them in 980 mL of distilled water, sterilizing at 121° C. for 15 min, and adding 20 mL of filtered sterilized glucose or prebiotic solution to make the final concentration of glucose or prebiotics 2% (i.e., 20 g / L).

[0073] The microorganisms used in this example and their sources are shown in Table 1.

[0074] Table 1: Microbial sources and numbers

[0075]

[0076] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0077] 1. Effects of combined fermentation of different strains and prebiotics on vitamin B1

[0078] Example 1: Experimental study on the effect of different combinations of bifidobacteria and prebiotics on vitamin B1

[0079] In this embodiment, the effects of different bifidobacteria and prebiotic combination fermentations on vitamin B1 production are tested, wherein the bifidobacteria are animal Bifidobacterium lactis subspecies strain BB-12, animal Bifidobacterium lactis subspecies strain BL-04, animal Bifidobacterium lactis subspecies strain B420, animal Bifidobacterium lactis subspecies strain HN019, animal Bifidobacterium lactis subspecies Y6, animal Bifidobacterium lactis subspecies CECT8145, Bifidobacterium longum subspecies longum W68, and Bifidobacterium bifidum Y22, and the prebiotics are glucose, fructooligosaccharides, galacto-oligosaccharides, and isomaltooligosaccharides, and the specific testing methods are as follows.

[0080] 1. Strain recovery culture

[0081] The recovery culture method of the strain in this experiment includes: taking a small amount of freeze-dried bacterial powder of different strains and inoculating it into MRSc liquid culture medium in a clean bench, culturing it in a 37 ℃ incubator for 24 hours, and storing the cultured bacterial solution for later use. Another part of the bacterial solution was mixed with an equal volume of 50% glycerol and stored at -80 ℃.

[0082] 2. Fermentation culture of strains

[0083] First, the above strains were streaked and inoculated on plates containing MRSc medium. After culturing at 37 °C for 48 h, single colonies were picked and inoculated in MRSc liquid medium (10 g beef extract, 10 g trypsin, 5 g yeast extract powder, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g triammonium citrate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate tetrahydrate, 5 g sodium acetate, 1 mL Tween-80, and 0.5 g cysteine ​​hydrochloride dissolved in 980 mL distilled water, fixed to 1 L, and sterilized at 121 °C for 15 min) and cultured for 24 h. Then, a 1% inoculation amount (inoculation concentration of approximately 1×10 9 cfu / mL), transferred to fresh MRSc liquid culture medium, cultured at 37°C for 24 h, and then transferred to MRSc culture medium containing glucose (GLU) or fructooligosaccharide (FOS) or galacto-oligosaccharide (GOS) or isomaltooligosaccharide (IMO) with a final concentration of 20 g / L for further culture at 37°C for 24 h.

[0084] The cultured bacterial solution was centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was collected, filtered with a sterile 0.22 μm filter membrane, dispensed into sterile cryopreservation tubes, and stored at -80°C for later use.

[0085] 3. Fermentation broth sample pretreatment

[0086] Take 1 mL of the above supernatant sample and freeze-dry it, add 10 μL of internal standard, 400 μL of pre-cooled methanol / acetonitrile (1 / 1, v / v) solution containing 0.3% formic acid, add black ceramic beads, homogenize twice (20 s each time), vortex mix for 30 s, incubate at 4 ℃ for 10 min to precipitate protein; then centrifuge at 4 ℃, 14000 rcf for 10 min, take 400 μL of supernatant and add 400 μL of pre-cooled gold-labeled water, vortex mix for 30 s, aspirate 800 μL of supernatant for solid phase extraction, blow the filtrate to dryness, and store at -80 ℃ for testing.

[0087] 4. Determination of vitamin B1 in fermentation broth

[0088] All 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 column temperature was 45 °C, the mobile phase A was 5 mM ammonium formate and 0.3% formic acid in water, the mobile phase B was pure methanol, the flow rate was 300 μL / min, and the injection volume was 5 μL.

[0089] The relevant liquid phase gradient is as follows: 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%. A QC sample is set every certain number of experimental samples in the sample queue to detect and evaluate the stability and repeatability of the system; the sample queue is set with a standard mixture of the target substance for the correction of the chromatographic retention time.

[0090] Mass spectrometry analysis was performed using a 5500 QTRAP 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 inter-group T-test significance.

[0091] 5. Results of vitamin B1 content test in the fermentation supernatant of different bifidobacteria and prebiotic combinations

[0092] After bifidobacteria were fermented under different prebiotic conditions, the content of vitamin B1 in the supernatant was shown in Table 2.

[0093] Table 2: Content of vitamin B1 in the fermentation supernatant of Bifidobacterium strains (μg / L)

[0094]

[0095] From the above measurement data, it can be seen that compared with glucose, the level of vitamin B1 in the supernatant of animal Bifidobacterium lactis BB-12 was significantly increased after fermentation with oligofructose and oligoisomaltose as prebiotics, but the level of vitamin B1 in the supernatant was decreased after fermentation with oligofructose and oligoisomaltose, but not significantly; the level of vitamin B1 in the supernatant of animal Bifidobacterium lactis BL-04 was extremely significantly increased after fermentation with oligofructose and oligoisomaltose as prebiotics; after fermentation of oligofructose and oligogalactose by animal Bifidobacterium lactis HN019, the level of vitamin B1 in the fermentation supernatant was increased but not significantly. Compared with glucose, the level of vitamin B1 in the fermentation supernatant was significantly increased after fermentation of galacto-oligosaccharides and isomaltooligosaccharides by Bifidobacterium animalis subsp. lactis BL-04, fermentation of fructo-oligosaccharides, galacto-oligosaccharides and isomaltooligosaccharides by Bifidobacterium longum subsp. longum W68, and fermentation of fructo-oligosaccharides by Bifidobacterium animalis subsp. lactis CECT8145.

[0096] Secondly, compared with glucose, the level of vitamin B1 in the supernatant was significantly increased after the combined fermentation of Bifidobacterium longum subspecies longum W68 and fructo-oligosaccharides or galacto-oligosaccharides. The level of vitamin B1 in the supernatant increased after fermentation of isomaltooligosaccharides, but not significantly. This shows that the combined fermentation of Bifidobacterium longum subspecies longum W68 and fructo-oligosaccharides or galacto-oligosaccharides can better increase the vitamin B1 in the fermentation supernatant, among which galacto-oligosaccharides have a better effect.

[0097] Finally, the level of vitamin B1 in the supernatant of Bifidobacterium bifidum Y22 increased after fermentation in a galacto-oligosaccharide-containing fermentation broth.

[0098] The above results show that except for Bifidobacterium longum subspecies longum Y6 and Bifidobacterium bifidum Y22, the fermentation of oligofructose by other strains of Bifidobacterium longum subspecies can increase the level of vitamin B1 in the fermentation supernatant, among which the combination of Bifidobacterium longum subspecies BL-04 and oligofructose has the best effect, which is increased by 6036.1% relative to the glucose group. In addition, the combined fermentation of Bifidobacterium longum subspecies longum W68 with oligofructose or oligogalactose can also effectively increase the level of vitamin B1 in the fermentation supernatant, among which oligogalactose has a better effect, which is increased by 1750.0% relative to the glucose group.

[0099] Example 2: Experimental study on the effect of different plant lactobacillus strains and prebiotic combination fermentation on vitamin B1

[0100] This example tests the effects of different Lactobacillus plantarum strains and prebiotic combination fermentations on vitamin B1 production, wherein the Lactobacillus plantarum strains include Lactobacillus plantarum FEED8, Lactobacillus plantarum LP45 and Lactobacillus plantarum CN2018, and the prebiotics are glucose, fructooligosaccharides, galacto-oligosaccharides and isomaltooligosaccharides, respectively. The specific testing method refers to the method section of Example 1 above.

[0101] The content of vitamin B1 in the supernatant after fermentation of different Lactobacillus plantarum strains under different prebiotic conditions is shown in Table 3.

[0102] Table 3: Content of vitamin B1 in the fermentation supernatant of Lactobacillus plantarum strains (μg / L)

[0103]

[0104] It can be seen from the above measurement data that when Lactobacillus plantarum ferments to produce vitamin B1, its preferred carbon source is glucose.

[0105] Example 3: Experimental study on the effect of different Lactobacillus acidophilus strains and prebiotic combination fermentation on vitamin B1

[0106] This example tests the effects of different Lactobacillus acidophilus strains and prebiotic combination fermentations on vitamin B1 production, wherein the Lactobacillus acidophilus strains include Lactobacillus acidophilus K43, Lactobacillus acidophilus NCFM and Lactobacillus acidophilus LA-5, and the prebiotics are glucose, oligofructose, galacto-oligosaccharide and isomaltooligosaccharide, respectively. The specific test method refers to the method section of Example 1 above.

[0107] The content of vitamin B1 in the supernatant after fermentation of different Lactobacillus acidophilus strains under different prebiotic conditions is shown in Table 4.

[0108] Table 4: Content of vitamin B1 in the fermentation supernatant of Lactobacillus acidophilus strains (μg / L)

[0109]

[0110] It can be seen from the above measurement data that when the lactobacillus is Lactobacillus acidophilus, the combination with glucose can significantly increase the level of vitamin B1 in the supernatant, among which the combination of Lactobacillus acidophilus LA-5 and glucose has the best effect.

[0111] Example 4: Experimental study on the effect of combined fermentation of Lactobacillus gasseri, Lactobacillus reuteri and prebiotics on vitamin B1

[0112] This example tests the effects of combined fermentation of Lactobacillus gasseri, Lactobacillus reuteri and prebiotics on the production of vitamin B1, wherein the Lactobacillus gasseri is Lactobacillus gasseri BDUP, the Lactobacillus reuteri is Lactobacillus reuteri K07, and the prebiotics are glucose, oligofructose, galacto-oligosaccharide and isomaltooligosaccharide, respectively. The specific test method refers to the method part of the above Example 1.

[0113] After Lactobacillus gasseri and Lactobacillus reuteri were fermented under different prebiotic conditions, the content of vitamin B1 in the supernatant was shown in Table 5.

[0114] Table 5: Content of vitamin B1 in the fermentation supernatant of Lactobacillus gasseri and Lactobacillus reuteri (μg / L)

[0115]

[0116] From the above test data, it can be seen that compared with glucose, the level of vitamin B1 in the supernatant of Lactobacillus gasseri BDUP increased after fermentation with oligofructose, galacto-oligosaccharide and isomaltooligosaccharide as prebiotics. Therefore, the combination of Lactobacillus gasseri BDUP with oligofructose, galacto-oligosaccharide and isomaltooligosaccharide cannot significantly increase the level of vitamin B1 in the fermentation broth.

[0117] In addition, compared with glucose, the level of vitamin B1 in the supernatant of Lactobacillus reuteri K07 was significantly reduced after fermentation with oligofructose, galacto-oligosaccharide and isomaltooligosaccharide as prebiotics. Therefore, the combination of Lactobacillus reuteri K07 with oligofructose, galacto-oligosaccharide and isomaltooligosaccharide cannot increase the level of vitamin B1 in the fermentation broth.

[0118] Example 5: Experimental study on the effect of fermentation of different Lactobacillus rhamnosus strains and prebiotics on vitamin B1

[0119] This example tests the effects of different Lactobacillus rhamnosus strains and prebiotic combination fermentations on vitamin B1 production, wherein the Lactobacillus rhamnosus strains include Lactobacillus rhamnosus YGG and Lactobacillus rhamnosus HN001, and the prebiotics are glucose, oligofructose, galacto-oligosaccharide, isomaltooligosaccharide and 2'-fucosyllactose, respectively. The specific test method refers to the method section of Example 1 above.

[0120] The content of vitamin B1 in the supernatant after fermentation of different Lactobacillus rhamnosus strains under different prebiotic conditions is shown in Table 6.

[0121] Table 6: Content of vitamin B1 in the fermentation supernatant of Lactobacillus rhamnosus strains (μg / L)

[0122]

[0123] From the above determination data, it can be seen that compared with glucose, the level of vitamin B1 in the supernatant of Lactobacillus rhamnosus YGG increased significantly after fermentation with oligofructose, galacto-oligosaccharide, isomalto-oligosaccharide or 2'-fucosyllactose as prebiotics; the level of vitamin B1 in the supernatant of Lactobacillus rhamnosus HN001 increased significantly after fermentation with oligofructose, galacto-oligosaccharide or 2'-fucosyllactose as prebiotics. The above results show that the combination of Lactobacillus rhamnosus strain and oligofructose or 2'-fucosyllactose has the best effect on improving the level of vitamin B1 in the fermentation supernatant.

[0124] Example 6: Experiment on the effect of different Lactobacillus casei strains and prebiotic combination fermentation on vitamin B1

[0125] This example tests the effects of different Lactobacillus paracasei, Lactobacillus casei strains and prebiotic combination fermentations on vitamin B1 production, wherein the Lactobacillus paracasei strains include Lactobacillus paracasei strain K9 and Lactobacillus paracasei strain Lpc-37, the Lactobacillus casei strain is Lactobacillus casei K35, and the prebiotics are glucose, oligofructose, galacto-oligosaccharide, isomaltooligosaccharide and 2'-fucosyllactose, respectively. The specific test method refers to the method section of Example 1 above.

[0126] The content of vitamin B1 in the supernatant of different Lactobacillus casei strains after fermentation under different prebiotic conditions is shown in Table 7.

[0127] Table 7: Content of vitamin B1 in the fermentation supernatant of Lactobacillus paracasei and Lactobacillus casei strains (μg / L)

[0128]

[0129] From the above determination data, it can be seen that compared with glucose, after fermentation with oligofructose, oligoisomaltose or 2'-fucosyllactose as prebiotics, the level of vitamin B1 in the supernatant of Lactobacillus paracasei K9 was significantly increased, and after fermentation with oligofructose or 2'-fucosyllactose as prebiotics, the level of vitamin B1 in the supernatant of Lactobacillus paracasei Lpc-37 was significantly increased; but when galacto-oligosaccharide was prebiotics, the level of vitamin B1 in the fermentation supernatant was not significantly increased. The above results show that the combination of Lactobacillus paracasei strains with oligofructose, oligoisomaltose or 2'-fucosyllactose can increase the level of vitamin B1 in the fermentation supernatant, and the best effect is achieved when combined with 2'-fucosyllactose.

[0130] Compared with glucose, the level of vitamin B1 in the supernatant of Lactobacillus casei K35 increased significantly after fermentation with oligofructose, oligoisomaltose or 2'-fucosyllactose as prebiotics, but the content of vitamin B1 in the fermentation supernatant decreased significantly when galacto-oligosaccharide was used as prebiotics. The above results show that Lactobacillus casei K35, in combination with oligofructose, oligoisomaltose or 2'-fucosyllactose, can increase the level of vitamin B1 in the fermentation supernatant, and the best effect is achieved when combined with 2'-fucosyllactose.

[0131] In summary, compared with the glucose group, the vitamin B1 level in the fermentation supernatant of some strains was significantly increased after lactobacillus was combined with different prebiotics, among which the vitamin B1 level in the supernatant of Lactobacillus rhamnosus YGG combined with fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides or 2'-fucosyllactose, the vitamin B1 level in the supernatant of Lactobacillus rhamnosus HN001 combined with fructooligosaccharides, galacto-oligosaccharides or 2'-fucosyllactose, the vitamin B1 level in the supernatant of Lactobacillus paracasei K9 combined with fructooligosaccharides, isomaltooligosaccharides or 2'-fucosyllactose, the vitamin B1 level in the supernatant of Lactobacillus paracasei Lpc-37 combined with isomaltooligosaccharides or 2'-fucosyllactose, and the vitamin B1 level in the supernatant of Lactobacillus casei K35 combined with fructooligosaccharides, isomaltooligosaccharides or 2'-fucosyllactose was extremely significantly increased.

[0132] 2. Effects of combined fermentation of different strains and prebiotics on strain growth

[0133] Example 7: Effects of different prebiotics on the growth of Bifidobacterium

[0134] In order to explore the effects of different prebiotics on the growth of probiotics, this example detected the effects of different prebiotics on the growth of bifidobacteria. The specific method is as follows:

[0135] In this experiment, glucose was used as the control, and oligofructose (FOS), galacto-oligosaccharide (GOS), xylooligosaccharide (XOS), isomaltooligosaccharide (IMO), 2'-fucosyllactose (2'-FL), inulin (Insulin), polydextrose (PDX) and resistant dextrin (RD) were selected as the sole carbon source to prepare MRSc medium, in which the concentration of glucose or prebiotics was 20 g / L. Subsequently, the Bifidobacterium strains were inoculated into the prepared MRSc medium containing different carbon sources at a 1% inoculation amount, and after anaerobic culture at 37 °C for 24 h, the absorbance of the bacterial solution at 600 nm was measured by a spectrophotometer.

[0136] Test results such as Figure 1 As shown, glucose, fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, inulin, polydextrose and resistant dextrin all have a certain promoting effect on bifidobacteria, among which glucose, fructooligosaccharides, galacto-oligosaccharides and isomaltooligosaccharides have the most significant growth-promoting effect.

[0137] Example 8: Effects of different prebiotics on the growth of Lactobacillus

[0138] In order to explore the effects of different prebiotics on the growth of probiotics, this example detected the effects of different prebiotics on the growth of Lactobacillus. The specific method is as follows:

[0139] In this experiment, glucose was used as the control, and oligofructose (FOS), galacto-oligosaccharide (GOS), xylooligosaccharide (XOS), isomaltooligosaccharide (IMO), 2'-fucosyllactose (2'-FL), inulin, polydextrose (PDX) and resistant dextrin (RD) were selected as the sole carbon sources to prepare MRSc culture medium, in which the concentration of glucose or prebiotics was 20 g / L. Then, the inoculum was inoculated with 1% (inoculation concentration was about 1×10 9 cfu / mL) were inoculated into the prepared MRSc medium containing different carbon sources and cultured at 37 ℃ for 24 h. The absorbance of the bacterial solution at 600 nm was measured using a spectrophotometer.

[0140] Test results such as Figure 2As shown, it is shown that glucose, oligofructose, galacto-oligosaccharide, isomaltooligosaccharide, inulin, polydextrose and resistant dextrin all have a certain promoting effect on lactobacillus, among which glucose, oligofructose, galacto-oligosaccharide and isomaltooligosaccharide have the most significant growth promoting effect. In addition, Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus casei can slightly utilize 2'-fucosyllactose. Therefore, oligofructose, galacto-oligosaccharide and isomaltooligosaccharide are selected for subsequent experiments, while Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus casei increase 2'-fucosyllactose.

[0141] 3. Effects of combined fermentation of different strains and prebiotics on biofilm formation

[0142] Example 9: Effects of different prebiotics on the biofilm formation ability of Bifidobacterium

[0143] This example detects the effects of different prebiotics on the biofilm formation ability of Bifidobacterium, and the specific method is as follows:

[0144] (1) After activating and subculturing the Bifidobacterium to be tested, the inoculum was inoculated at a concentration of 1% (inoculation concentration is about 1×10 9 cfu / mL) were inoculated into a sterile 96-well plate containing MRSc medium containing 2% (final concentration of 20 g / L) glucose or prebiotics, and cultured anaerobically at 37 °C for 12 to 24 h. The medium without inoculation of bacteria was used as the control well.

[0145] (2) Discard the culture medium, wash the plate 2-3 times with sterile PBS, and dry the plate at 37 °C for 1 h.

[0146] (3) Add 200 μL of 0.1% crystal violet solution to the well plate and stain at room temperature for 30 min;

[0147] (4) Discard the staining solution and wash three times with sterile PBS;

[0148] (5) Dry the plate at room temperature, then add 200 μL of 96% ethanol solution and leave at room temperature for 30 min.

[0149] (6) Measure the absorbance (540-620 nm) using an ELISA reader.

[0150] Test results such as Figure 3 As shown, it can be seen that compared with glucose, the biofilm formation ability of bifidobacteria after fermenting oligofructose is generally increased. The biofilm formation ability of animal Bifidobacterium lactis subspecies BB420, BB6, Bifidobacterium longum BB68 and Bifidobacterium bifidum BB22 after fermenting oligogalactose is significantly improved.

[0151] Example 10: Effects of different prebiotics on the biofilm formation ability of Lactobacillus

[0152] This example detects the effects of different prebiotics on the biofilm formation ability of Lactobacillus, and the specific method is as follows:

[0153] (1) After activating and subculturing the lactobacilli to be tested, inoculate them at a 1% inoculum into a sterile 96-well plate containing MRSc medium containing 2% glucose or prebiotics, and culture them at 37°C for 12 to 24 h. The medium without inoculation of the bacteria was used as the control well.

[0154] (2) Discard the culture medium, wash the plate 2-3 times with sterile PBS, and dry the plate at 37 °C for 1 h.

[0155] (3) Add 200 μL of 0.1% crystal violet solution to the well plate and stain at room temperature for 30 min;

[0156] (4) Discard the staining solution and wash three times with sterile PBS;

[0157] (5) Dry the plate at room temperature, then add 200 μL of 96% ethanol solution and leave at room temperature for 30 min.

[0158] (6) Measure the absorbance (540-620 nm) using an ELISA reader.

[0159] Test results such as Figure 4 As shown, it can be seen that compared with glucose, the biofilm formation ability of Lactobacillus plantarum after fermenting oligofructose generally increases; the biofilm formation ability of Lactobacillus acidophilus, Lactobacillus gasseri and Lactobacillus reuteri after fermenting oligofructose isomaltose significantly increases; the biofilm formation ability of Lactobacillus rhamnosus after fermenting oligofructose is significantly increased; the biofilm formation ability of Lactobacillus paracasei after fermenting oligofructose, oligofructose isomaltose and 2'-fucosyllactose is significantly increased; the biofilm formation ability of Lactobacillus casei after fermenting oligofructose, galacto-oligosaccharide, oligoisomaltose and 2'-fucosyllactose is significantly increased.

[0160] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An application of a synbiotic combination in fermentation synthesis of vitamin B1, characterized in that: The synbiotic combination comprises: A1), Lactobacillus, and A2), 2'-fucosyllactose; The inoculation amount of Lactobacillus is 1% to 5%, and the inoculation concentration is 1×10 7 cfu / mL~1×10 11 cfu / mL; the concentration of 2'-fucosyllactose is 10 g / L~50 g / L; The lactobacillus is selected from at least one of Lactobacillus rhamnosus YGG, Lactobacillus rhamnosus HN001, Lactobacillus paracasei K9, Lactobacillus paracasei Lpc-37, and Lactobacillus casei K35.

2. Application of a fermentation method in the synthesis of vitamin B1, characterized in that: The method comprises adding a synbiotic combination to a fermentation broth for fermentation, wherein the synbiotic combination comprises: A1), Lactobacillus, and A2), 2'-fucosyllactose; The inoculation amount of Lactobacillus is 1% to 5%, and the inoculation concentration is 1×10 7 cfu / mL~1×10 11 cfu / mL; the concentration of 2'-fucosyllactose is 10 g / L~50 g / L; The lactobacillus is selected from at least one of Lactobacillus rhamnosus YGG, Lactobacillus rhamnosus HN001, Lactobacillus paracasei K9, Lactobacillus paracasei Lpc-37, and Lactobacillus casei K35.

3. The use according to claim 2, characterized in that: The fermentation temperature is 37±2° C., and / or the fermentation time is 1 to 3 days.

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