Composite bifidobacterium preparation and use thereof in yogurt fermentation

Abstract

Provided in the present application are a composite Bifidobacterium preparation and the use thereof in yogurt fermentation. The strains in the composite Bifidobacterium preparation consist of Bifidobacterium animalis subsp. lactis strain BLa36, Bifidobacterium longum strain BL21, Bifidobacterium breve strain BBr60, Bifidobacterium adolescentis strain BAC30, and Bifidobacterium infantis strain Bl45. In the present application, the five specific types of different Bifidobacterium strains are compounded. There are potential interactions between the five strains, enabling the strains to cooperate with each other and produce synergistic effects in terms of enhancing the gastric juice tolerance of the strains and improving the overall survival rate of fermentation strains. When used in combination with a basic fermentation strain for yogurt fermentation, the composite Bifidobacterium preparation can significantly improve the acid tolerance of lactic acid bacteria in yogurt, thereby leading to a substantial increase in the number of live bacteria that enter the human intestinal tract and exert functions therein. Furthermore, the composite Bifidobacterium preparation can significantly promote intestinal peristalsis and gastric motility, ameliorate imbalances in intestinal flora, and promote intestinal health.

Description

A compound bifidobacterium agent and application thereof in yogurt fermentation TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotic agents, and relates to a compound bifidobacterium agent and application thereof in yogurt fermentation. BACKGROUND

[0002] A large number of microorganisms residing in the intestinal tract are important participants in host metabolism, and play an important role in food digestion and nutrient absorption, immune regulation, disease prevention and the like of the host. When a sufficient amount of probiotics is ingested, the probiotics can have a beneficial effect on the health of the host. The probiotics produce beneficial metabolic products, while inhibiting the excessive growth of harmful bacteria, and promote the balance of the intestinal environment. Therefore, additional supplementation of probiotics is considered to be an important way to restore the balance of the intestinal tract. When the intestinal microecological balance is destroyed, symptoms such as irritable bowel syndrome, constipation and diarrhea can be caused.

[0003] Yogurt is a good carrier of probiotics, and ordinary yogurt usually only contains basic fermentation strains, Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Although these strains play a certain role in the fermentation process, their effect is relatively limited. The main reason is that Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are difficult to tolerate the low-pH gastric acid environment, so the number of live bacteria of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus entering the intestinal tract and playing a role is extremely limited. Adding probiotics to yogurt is an important way to increase the number of beneficial live bacteria of yogurt in the intestinal tract.

[0004] In order to improve the probiotic supplementation effect of yogurt, more types of acid-tolerant probiotics, such as bifidobacterium, Lactobacillus acidophilus and Lactobacillus rhamnosus, are tried to be developed. These probiotics have stronger acid tolerance, can better resist the erosion of gastric acid and bile, increase the survival rate of reaching the intestinal tract, and also need to consider the problems of taste disorder and undesirable texture of yogurt caused by the probiotics. Adding a high dose of probiotics to yogurt has always been a major challenge. SUMMARY

[0005] The present application provides a compound bifidobacterium agent and application thereof in yogurt fermentation.

[0006] In a first aspect, the application provides a compound Bifidobacterium bacterial agent, wherein the strains in the compound Bifidobacterium bacterial agent are composed of Bifidobacterium animalis subsp. lactis BLa36 strain with a preservation number of CGMCC No. 24029, Bifidobacterium longum BL21 strain with a preservation number of CGMCC No. 10452, Bifidobacterium breve BBr60 strain with a preservation number of CGMCC No. 12915, Bifidobacterium adolescentis BAC30 strain with a preservation number of CGMCC No. 19884, and Bifidobacterium infantis BI45 strain with a preservation number of CGMCC No. 15134.

[0007] The application develops a brand-new probiotic compound mode, which is to compound five specific different types of Bifidobacterium strains. It is found that there is a potential interaction among the five strains, which can cooperate with each other to synergistically improve the gastric juice tolerance of the strains and the overall survival rate of the fermentation strains. When the five strains are used for yogurt fermentation together with the basic fermentation strains, the acid tolerance of the lactic acid bacteria in the yogurt can be significantly improved, the number of live bacteria entering the human intestinal tract and playing a role can be significantly increased, the texture of the prepared yogurt is delicate, the taste is rich, and the aroma is rich. The application can significantly promote intestinal peristalsis, promote gastric motility, improve the imbalance of intestinal flora, and promote intestinal health. Compared with other Bifidobacterium compound modes, the compound Bifidobacterium bacterial agent involved in the application is more excellent in the above-mentioned functions.

[0008] Preferably, the ratio of the viable bacterial counts of the BLa36 strain, the BL21 strain, the BBr60 strain, the BAC30 strain, and the BI45 strain is (1-10):(1-10):(1-10):(1-5):(1-5).

[0009] Specific point values in (1-10) can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. Specific point values in (1-5) can be selected from 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc. Other specific point values in the above numerical ranges can be selected, which will not be described one by one here.

[0010] Based on the potential mutual cooperation relationship among the five Bifidobacterium strains, when the specific viable bacteria number ratio relationship is met, the effect of improving gastric juice tolerance and overall survival rate of the strains, and improving yogurt texture, taste, aroma, promoting intestinal peristalsis, and promoting gastric motility is more prominent.

[0011] Preferably, the total viable bacteria number in the compound Bifidobacterium bacterial agent is not less than 1×10 11 CFU / mL or 1×10 11 CFU / g, for example, 1×10 11 CFU / mL (CFU / g), 5×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), 5×10 12 CFU / mL (CFU / g), etc.; other specific point values within the numerical range can be selected, which will not be repeated here.

[0012] Preferably, the dosage form of the compound Bifidobacterium bacterial agent includes a solution, a freeze-dried powder, a capsule, a tablet, or a granule.

[0013] When the compound Bifidobacterium bacterial agent is a freeze-dried powder, it is prepared by the following preparation method:

[0014] The relevant strains are inoculated into the culture medium for activation and fermentation culture to obtain fermentation liquor; the fermentation liquor is centrifuged and mixed with a freeze-drying protective agent for freeze-drying to obtain bacterial powder of each strain; and the bacterial powder of each strain is mixed according to the viable bacteria number ratio to obtain the compound Bifidobacterium bacterial agent.

[0015] Preferably, the freeze-drying protective agent includes any one or a combination of at least two of skimmed milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol, or xylitol.

[0016] In the second aspect, the application provides a compound probiotic agent, which includes the compound Bifidobacterium bacterial agent of the first aspect and a basic bacterial agent; the strains in the basic bacterial agent are composed of Streptococcus thermophilus ST81 strain with a preservation number of CGMCC No. 15752 and Lactobacillus delbrueckii subsp. bulgaricus LB42 strain with a preservation number of CGMCC No. 15751.

[0017] The application also finds that the composite Bifidobacterium bacterial agent involved in the first aspect, in combination with the Streptococcus thermophilus ST81 strain and the Lactobacillus delbrueckii bulgaricus LB42 strain, is used for yogurt fermentation, which can significantly increase the total number of lactic acid bacteria in the yogurt, and the prepared yogurt has delicate texture, rich taste, rich aroma, and can significantly promote intestinal peristalsis, promote gastric motility, improve intestinal flora imbalance, and promote intestinal health.

[0018] Preferably, the ratio of viable bacteria of the ST81 strain to the LB42 strain is 30:(0.001-10), such as 30:0.001, 30:0.01, 30:0.1, 30:0.5, 30:1, 30:2, 30:3, 30:4, 30:5, 30:6, 30:8, 30:10, and other specific point values in the numerical range can be selected, which will not be repeated here.

[0019] The total number of viable bacteria in the basic bacterial agent is not less than 1×10 11 CFU / mL or 1×10 11 CFU / g, such as 1×10 11 CFU / mL (CFU / g), 5×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), and other specific point values in the numerical range can be selected, which will not be repeated here.

[0020] The mass ratio of the composite Bifidobacterium bacterial agent to the basic bacterial agent is (10-300):(10-40); wherein the specific point values in (10-300) can be selected as 10, 20, 30, 50, 70, 80, 100, 150, 200, 250, 300, etc.; wherein the specific point values in (10-40) can be selected as 10, 15, 20, 25, 30, 35, 40, etc.; other specific point values in the numerical range can be selected, which will not be repeated here.

[0021] The dosage form of the composite probiotic agent includes solution, lyophilized powder, capsule, tablet or granule.

[0022] In a third aspect, the application provides the use of the composite Bifidobacterium bacterial agent of the first aspect or the composite probiotic agent of the second aspect in yogurt fermentation.

[0023] Preferably, the method for yogurt fermentation comprises:

[0024] (1) mixing raw cow milk with a composite prebiotic, sterilizing and cooling to serve as a fermentation base;

[0025] (2) inoculating the composite probiotic agent into the fermentation base for fermentation.

[0026] Preferably, the complex prebiotic comprises any one or a combination of at least two of fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomalto-oligosaccharides, soybean oligosaccharides, inulin, polydextrose, alpha-lactalbumin or lactoferrin.

[0027] Preferably, the complex prebiotic is 0.01-1% of the fermentation base, such as 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc.

[0028] Preferably, the inoculation amount of the complex probiotic agent is 20-300g / T, such as 20g / T, 50g / T, 70g / T, 80g / T, 100g / T, 150g / T, 200g / T, 250g / T, 300g / T, etc.

[0029] Preferably, the fermentation temperature is 40-43℃, such as 40℃, 41℃, 42℃, 43℃, etc.; the fermentation time is 5-10h, such as 5h, 6h, 7h, 8h, 9h, 10h, etc.; the end point of fermentation is that the acidity reaches 70-80°T (such as 70°T, 72°T, 75°T, 78°T, 80°T, etc.) or pH = 4.3-4.5 (such as pH = 4.3, pH = 4.4, pH = 4.5, etc.).

[0030] Other specific point values within the above numerical ranges can be selected, which will not be repeated here.

[0031] In a fourth aspect, the application provides use of the complex bifidobacterium agent of the first aspect or the complex probiotic agent of the second aspect in the preparation of a product for promoting intestinal peristalsis and promoting intestinal health.

[0032] The product includes food, health products or pharmaceuticals.

[0033] Compared with the prior art, the application has the following beneficial effects:

[0034] The application develops a brand-new probiotic compound mode, which is to compound five specific different types of bifidobacterium strains, and find that there is potential interaction among the five strains, which can cooperate with each other, synergistically improve the gastric juice tolerance of the strains and the overall survival rate of the fermentation strains, and use the compound basic fermentation strains for yogurt fermentation, which can significantly improve the acid tolerance of lactic acid bacteria in yogurt, significantly increase the number of live bacteria entering the human intestine and playing a role; and the prepared yogurt has delicate texture, rich taste and rich aroma; and can significantly promote intestinal peristalsis, promote gastric motility, improve intestinal flora imbalance, and promote intestinal health. Compared with other bifidobacterium compound modes, the compound bifidobacterium agent involved in the application is more excellent in the above-mentioned functions.

[0035] The classification name of the BLa36 strain involved in the application is Bifidobacterium animalis subsp. lactis, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is December 2, 2021, the preservation number is CGMCC No. 24029, and the address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0036] The classification name of the BL21 strain involved in the application is Bifidobacterium longum, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is January 27, 2015, the preservation number is CGMCC No. 10452, and the address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0037] The classification name of the BBr60 strain involved in the application is Bifidobacterium breve, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is August 29, 2016, the preservation number is CGMCC No. 12915, and the address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0038] The classification name of the BAC30 strain involved in the application is Bifidobacterium adolescentis, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is May 28, 2020, the preservation number is CGMCC No. 19884, and the address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0039] The classification name of the BI45 strain involved in the present application is Bifidobacterium infantis, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is December 27, 2017, the preservation number is CGMCC No.15134, and the address is No.3, Beichen West Road, Chaoyang District, Beijing.

[0040] The classification name of the ST81 strain involved in the present application is Streptococcus thermophilus, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is May 11, 2018, the preservation number is CGMCC No.15752, and the address is No.3, Beichen West Road, Chaoyang District, Beijing.

[0041] The classification name of the LB42 strain involved in the present application is Lactobacillus delbrueckii subsp.bulgaricus, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is May 11, 2018, the preservation number is CGMCC No.15751, and the address is No.3, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0042] Fig. 1 is a statistical diagram of the abundance of Firmicutes in the cecum of mice in each group.

[0043] Fig. 2 is a statistical diagram of the abundance of Bacteroidetes in the cecum of mice in each group.

[0044] Fig. 3 is a statistical diagram of the ratio of Firmicutes to Bacteroidetes (F / B) in the cecum of mice in each group.

[0045] Fig. 4 is a statistical diagram of the abundance of Lachnospira in the cecum of mice in each group.

[0046] Fig. 5 is a statistical diagram of the abundance of Alistipes in the cecum of mice in each group.

[0047] Fig. 6 is a statistical diagram of the abundance of Acinetobacter in the cecum of mice in each group. DETAILED DESCRIPTION

[0048] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application, and should not be regarded as specific limitations on the present application.

[0049] The medium formula involved in the following examples is as follows:

[0050] MRS medium (g / L): 10 g / L of proteose peptone, 10 g / L of beef extract, 20 g / L of glucose, 2 g / L of sodium acetate, 5 g / L of yeast powder, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of K2PO4·3H2O, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4, 1 mL / L of polysorbate 80 (Tween 80), 0.5 g / L of cysteine hydrochloride.

[0051] Preparation Example 1

[0052] The present preparation example provides ten kinds of bacterial powder products, and the specific preparation method is as follows:

[0053] The BLa36 strain, BL21 strain, BBr60 strain, BAC30 strain, BI45 strain, commercially available ATCC55814 strain, commercially available ATCC15700 strain, commercially available ATCC15703 strain, ST81 strain, and LB42 strain were inoculated into MRS liquid medium, activated at 37°C for 24 h, continuously activated for 2 times, and activated liquid was obtained; the activated liquid was inoculated into MRS liquid medium at an inoculation amount of 3% (v / v), and cultured at 37°C for 18 h to obtain bacterial liquid; the bacterial liquid was centrifuged at 4000 rpm for 5 min at 4°C, filtered, mixed with 5 times the mass of protective agent (20% skim milk), and freeze-dried to obtain BLa36 bacterial powder, BL21 bacterial powder, BBr60 bacterial powder, BAC30 bacterial powder, BI45 bacterial powder, ATCC55814 bacterial powder, ATCC15700 bacterial powder, ATCC15703 bacterial powder, ST81 bacterial powder, and LB42 bacterial powder. The detection results showed that the viable bacterial counts were 5×10 11 CFU / g, 1×10 12 CFU / g, 1×10 11 CFU / g, 1×10 12 CFU / g, 6×10 11 CFU / g, 1×10 11 CFU / g, 5×10 11 CFU / g, 1×10 12 CFU / g, 1×10 11 CFU / g, 1×10 11 CFU / g, and 1×10

[0054] Preparation Example 2

[0055] The present preparation example provides the following compound bifidobacterium agent product:

[0056] (1) Five-bacteria compound probiotic agent 1: BLa36 bacterial powder, BL21 bacterial powder, BBr60 bacterial powder, BAC30 bacterial powder and BI45 bacterial powder with a mass ratio of 2:2:2:1:1 are uniformly mixed to obtain the compound probiotic agent.

[0057] (2) Five-bacteria compound probiotic agent 2: BLa36 bacterial powder, BL21 bacterial powder, BBr60 bacterial powder, BAC30 bacterial powder and BI45 bacterial powder with a mass ratio of 1:1:2:1:1 are uniformly mixed to obtain the compound probiotic agent.

[0058] (3) Five-bacteria compound probiotic agent 3: BLa36 bacterial powder, commercially available ATCC55814 bacterial powder, commercially available ATCC15700 bacterial powder, commercially available ATCC15703 bacterial powder and BI45 bacterial powder with a mass ratio of 2:2:2:1:1 are uniformly mixed to obtain the compound probiotic agent.

[0059] (4) Four-bacteria compound probiotic agent 1: BL21 bacterial powder, BBr60 bacterial powder, BAC30 bacterial powder and BI45 bacterial powder with a mass ratio of 2:2:1:1 are uniformly mixed to obtain the compound probiotic agent.

[0060] (5) Four-bacteria compound probiotic agent 2: BLa36 bacterial powder, BBr60 bacterial powder, BAC30 bacterial powder and BI45 bacterial powder with a mass ratio of 2:2:1:1 are uniformly mixed to obtain the compound probiotic agent.

[0061] (6) Four-bacteria compound probiotic agent 3: BLa36 bacterial powder, BL21 bacterial powder, BAC30 bacterial powder and BI45 bacterial powder with a mass ratio of 2:2:1:1 are uniformly mixed to obtain the compound probiotic agent.

[0062] (7) Four-bacteria compound probiotic agent 4: BLa36 bacterial powder, BL21 bacterial powder, BBr60 bacterial powder and BI45 bacterial powder with a mass ratio of 2:2:2:1 are uniformly mixed to obtain the compound probiotic agent.

[0063] (8) Four-bacteria compound probiotic agent 5: BLa36 bacterial powder, BL21 bacterial powder, BBr60 bacterial powder and BAC30 bacterial powder with a mass ratio of 2:2:2:1 are uniformly mixed to obtain the compound probiotic agent.

[0064] (9) Three-bacteria compound probiotic agent: BLa36 bacterial powder, BL21 bacterial powder and BBr60 bacterial powder with a mass ratio of 1:1:1 are uniformly mixed to obtain the compound probiotic agent.

[0065] Preparation Example 3

[0066] The present preparation example provides the following basic fermentation bacterial agent products:

[0067] (1) Basic fermentation bacterial agent 1: ST81 bacterial powder and LB42 bacterial powder with a mass ratio of 10:1 are uniformly mixed to obtain the compound probiotic agent.

[0068] (2) Basic fermentation agent 2: ST81 bacterial powder and LB42 bacterial powder with a mass ratio of 3:1 were mixed uniformly, and the mixture was obtained.

[0069] Example 1

[0070] This example provides a yogurt product, and the preparation method is as follows:

[0071] (1) White granulated sugar and a composite prebiotic (xylo-oligosaccharide, fructo-oligosaccharide, and isomalto-oligosaccharide with an equal mass ratio) were added to raw cow milk to dissolve fully, wherein the raw cow milk accounted for 94% of the total weight, the white granulated sugar accounted for 5%, and the composite prebiotic accounted for 1%. Then, the mixture was sterilized at 95°C for 15 min, and then quickly cooled to 40°C in cold water as a fermentation base for standby;

[0072] (2) The basic fermentation agent 1 prepared in Preparation Example 3 (inoculation amount of 30 g / T) and the five-bacterial compound probiotic agent 1 prepared in Preparation Example 2 (inoculation amount of 100 g / T) were inoculated into the sterilized fermentation base, and the fermentation temperature was 43°C. The fermentation was stopped when the acidity reached 75°T or pH = 4.4, and then the mixture was stored at 4°C.

[0073] The viable Bifidobacterium count in the yogurt was counted according to the method in QB / T 4575-2013, and the results showed that the total viable Bifidobacterium count was 8 x 10 8 CFU / g of yogurt.

[0074] Example 2

[0075] This example provides a yogurt product, and the preparation method is as follows:

[0076] (1) White granulated sugar and a composite prebiotic (xylo-oligosaccharide, fructo-oligosaccharide, and isomalto-oligosaccharide with an equal mass ratio) were added to raw cow milk to dissolve fully, wherein the raw cow milk accounted for 94% of the total weight, the white granulated sugar accounted for 5%, and the composite prebiotic accounted for 1%. Then, the mixture was sterilized at 95°C for 15 min, and then quickly cooled to 40°C in cold water as a fermentation base for standby;

[0077] (2) The basic fermentation agent 2 prepared in Preparation Example 3 (inoculation amount of 30 g / T) and the five-bacterial compound probiotic agent 2 prepared in Preparation Example 2 (inoculation amount of 100 g / T) were inoculated into the sterilized fermentation base, and the fermentation temperature was 40°C. The fermentation was stopped when the acidity reached 75°T or pH = 4.4, and then the mixture was stored at 4°C.

[0078] The viable Bifidobacterium count in the yogurt was counted according to the method in QB / T 4575-2013, and the results showed that the total viable Bifidobacterium count was 4 x 10 8 CFU / g of yogurt.

[0079] Comparative Example 1

[0080] This comparative example provides a yogurt product, the preparation method of which is different from that of Example 1 only in that the five-strain probiotic agent 1 prepared in Preparation Example 2 is replaced with the four-strain probiotic agent 1 prepared in Preparation Example 2 in equal amount. Other conditions remain unchanged.

[0081] Comparative Example 2

[0082] This comparative example provides a yogurt product, the preparation method of which is different from that of Example 1 only in that the five-strain probiotic agent 1 prepared in Preparation Example 2 is replaced with the four-strain probiotic agent 2 prepared in Preparation Example 2 in equal amount. Other conditions remain unchanged.

[0083] Comparative Example 3

[0084] This comparative example provides a yogurt product, the preparation method of which is different from that of Example 1 only in that the five-strain probiotic agent 1 prepared in Preparation Example 2 is replaced with the four-strain probiotic agent 3 prepared in Preparation Example 2 in equal amount. Other conditions remain unchanged.

[0085] Comparative Example 4

[0086] This comparative example provides a yogurt product, the preparation method of which is different from that of Example 1 only in that the five-strain probiotic agent 1 prepared in Preparation Example 2 is replaced with the four-strain probiotic agent 4 prepared in Preparation Example 2 in equal amount. Other conditions remain unchanged.

[0087] Comparative Example 5

[0088] This comparative example provides a yogurt product, the preparation method of which is different from that of Example 1 only in that the five-strain probiotic agent 1 prepared in Preparation Example 2 is replaced with the four-strain probiotic agent 5 prepared in Preparation Example 2 in equal amount. Other conditions remain unchanged.

[0089] Comparative Example 6

[0090] This comparative example provides a yogurt product, the preparation method of which is different from that of Example 1 only in that the five-strain probiotic agent 1 prepared in Preparation Example 2 is replaced with the five-strain probiotic agent 3 prepared in Preparation Example 2 in equal amount. Other conditions remain unchanged.

[0091] Comparative Example 7

[0092] This comparative example provides a yogurt product, the preparation method of which is different from that of Example 1 only in that the five-strain probiotic agent 1 prepared in Preparation Example 2 is replaced with the three-strain probiotic agent prepared in Preparation Example 2 in equal amount. Other conditions remain unchanged.

[0093] Comparative Example 8

[0094] The comparative example provides a yogurt product, the preparation method of which is only different from that of Example 1 in that no five-bacterial complex probiotic agent 1 prepared in Preparation Example 2 is inoculated in step (2), and other conditions remain unchanged.

[0095] Test Example 1

[0096] Evaluation of in vitro gastric juice tolerance:

[0097] (1) Solution preparation:

[0098] Preparation of electrolyte solution A: weigh 0.064 g of potassium chloride, 0.015 g of potassium dihydrogen phosphate, 0.263 g of sodium bicarbonate, 0.345 g of sodium chloride, 0.003 g of magnesium chloride hexahydrate, 0.006 g of ammonium carbonate, 1.5 g of tryptone, and 0.05 g of L-cysteine hydrochloride, add 95 mL of distilled water to dissolve thoroughly, adjust the pH to 3.0, and dilute to 100 mL.

[0099] Preparation of electrolyte solution B: weigh 0.022 g of calcium chloride dihydrate, dissolve in water and dilute to 100 mL.

[0100] Dissolution solution: weigh 8.5 g of sodium chloride and 0.5 g of L-cysteine hydrochloride monohydrate, add 1000 mL of distilled water to dissolve thoroughly.

[0101] The above three solutions are sterilized at 121℃ for 15 min and then used.

[0102] Take 8.0 mL of electrolyte solution A and 1.0 mL of electrolyte solution B in a beaker, add an amount of pepsin corresponding to 4000 U, adjust the pH according to the different simulated gastric juice conditions in Table 1 using 1 mol / L hydrochloric acid solution or 1 mol / L sodium hydroxide solution, dilute to 10 mL, mix well, filter through a 0.22 μm sterile filter membrane to prepare a simulated gastric juice, and use it immediately.

[0103] Table 1

[0104] (2) Detection of Bifidobacterium:

[0105] Take 1.0 g of the yogurt prepared in Examples 1-2 and Comparative Example 1-7 respectively in a sterile homogenization bag containing 99 mL of dissolution solution, mix thoroughly, pass through the homogenizer at 10 times per second for 2 min, until the sample is evenly dispersed, to prepare a sample bacterial solution, which is prepared immediately and used, and the total bacterial concentration of Bifidobacterium in the sample bacterial solution is determined according to GB 4789.35-2023, and is recorded as N1 (unit: CFU / mL).

[0106] Take 5.0 mL simulated gastric juice and 5.0 mL sample bacterial solution in a 50 mL centrifuge tube, vortex mix, slowly add about 5 mL mineral oil on the solution with a rubber dropper, form an oil seal layer on the solution, place the centrifuge tube in a 37℃ constant temperature water bath incubator for a specified treatment time, remove the mineral oil with a rubber dropper, measure the total concentration of Bifidobacterium, recorded as N1'(unit: CFU / mL).

[0107] Repeat the above operation twice, where the total concentration of Bifidobacterium before treatment is recorded as N2 and N3, and the total concentration of Bifidobacterium after treatment is recorded as N2' and N3'.

[0108] The gastric juice resistance is evaluated by the survival rate of the strain, and the calculation formula is: Survival rate (%) = (2N1' / N1+2N2' / N2+2N3' / N3) / 3x100%

[0109] The average survival rate of each group is shown in Table 2:

[0110] Table 2

[0111] From the data results in Table 2, it can be seen that the yogurt product obtained by using the composite Bifidobacterium bacterial agent with the basic fermentation bacterial agent for fermentation has excellent gastric acid resistance, and the survival rate of Bifidobacterium can still reach 83.7% at pH=2. Compared with the data of Comparative Examples 1-7, if the strain compounding method of the composite Bifidobacterium bacterial agent is changed, the survival rate of Bifidobacterium in acidic environment is significantly reduced, which indicates that it affects the gastric acid resistance of yogurt.

[0112] (3) Detection of lactic acid bacteria:

[0113] Take 1.0 g of yogurt prepared in Example 1-2 and Comparative Example 1-8 respectively under sterile operation, place in a sterile homogenization bag containing 99 mL of dissolution solution, mix thoroughly, then pass through the homogenizer at 10 times / sec for 2 min, until the sample is evenly dispersed, prepare the sample bacterial solution, use it immediately, and measure the total concentration of lactic acid bacteria in the sample bacterial solution according to GB 4789.35-2023, recorded as N1(unit: CFU / mL).

[0114] Take 5.0 mL simulated gastric juice and 5.0 mL sample bacterial solution in a 50 mL centrifuge tube, vortex mix, slowly add about 5 mL mineral oil on the solution with a rubber dropper, form an oil seal layer on the solution, place the centrifuge tube in a 37℃ constant temperature water bath incubator for a specified treatment time, remove the mineral oil with a rubber dropper, measure the total concentration of Bifidobacterium, recorded as N1'(unit: CFU / mL).

[0115] The above operation was repeated twice, and the total lactic acid bacteria concentration determination results before treatment were recorded as N2 and N3, and the total lactic acid bacteria concentration determination results after treatment were recorded as N2' and N3'.

[0116] The gastric juice resistance was evaluated by the survival rate of the strains, and the calculation formula was: Survival rate (%) = (2N1' / N1+2N2' / N2+2N3' / N3) / 3x100%

[0117] The average survival rate of lactic acid bacteria (strains in the compound bifidobacterium agent and the basic fermentation agent are lactic acid bacteria) in each group is shown in Table 3:

[0118] Table 3

[0119] As can be seen from the data results in Table 3, the yogurt product obtained by using the compound bifidobacterium agent in combination with the basic fermentation agent for fermentation has excellent gastric acid resistance, wherein the survival rate of all lactic acid bacteria can still reach 81.6% at pH=2, while the survival rate of lactic acid bacteria in the yogurt product obtained by using only the basic fermentation agent for fermentation is only 8.7% at pH=2. Compared with the data of Comparative Examples 1-7, if the strain compounding mode of the compound bifidobacterium agent is changed, the survival rate of lactic acid bacteria in the acidic environment is significantly reduced, which indicates that it affects the gastric acid resistance of yogurt.

[0120] Test Example 2

[0121] Evaluation of the effect on intestinal motility of mice:

[0122] (1) Test animals:

[0123] 8-week-old BALB / c male mice (20±2g) 120, clean and quiet feeding environment, temperature 20-24℃, humidity 50-60%, mice purchased from Spafas (Beijing) Biotechnology Co., Ltd.

[0124] (2) Animal grouping and intervention method:

[0125] After all the mice were adapted for 7 days, the mice were randomly divided into 12 groups: blank control group NC, model group MC, yogurt groups prepared from Examples 1-2 and Comparative Examples 1-8. On the first day of the test, the blank control group and the model group were given 200μL of normal saline by gavage, and the other yogurt groups were given 200μL of yogurt by gavage, once a day during the test period. The modeling method was as follows: on the 15th-17th day of the test, except for the blank control group, the mice in the other groups were given 10mg / kg.bw loperamide by gavage, and 4h later, they were given normal saline or yogurt by gavage. The specific situation is shown in Table 4.

[0126] Table 4

[0127] (3) Mouse safety evaluation

[0128] During the animal experiment, the body weight and food intake were measured twice a week, and the health status of the mice was observed. The results showed that before modeling, the body weight of the mice in each group showed an upward trend, indicating that the intervention sample had no side effects on the weight gain of the mice, and there was no significant difference in the body weight change of the mice in each group, and the mice in each group gained more than 5%, indicating that the mice were healthy and grew well. The food utilization rate of the mice in each group was similar to that of the control, and the food utilization rate was higher than 3%, indicating that the mice in each group had good food utilization.

[0129] (4) Effect evaluation:

[0130] (4.1) Time of first black stool:

[0131] On the 18th day of the experiment, after fasting for 16 hours, the control group was given physiological saline by gavage, and the other groups of mice were given loperamide by gavage. After 1 hour, 0.2 mL of ink was given to each mouse by gavage. From the gavage of ink, the time of the first black stool of each mouse (min) was recorded, and the time of the last mouse in the model group was taken as the termination time.

[0132] The statistical results are shown in Table 5. Compared with the blank control group, the time of the first black stool of the mice in the model group was significantly prolonged. After intervention with yogurt in each group, the time of the first black stool of the mice in each group was shortened to varying degrees, and the effect of Example 1 and Example 2 groups had a significant advantage, indicating that the probiotic agent involved in the present application had the effect of promoting intestinal peristalsis and lubricating the intestines and defecation.

[0133] (4.2) Small intestine propulsion rate:

[0134] On the 19th day of the experiment, the control group was given physiological saline by gavage, and the other groups of mice were given loperamide by gavage. After 1 hour, all mice were given ink (0.2 mL per mouse) by gavage. After 25 minutes, the mice were sacrificed, and the intestinal tube from the pylorus to the ileocecal junction was cut. The small intestine was gently stretched into a straight line, and the length of the intestinal tube was measured as the "total length of the small intestine". The length of the ink propulsion was from the pylorus to the front of the ink, and the small intestine propulsion rate (%) = ink propulsion length / total length of small intestine x 100%.

[0135] The statistical results are shown in Table 5. Compared with the blank control group, the small intestine propulsion rate of the mice in the model group was significantly reduced. After intervention with yogurt in each group, the small intestine propulsion rate of the mice in each group was restored to varying degrees, and the recovery effect of Example 1 and Example 2 groups had a significant advantage, indicating that the probiotic agent involved in the present application had the effect of promoting intestinal peristalsis and lubricating the intestines and defecation.

[0136] Table 5

[0137] (4.3) Analysis of gastrointestinal regulatory peptides:

[0138] After the experiment, the mice were dissected, blood was collected to separate serum, and the contents of four kinds of gastrointestinal regulatory peptides (Gas, SP, VIP, ET-1) in the serum of mice were determined by ELISA kit, and the statistical results are shown in Table 6 respectively. Gas and SP are excitatory peptide neurotransmitters, while ET-1 and VIP are inhibitory peptide neurotransmitters. After intervention of each group, Gas and SP were improved compared with the model group, while ET-1 and VIP were significantly decreased, and the effects of Example 1 group and Example 2 group were the best, indicating that the yogurt prepared by the probiotic agent involved in the application can promote intestinal peristalsis.

[0139] Table 6

[0140] (4.4) Inflammatory factor analysis:

[0141] The contents of inflammatory factors (IL-6, IL-10, TNF-α) in the serum of mice were determined by ELISA kit, and the statistical results are shown in Table 7 respectively. After intervention of each group, the anti-inflammatory factor IL-10 was improved compared with the model group, while the pro-inflammatory factors IL-6 and TNF-α were decreased, and the effects of Example 1 group and Example 2 group were the best.

[0142] Table 7

[0143] (4.5) Intestinal flora metagenome analysis:

[0144] The cecum of mice in the blank control group, the model group, Example 1 group and Comparative Example 8 group was intercepted, and the intestinal flora metagenome was analyzed by 16S rDNA.

[0145] Firmicutes and Bacteroidetes accounted for the main proportion of intestinal flora, and the two flora and the ratio of Firmicutes to Bacteroidetes (F / B) were specifically analyzed. F / B is related to maintaining the balance in the body. The statistical results are shown in Figures 1-3. Compared with the blank control group, the number of Firmicutes in the model group was reduced, and the number of Bacteroidetes was increased, indicating that with the constipation of the mice, the beneficial flora in the cecum was less, and the number of some pathogenic bacteria was increased. Compared with the model group, the number of Firmicutes in the intervention group was increased, and the number of Bacteroidetes was significantly reduced, and the F / B was higher than that of the model group, indicating that the intake of the probiotic yogurt of the application has a promoting effect on the balance of intestinal flora.

[0146] Lachnospira can hydrolyze starch and other sugars to produce butyrate and other short-chain fatty acids; Alistipes helps human digestion, nutrient absorption and regulation; Acinetobacter belongs to gram-negative bacteria, which can cause infection when the body's resistance decreases, can colonize in human skin, wounds, respiratory tract and gastrointestinal tract, and can also exist in oral biofilm, and can easily cause pneumonia after being inhaled into the lower respiratory tract. The results of statistical analysis of the three bacterial flora at the genus level are shown in Figures 4-6. Compared with the model group, the number of Lachnospira and Alistipes in the Example 1 group and the Comparative Example 8 group increased, and the number of Acinetobacter decreased, and the effect of the Example 1 group was significantly better, indicating that the intake of the probiotic yogurt of the present application can reduce the number of harmful bacteria in the intestinal tract and increase the number of beneficial bacteria, and also indicates at the genus level that the balance of intestinal flora can be improved.

[0147] The applicant declares that the technical solutions of the present application are illustrated by the above examples, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0148] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0149] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A compound bifidobacterium agent, wherein the strains are composed of Bifidobacterium animalis subsp. lactis BLa36 strain with the preservation number of CGMCC No. 24029, Bifidobacterium longum BL21 strain with the preservation number of CGMCC No. 10452, Bifidobacterium breve BBr60 strain with the preservation number of CGMCC No. 12915, Bifidobacterium adolescentis BAC30 strain with the preservation number of CGMCC No. 19884, and Bifidobacterium infantis BI45 strain with the preservation number of CGMCC No. 15134.

2. The complex Bifidobacterium bacterial agent of claim 1, wherein, The ratio of viable bacterial count of the BLa36 strain, the BL21 strain, the BBr60 strain, the BAC30 strain and the BI45 strain is (1-10) :(1-10) :(1-10) :(1-5) :(1-5).

3. The composite Bifidobacterium agent according to claim 1, wherein, The total number of viable bacteria in the complex bifidobacterium agent is not less than 1×10 11 CFU / mL or 1×10 11 CFU / g.

4. The Bifidobacterium composite agent of claim 1, characterized by, The dosage form of the compound bifidobacterium agent includes solution, lyophilized powder, capsule, tablet or granule. 5.A compound probiotic agent, comprising the compound bifidobacterium agent of any one of claims 1-4 and a base agent; the strains in the base agent are composed of Streptococcus thermophilus ST81 strain with the preservation number of CGMCC No. 15752 and Lactobacillus delbrueckii subsp. bulgaricus LB42 strain with the preservation number of CGMCC No. 15751.

6. The complex probiotic agent of claim 5, wherein, The ratio of viable bacterial count of the ST81 strain and the LB42 strain is 30 :(0.001-10) ; The total number of viable bacteria in the base inoculant is not less than 1 x 10 11 CFU / mL or 1 x 10 11 CFU / g; The mass ratio of the compound bifidobacterium agent and the base agent is (10-300) :(10-40) ; The dosage form of the compound probiotic agent includes solution, lyophilized powder, capsule, tablet or granule. 7.The compound bifidobacterium agent of any one of claims 1-4 or the compound probiotic agent of any one of claims 5-6 is applied in yogurt fermentation.

8. Use according to claim 7, wherein, The method of the yogurt fermentation comprises: (1) mixing raw cow milk with a compound prebiotic, sterilizing and cooling to serve as a fermentation base; (2) inoculating the compound probiotic agent into the fermentation base for fermentation.

9. Use according to claim 8, wherein, The compound prebiotic includes any one or a combination of at least two of fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, isomaltooligosaccharide, soybean oligosaccharide, inulin, polydextrose, α-lactalbumin or lactoferrin; Preferably, the compound prebiotic is 0.01-1% of the fermentation base; Preferably, the inoculation amount of the compound probiotic agent is 20-300 g / T; Preferably, the fermentation temperature is 40-43°C, the fermentation time is 5-10h, and the end of fermentation is reached when the acidity is 70-80°T or pH=4.3-4.

5.

10. Use of the complex Bifidobacterium agent of any one of claims 1-4 or the complex probiotic agent of any one of claims 5-6 for the preparation of a product for promoting intestinal peristalsis and intestinal health.

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