Method for improving survival rate and storage stability of bifidobacterium fermentation liquor

Abstract

The invention discloses a method for improving the survival rate and storage stability of bifidobacterium fermentation liquor, which comprises the following steps: firstly, preparing a special fermentation culture medium; performing fermentation culture on the bifidobacterium by adopting a special fermentation culture medium; inoculating into a special fermentation culture medium according to the mass ratio of 2.5%, fermenting at 37 DEG C, and culturing at constant temperature for 10-12 hours to obtain bifidobacterium fermentation liquor; the special fermentation culture medium is prepared by introducing 0.4-0.6 g / L of a composite additive and 0.4-0.6 g / L of anthocyanin into an MRS optimized culture medium; wherein the mass ratio of the composite additive to the anthocyanin is 1: 1; the number of viable bacteria in the bifidobacterium fermentation liquor obtained through culture by the method is large, the high bifidobacterium survival rate can be kept after freeze-drying storage, and the corresponding probiotic effect of the bifidobacterium freeze-dried powder can be effectively kept under the condition that the large number of viable bacteria is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of bifidobacterium cultivation, in particular to a method for improving the survival rate and storage stability of bifidobacterium fermentation liquid. Background Art

[0002] Bifidobacterium is an important probiotic belonging to the Bacteria kingdom, Actinomycetes, Actinomycetes, Bifidobacteriales, Bifidobacteriaceae, and Bifidobacterium genus. Its name comes from its morphological characteristics: the cells are rod-shaped, sometimes forked at one end, resembling the letters "Y" or "V."

[0003] Bifidobacteria are obligate anaerobes and are extremely sensitive to oxygen. Their optimal growth temperature is typically between 37°C and 41°C, but they can also grow within certain ranges (such as 25°C to 28°C or 43°C to 45°C). Bifidobacteria metabolize glucose through heterofermentation. They lack aldolase and glucose-6-phosphate dehydrogenase, but do possess phosphoketolase activity. They can ferment glucose to produce organic acids such as lactic acid and acetic acid, which play an important role in intestinal health.

[0004] Physiological functions and applications of Bifidobacterium:

[0005] * Maintaining intestinal health: Bifidobacteria, as beneficial bacteria in the intestine, can compete with harmful bacteria for survival resources, inhibiting their growth and reproduction, thereby maintaining a balanced intestinal flora. At the same time, they can also enhance the intestinal mucosal barrier function and reduce the risk of intestinal infection.

[0006] * Enhance immunity: Bifidobacteria can activate the body's immune system, increase the activity of immune cells, and promote the production of immunoglobulins, thereby enhancing the body's defense against pathogenic microorganisms.

[0007] *Improve the intestinal environment: By producing substances such as organic acids and bacteriocins, bifidobacteria can lower the intestinal pH, inhibit the growth of harmful bacteria, and promote intestinal peristalsis to reduce the occurrence of constipation.

[0008] * Promote digestion and absorption: Bifidobacteria can break down complex components in food, such as cellulose and protein, making them easier for the body to absorb and utilize. They can also synthesize nutrients such as B vitamins and vitamin K to help maintain good health.

[0009] *Other effects: Bifidobacteria also possess various physiological functions, including anti-tumor and anti-aging. For example, they can reduce damage to the body by absorbing carcinogens; protect liver health by regulating liver endotoxin levels; and improve human nutritional status by alleviating lactose intolerance.

[0010] Numerous scientific experiments have confirmed that regular intake of bifidobacteria and their products can maintain intestinal health. However, in order for bifidobacteria products to achieve their desired probiotic effects, the number of live bacteria in the product must be sufficient. However, in the existing technology, the number of live bacteria is often low during the fermentation process of bifidobacteria, resulting in low yield and failure to achieve the desired probiotic effect.

[0011] Therefore, the present invention solves the corresponding problems by providing a method for improving the survival rate of bifidobacterium fermentation broth. Summary of the Invention

[0012] The purpose of the present invention is to provide a method for improving the survival rate of bifidobacterium fermentation broth to solve the deficiencies in the prior art.

[0013] The technical solution adopted by the present invention is as follows:

[0014] A method for improving the survival rate of bifidobacterium fermentation broth, comprising: first, preparing a special fermentation medium;

[0015] Use special fermentation medium to ferment and culture Bifidobacterium;

[0016] Inoculate 2.5% by mass into a special fermentation medium, keep the fermentation temperature at 37°C, and culture at a constant temperature for 10-12 hours to obtain a bifidobacterium fermentation liquid;

[0017] The special fermentation medium is a medium in which 0.4-0.6 g / L of compound additives and 0.4-0.6 g / L of anthocyanidins are introduced into the MRS optimized medium;

[0018] Among them, the mass ratio of the composite additive to anthocyanin is 1:1.

[0019] As a further technical solution: the bifidobacterium is Bifidobacterium V9.

[0020] As a further technical solution: the preparation method of the composite additive is:

[0021] First, hydroxyethyl starch is added to a sodium hydroxide solution, and the mixture is stirred and mixed to obtain a mixed solution;

[0022] Add curcumin to the mixture, adjust the temperature to 45°C, keep stirring and react for 15-18 hours, and then adjust the pH of the reaction system to neutral to obtain a preliminary reaction solution;

[0023] The obtained preliminary reaction solution was added dropwise into anhydrous ethanol for alcohol precipitation;

[0024] The precipitate is collected by filtration, then added into deionized water, stirred and mixed evenly, then dialyzed through a dialysis bag, and freeze-dried to obtain a composite additive.

[0025] As a further technical solution: the mass fraction of the sodium hydroxide solution is 3%;

[0026] The mixing ratio of the hydroxyethyl starch and the sodium hydroxide solution is 3.6 g:200 mL.

[0027] As a further technical solution: the mass ratio of curcumin to hydroxyethyl starch is 1:18-20.

[0028] As a further technical solution: the anthocyanin extraction method is:

[0029] Peel and deseed the purple grapes, leaving the skins and seeds;

[0030] The grape skins and seeds were cleaned and then placed in a drying oven for 10 hours under constant temperature drying to obtain a dried material.

[0031] Among them, the constant temperature drying temperature is 80℃;

[0032] The obtained dried material is crushed by a crusher to obtain a mixed crushed material;

[0033] The obtained mixed crushed material is added to petroleum ether for degreasing, and then filtered and dried to obtain a defatted material;

[0034] Among them, the drying temperature is 120℃ and the time is 20min;

[0035] The defatted material was mixed with the extractant, stirred for 4 hours, then ultrasonicated for 30 minutes, and finally centrifuged at high speed for 20 minutes, and the supernatant was collected;

[0036] The supernatant is spray-dried to obtain the desired anthocyanidins.

[0037] As a further technical solution: the mass ratio of the grape skin to the grape seed is 1:2-3;

[0038] The average particle size of the mixed crushed material is 80 mesh;

[0039] The mixing ratio of the mixed crushed material and petroleum ether is 15-20g:200mL.

[0040] As a further technical solution: the ultrasonic frequency is 50kHz;

[0041] The high-speed centrifugal speed is 8000 r / min.

[0042] As a further technical solution: the weight ratio of the degreasing material to the extractant is 1:5;

[0043] The extractant is an ethanol solution;

[0044] The mass fraction of the ethanol solution is 80%.

[0045] A method for improving the storage stability of bifidobacterium fermentation broth comprises the following steps:

[0046] (1) The fermented bacterial broth was centrifuged at 5000 g for 15-18 min to collect the bacterial sludge;

[0047] (2) Add the obtained bacterial sludge to the lyophilization protective agent in a 1:1 mass ratio, mix well, let it stand for 20 minutes, pre-freeze at -78°C overnight, and freeze-dry in a vacuum freeze dryer after pre-freezing. The freezing temperature is -55°C, the vacuum degree is 12-15 Pa, and the time is 24 hours to obtain lyophilized powder;

[0048] The freeze-drying protective agent comprises, by weight percentage, 4.5% glycerol, 1.2% ascorbic acid, 0.8% trehalose, 0.2% D-mannitol, 1.5% glycine, 3% leucine, 10% skim milk powder, and the rest is pure water.

[0049] Beneficial effects:

[0050] The bifidobacterium fermentation liquid obtained by the method of the present invention has a large number of viable bacteria, and can maintain a high survival rate of bifidobacteria after freeze-drying and storage. While ensuring a large number of viable bacteria, the bifidobacterium freeze-dried powder can effectively maintain the corresponding probiotic effect.

[0051] The present invention can greatly promote the growth of bifidobacteria and significantly enhance the vitality of bifidobacteria by introducing a composite additive and a specially prepared anthocyanin into the culture medium. Its mechanism of action is mainly to enhance the metabolic activity of bifidobacteria themselves and optimize fermentation conditions including pH value, redox potential, etc., so that bifidobacteria can grow and reproduce in a more suitable environment, thereby improving fermentation efficiency and survival rate.

[0052] By fermenting and cultivating stronger bifidobacteria, the storage survival rate of bifidobacterium freeze-dried powder can be greatly improved during the subsequent freeze-drying storage process. After a long period of storage, it can still maintain a high survival rate, which can ensure a sufficient number of live bifidobacteria, thereby achieving its corresponding probiotic effect.

[0053] In order to further improve the integrity of the bifidobacterium under the protection of the prepared freeze-drying protectant, the cell membrane can be further stabilized, and the damage to the bifidobacterium cells caused by stress during the freeze-drying process can be greatly reduced, thereby maintaining the activity of the bifidobacterium bacteria; specifically, the cell membrane of the bifidobacterium obtained by the culture of the present invention is more depressed and directly interacts with sugars, amino acids, etc., thereby forming a protective layer, which can effectively prevent the sublimation and escape of the effective components of the bifidobacterium, and can promote the retention of more effective components of the bifidobacterium. In this way, through the action of the protective layer, the damage during the freeze-drying process can be greatly reduced.

[0054] During storage, bifidobacteria may be oxidized by oxygen, resulting in decreased activity or even death. However, some components of the freeze-dried protective agent used in the present invention have antioxidant properties (such as ascorbic acid), which can scavenge free radicals and other oxidizing substances, delay the aging process of bacteria, and thus improve their storage stability.

[0055] In summary, the present invention mainly enhances the viability of bifidobacteria and improves the storage stability of bifidobacterium freeze-dried powder by cooperating with the multiple mechanisms of freeze-drying protectants. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Statistical graph of the survival rate of Bifidobacterium species in each group. DETAILED DESCRIPTION

[0057] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0058] The culture medium involved in the following examples is as follows:

[0059] MRS optimized medium: peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, Tween-80 1.0 mL / L, diammonium hydrogen citrate 2 g / L, potassium hydrogen phosphate 2.6 g / L, manganese sulfate 0.25 g / L, magnesium sulfate 0.58 g / L, cysteine ​​hydrochloride 0.5 g / L, and distilled water 1000 g / L.

[0060] The yeast extract powder is FM985.

[0061] The MRS optimized medium was sterilized at 121°C for 30 min before use.

[0062] The following are specific embodiments

[0063] Example 1

[0064] A method for improving the survival rate of bifidobacterium fermentation broth, comprising: first, preparing a special fermentation medium;

[0065] Use special fermentation medium to ferment and culture Bifidobacterium;

[0066] Inoculate 2.5% by mass into a special fermentation medium, keep the fermentation temperature at 37°C, and culture at this constant temperature for 10 hours to obtain a bifidobacterium fermentation liquid;

[0067] The special fermentation medium is a medium in which 0.4 g / L of compound additives and 0.4 g / L of anthocyanin are introduced into the MRS optimized medium;

[0068] Among them, the mass ratio of the composite additive to anthocyanin is 1:1.

[0069] The bifidobacterium was Bifidobacterium V9.

[0070] The preparation method of the composite additive is as follows:

[0071] First, hydroxyethyl starch is added to a sodium hydroxide solution, and the mixture is stirred and mixed to obtain a mixed solution;

[0072] Add curcumin to the mixed solution, adjust the temperature to 45°C, keep stirring and react for 15 hours, and then adjust the pH of the reaction system to neutral to obtain a preliminary reaction solution;

[0073] The obtained preliminary reaction solution was added dropwise into anhydrous ethanol for alcohol precipitation;

[0074] The precipitate is collected by filtration, then added into deionized water, stirred and mixed evenly, then dialyzed through a dialysis bag, and freeze-dried to obtain a composite additive.

[0075] The mass fraction of sodium hydroxide solution is 3%;

[0076] The mixing ratio of the hydroxyethyl starch and the sodium hydroxide solution is 3.6 g:200 mL.

[0077] The mass ratio of curcumin to hydroxyethyl starch is 1:18.

[0078] The anthocyanin extraction method is:

[0079] Peel and deseed the purple grapes, leaving the skins and seeds;

[0080] The grape skins and seeds were cleaned and then placed in a drying oven for 10 hours under constant temperature drying to obtain a dried material.

[0081] Among them, the constant temperature drying temperature is 80℃;

[0082] The obtained dried material is crushed by a crusher to obtain a mixed crushed material;

[0083] The obtained mixed crushed material is added to petroleum ether for degreasing, and then filtered and dried to obtain a defatted material;

[0084] Among them, the drying temperature is 120℃ and the time is 20min;

[0085] The defatted material was mixed with the extractant, stirred for 4 hours, then ultrasonicated for 30 minutes, and finally centrifuged at high speed for 20 minutes, and the supernatant was collected;

[0086] The supernatant is spray-dried to obtain the desired anthocyanidins.

[0087] The mass ratio of grape skin to grape seed is 1:2;

[0088] The average particle size of the mixed crushed material is 80 mesh;

[0089] The mixing ratio of the mixed crushed material and petroleum ether is 15g:200mL.

[0090] The ultrasonic frequency is 50kHz;

[0091] The high-speed centrifugal speed is 8000 r / min.

[0092] The mass ratio of the degreased material to the extractant is 1:5;

[0093] The extractant is an ethanol solution;

[0094] The mass fraction of the ethanol solution is 80%.

[0095] Example 2

[0096] A method for improving the survival rate of bifidobacterium fermentation broth, comprising: first, preparing a special fermentation medium;

[0097] Use special fermentation medium to ferment and culture Bifidobacterium;

[0098] Inoculate 2.5% by mass into a special fermentation medium, keep the fermentation temperature at 37°C, and culture at this constant temperature for 11 hours to obtain a bifidobacterium fermentation liquid;

[0099] The special fermentation medium is a medium in which 0.45 g / L of compound additives and 0.45 g / L of anthocyanin are introduced into the MRS optimized medium;

[0100] Among them, the mass ratio of the composite additive to anthocyanin is 1:1.

[0101] The bifidobacterium was Bifidobacterium V9.

[0102] The preparation method of the composite additive is as follows:

[0103] First, hydroxyethyl starch is added to a sodium hydroxide solution, and the mixture is stirred and mixed to obtain a mixed solution;

[0104] Add curcumin to the mixed solution, adjust the temperature to 45°C, keep stirring and react for 18 hours, and then adjust the pH of the reaction system to neutral to obtain a preliminary reaction solution;

[0105] The obtained preliminary reaction solution was added dropwise into anhydrous ethanol for alcohol precipitation;

[0106] The precipitate is collected by filtration, then added into deionized water, stirred and mixed evenly, then dialyzed through a dialysis bag, and freeze-dried to obtain a composite additive.

[0107] The mass fraction of sodium hydroxide solution is 3%;

[0108] The mixing ratio of the hydroxyethyl starch and the sodium hydroxide solution is 3.6 g:200 mL.

[0109] The mass ratio of curcumin to hydroxyethyl starch is 1:19.

[0110] The anthocyanin extraction method is:

[0111] Peel and deseed the purple grapes, leaving the skins and seeds;

[0112] The grape skins and seeds were cleaned and then placed in a drying oven for 10 hours under constant temperature drying to obtain a dried material.

[0113] Among them, the constant temperature drying temperature is 80℃;

[0114] The obtained dried material is crushed by a crusher to obtain a mixed crushed material;

[0115] The obtained mixed crushed material is added to petroleum ether for degreasing, and then filtered and dried to obtain a defatted material;

[0116] Among them, the drying temperature is 120℃ and the time is 20min;

[0117] The defatted material was mixed with the extractant, stirred for 4 hours, then ultrasonicated for 30 minutes, and finally centrifuged at high speed for 20 minutes, and the supernatant was collected;

[0118] The supernatant is spray-dried to obtain the desired anthocyanidins.

[0119] The mass ratio of grape skin to grape seed is 1:2.2;

[0120] The average particle size of the mixed crushed material is 80 mesh;

[0121] The mixing ratio of the mixed crushed material and petroleum ether is 16g:200mL.

[0122] The ultrasonic frequency is 50kHz;

[0123] The high-speed centrifugal speed is 8000 r / min.

[0124] The mass ratio of the degreased material to the extractant is 1:5;

[0125] The extractant is an ethanol solution;

[0126] The mass fraction of the ethanol solution is 80%.

[0127] Example 3

[0128] A method for improving the survival rate of bifidobacterium fermentation broth, comprising: first, preparing a special fermentation medium;

[0129] Use special fermentation medium to ferment and culture Bifidobacterium;

[0130] Inoculate 2.5% by mass into a special fermentation medium, keep the fermentation temperature at 37°C, and culture at this constant temperature for 11 hours to obtain a bifidobacterium fermentation liquid;

[0131] The special fermentation medium is a medium in which 0.5 g / L of compound additives and 0.5 g / L of anthocyanin are introduced into the MRS optimized medium;

[0132] Among them, the mass ratio of the composite additive to anthocyanin is 1:1.

[0133] The bifidobacterium was Bifidobacterium V9.

[0134] The preparation method of the composite additive is as follows:

[0135] First, hydroxyethyl starch is added to a sodium hydroxide solution, and the mixture is stirred and mixed to obtain a mixed solution;

[0136] Add curcumin to the mixed solution, adjust the temperature to 45°C, keep stirring and react for 16 hours, and then adjust the pH of the reaction system to neutral to obtain a preliminary reaction solution;

[0137] The obtained preliminary reaction solution was added dropwise into anhydrous ethanol for alcohol precipitation;

[0138] The precipitate is collected by filtration, then added into deionized water, stirred and mixed evenly, then dialyzed through a dialysis bag, and freeze-dried to obtain a composite additive.

[0139] The mass fraction of sodium hydroxide solution is 3%;

[0140] The mixing ratio of the hydroxyethyl starch and the sodium hydroxide solution is 3.6 g:200 mL.

[0141] The mass ratio of curcumin to hydroxyethyl starch is 1:18.

[0142] The anthocyanin extraction method is:

[0143] Peel and deseed the purple grapes, leaving the skins and seeds;

[0144] The grape skins and seeds were cleaned and then placed in a drying oven for 10 hours under constant temperature drying to obtain a dried material.

[0145] Among them, the constant temperature drying temperature is 80℃;

[0146] The obtained dried material is crushed by a crusher to obtain a mixed crushed material;

[0147] The obtained mixed crushed material is added to petroleum ether for degreasing, and then filtered and dried to obtain a defatted material;

[0148] Among them, the drying temperature is 120℃ and the time is 20min;

[0149] The defatted material was mixed with the extractant, stirred for 4 hours, then ultrasonicated for 30 minutes, and finally centrifuged at high speed for 20 minutes, and the supernatant was collected;

[0150] The supernatant is spray-dried to obtain the desired anthocyanidins.

[0151] The mass ratio of grape skin to grape seed is 1:2.5;

[0152] The average particle size of the mixed crushed material is 80 mesh;

[0153] The mixing ratio of the mixed crushed material and petroleum ether is 18g:200mL.

[0154] The ultrasonic frequency is 50kHz;

[0155] The high-speed centrifugal speed is 8000 r / min.

[0156] The mass ratio of the degreased material to the extractant is 1:5;

[0157] The extractant is an ethanol solution;

[0158] The mass fraction of the ethanol solution is 80%.

[0159] Example 4

[0160] A method for improving the survival rate of bifidobacterium fermentation broth, comprising: first, preparing a special fermentation medium;

[0161] Use special fermentation medium to ferment and culture Bifidobacterium;

[0162] Inoculate 2.5% by mass into a special fermentation medium, keep the fermentation temperature at 37°C, and culture at this constant temperature for 12 hours to obtain a bifidobacterium fermentation liquid;

[0163] The special fermentation medium is a medium in which 0.55 g / L of compound additives and 0.55 g / L of anthocyanin are introduced into the MRS optimized medium;

[0164] Among them, the mass ratio of the composite additive to anthocyanin is 1:1.

[0165] The bifidobacterium was Bifidobacterium V9.

[0166] The preparation method of the composite additive is as follows:

[0167] First, hydroxyethyl starch is added to a sodium hydroxide solution, and the mixture is stirred and mixed to obtain a mixed solution;

[0168] Add curcumin to the mixed solution, adjust the temperature to 45°C, keep stirring and react for 15 hours, and then adjust the pH of the reaction system to neutral to obtain a preliminary reaction solution;

[0169] The obtained preliminary reaction solution was added dropwise into anhydrous ethanol for alcohol precipitation;

[0170] The precipitate is collected by filtration, then added into deionized water, stirred and mixed evenly, then dialyzed through a dialysis bag, and freeze-dried to obtain a composite additive.

[0171] The mass fraction of sodium hydroxide solution is 3%;

[0172] The mixing ratio of the hydroxyethyl starch and the sodium hydroxide solution is 3.6 g:200 mL.

[0173] The mass ratio of curcumin to hydroxyethyl starch is 1:19.

[0174] The anthocyanin extraction method is:

[0175] Peel and deseed the purple grapes, leaving the skins and seeds;

[0176] The grape skins and seeds were cleaned and then placed in a drying oven for 10 hours under constant temperature drying to obtain a dried material.

[0177] Among them, the constant temperature drying temperature is 80℃;

[0178] The obtained dried material is crushed by a crusher to obtain a mixed crushed material;

[0179] The obtained mixed crushed material is added to petroleum ether for degreasing, and then filtered and dried to obtain a defatted material;

[0180] Among them, the drying temperature is 120℃ and the time is 20min;

[0181] The defatted material was mixed with the extractant, stirred for 4 hours, then ultrasonicated for 30 minutes, and finally centrifuged at high speed for 20 minutes, and the supernatant was collected;

[0182] The supernatant is spray-dried to obtain the desired anthocyanidins.

[0183] The mass ratio of grape skin to grape seed is 1:2.5;

[0184] The average particle size of the mixed crushed material is 80 mesh;

[0185] The mixing ratio of the mixed crushed material and petroleum ether is 18g:200mL.

[0186] The ultrasonic frequency is 50kHz;

[0187] The high-speed centrifugal speed is 8000 r / min.

[0188] The mass ratio of the degreased material to the extractant is 1:5;

[0189] The extractant is an ethanol solution;

[0190] The mass fraction of the ethanol solution is 80%.

[0191] Example 5

[0192] A method for improving the survival rate of bifidobacterium fermentation broth, comprising: first, preparing a special fermentation medium;

[0193] Use special fermentation medium to ferment and culture Bifidobacterium;

[0194] Inoculate 2.5% by mass into a special fermentation medium, keep the fermentation temperature at 37°C, and culture at this constant temperature for 12 hours to obtain a bifidobacterium fermentation liquid;

[0195] The special fermentation medium is a medium in which 0.6 g / L of compound additives and 0.6 g / L of anthocyanin are introduced into the MRS optimized medium;

[0196] Among them, the mass ratio of the composite additive to anthocyanin is 1:1.

[0197] The bifidobacterium was Bifidobacterium V9.

[0198] The preparation method of the composite additive is as follows:

[0199] First, hydroxyethyl starch is added to a sodium hydroxide solution, and the mixture is stirred and mixed to obtain a mixed solution;

[0200] Add curcumin to the mixed solution, adjust the temperature to 45°C, keep stirring and react for 18 hours, and then adjust the pH of the reaction system to neutral to obtain a preliminary reaction solution;

[0201] The obtained preliminary reaction solution was added dropwise into anhydrous ethanol for alcohol precipitation;

[0202] The precipitate is collected by filtration, then added into deionized water, stirred and mixed evenly, then dialyzed through a dialysis bag, and freeze-dried to obtain a composite additive.

[0203] The mass fraction of sodium hydroxide solution is 3%;

[0204] The mixing ratio of the hydroxyethyl starch and the sodium hydroxide solution is 3.6 g:200 mL.

[0205] The mass ratio of curcumin to hydroxyethyl starch is 1:20.

[0206] The anthocyanin extraction method is:

[0207] Peel and deseed the purple grapes, leaving the skins and seeds;

[0208] The grape skins and seeds were cleaned and then placed in a drying oven for 10 hours under constant temperature drying to obtain a dried material.

[0209] Among them, the constant temperature drying temperature is 80℃;

[0210] The obtained dried material is crushed by a crusher to obtain a mixed crushed material;

[0211] The obtained mixed crushed material is added to petroleum ether for degreasing, and then filtered and dried to obtain a defatted material;

[0212] Among them, the drying temperature is 120℃ and the time is 20min;

[0213] The defatted material was mixed with the extractant, stirred for 4 hours, then ultrasonicated for 30 minutes, and finally centrifuged at high speed for 20 minutes, and the supernatant was collected;

[0214] The supernatant is spray-dried to obtain the desired anthocyanidins.

[0215] The mass ratio of grape skin to grape seed is 1:3;

[0216] The average particle size of the mixed crushed material is 80 mesh;

[0217] The mixing ratio of the mixed crushed material and petroleum ether is 20g:200mL.

[0218] The ultrasonic frequency is 50kHz;

[0219] The high-speed centrifugal speed is 8000 r / min.

[0220] The mass ratio of the degreased material to the extractant is 1:5;

[0221] The extractant is an ethanol solution;

[0222] The mass fraction of the ethanol solution is 80%.

[0223] In the above embodiments:

[0224] A method for improving the storage stability of bifidobacterium fermentation broth comprises the following steps:

[0225] (1) The fermented bacterial broth was centrifuged at 5000 g for 15 min to collect the bacterial sludge;

[0226] (2) The obtained bacterial sludge was added to the lyophilization protective agent in a 1:1 mass ratio, mixed evenly, allowed to stand for 20 min, pre-frozen at -78 °C overnight, and then placed in a vacuum freeze dryer for freeze drying at a freezing temperature of -55 °C, a vacuum degree of 1 Pa, and a time of 24 h to obtain a lyophilized powder;

[0227] The freeze-drying protective agent comprises, by weight percentage, 4.5% glycerol, 1.2% ascorbic acid, 0.8% trehalose, 0.2% D-mannitol, 1.5% glycine, 3% leucine, 10% skim milk powder, and the rest is pure water.

[0228] Comparative Example 1:

[0229] Based on the technical solution of Example 1, no composite additives are added to the special fermentation medium, and the rest of the technical solutions remain unchanged.

[0230] Comparative Example 2:

[0231] Based on the technical solution of Example 1, no anthocyanin was added to the special fermentation medium, and the rest of the technical solutions remained unchanged.

[0232] Comparative Example 3:

[0233] Based on the technical solution of Example 1, no anthocyanin and composite additives were added to the special fermentation medium, and the rest of the technical solutions remained unchanged.

[0234] test:

[0235] The number of viable bifidobacteria in the fermentation broth of the embodiment and the comparative example was counted and compared. The test results are shown in Table 1:

[0236] Table 1

[0237] Viable bacteria count cfu / mL Example 1 <![CDATA[8.21×10 9 ]]> Example 2 <![CDATA[8.23×10 9 ]]> Example 3 <![CDATA[8.25×10 9 ]]> Example 4 <![CDATA[8.24×10 9 ]]> Example 5 <![CDATA[8.26×10 9 ]]> Comparative Example 1 <![CDATA[6.89×10 9 ]]> Comparative Example 2 <![CDATA[7.37×10 9 ]]> Comparative Example 3 <![CDATA[6.09×10 9 ]]>

[0238] As can be seen from Table 1, the special fermentation medium used in the present invention can ferment to obtain a fermentation liquid with a higher number of viable bifidobacteria.

[0239] Based on the sample of Example 1, the effects of different contents of the composite additive on the number of viable bacteria in the fermentation broth were tested:

[0240] Table 2

[0241] Concentration g / L Viable bacteria count cfu / mL 0 <![CDATA[6.89×10 9 ]]> 0.2 <![CDATA[7.72×10 9 ]]> 0.4 <![CDATA[8.24×10 9 ]]> 0.6 <![CDATA[8.26×10 9 ]]> 0.8 <![CDATA[8.12×10 9 ]]> 1.0 <![CDATA[8.10×10 9 ]]>

[0242] It can be seen from Table 2 that with the increase in the amount of compound additives added, the number of viable bacteria in the fermentation broth first increased, then tended to remain unchanged, and then fell slightly.

[0243] The bifidobacterium fermentation broth obtained in the examples and comparative examples was subjected to the method for improving storage stability of bifidobacterium fermentation broth, and the survival rate of the bacteria before and after treatment in each group was tested:

[0244] First, in a sterile environment, 0.2 g of the lyophilized powder of the example and the comparative example (stored under the same conditions for 45 days) was dissolved in 2 mL of sterile water and thoroughly mixed; then, the resulting bacterial suspension was gradiently diluted with sterile water, and after dilution, the bacterial suspension was evenly spread on the same solid culture medium using a coating rod;

[0245] Finally, the culture medium was placed in a constant temperature incubator at 37°C for 48 h, and viable bacteria were counted after the incubation.

[0246] Bacterial survival rate = (number of viable bacteria before freezing / number of viable bacteria after freezing) × 100%;

[0247] Table 3

[0248] Bacteria survival rate% Example 1 92.36 Example 2 92.83 Example 3 93.05 Example 4 92.17 Example 5 91.98 Comparative Example 1 83.46 Comparative Example 2 80.17 Comparative Example 3 72.69

[0249] It can be seen from Table 3 that after being treated by the method of the present invention, the bacterial strain survival rate can still be maintained at a high level after being stored for a long time.

[0250] The above description is only a preferred embodiment of the present invention, but the scope of implementation of the present invention is not limited to the embodiment shown. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which do not exceed the spirit covered by the description, should be within the scope of protection of the present invention.

Claims

1. A method for improving the survival rate of bifidobacterium fermentation broth, It is characterized in that First, a special fermentation medium is prepared; Use special fermentation medium to ferment and culture Bifidobacterium; Inoculate into a special fermentation medium at a mass ratio of 2.5%, ferment at a temperature of 37°C, and culture at a constant temperature for 10-12 hours to obtain a bifidobacterium fermentation liquid; The special fermentation medium is a medium in which 0.4-0.6 g / L of composite additives and 0.4-0.6 g / L of anthocyanin are introduced into the MRS optimized medium; Among them, the mass ratio of the composite additive to anthocyanin is 1:

1.

2. A method for improving the survival rate of bifidobacterium fermentation broth according to claim 1, Features: The bifidobacterium is bifidobacterium V9.

3. A method for improving the survival rate of bifidobacterium fermentation broth according to claim 1, Features: The preparation method of the composite additive is: First, hydroxyethyl starch is added to a sodium hydroxide solution, and the mixture is stirred and mixed to obtain a mixed solution; Add curcumin to the mixed solution, adjust the temperature to 45°C, keep the mixture warm and stir for 15-18 hours, and then adjust the pH of the reaction system to neutral to obtain a preliminary reaction solution; The obtained preliminary reaction solution is added dropwise into anhydrous ethanol for alcohol precipitation; The precipitate is collected by filtration, then added into deionized water, stirred and mixed evenly, then dialyzed through a dialysis bag, and then freeze-dried to obtain a composite additive.

4. A method for improving the survival rate of bifidobacterium fermentation broth according to claim 3, Features: The mass fraction of the sodium hydroxide solution is 3%; The mixing ratio of the hydroxyethyl starch and the sodium hydroxide solution is 3.6 g:200 mL.

5. A method for improving the survival rate of bifidobacterium fermentation broth according to claim 3, Features: The mass ratio of curcumin to hydroxyethyl starch is 1:18-20.

6. A method for improving the survival rate of bifidobacterium fermentation broth according to claim 1, Features: The anthocyanin extraction method is: Peel and deseed the purple grapes, leaving the grape skins and seeds; The obtained grape skins and grape seeds are cleaned, and then placed in a drying oven for constant temperature drying for 10 hours to obtain a dried material; Among them, the constant temperature drying temperature is 80℃; The obtained dried material is crushed by a crusher to obtain a mixed crushed material; The obtained mixed crushed material is added into petroleum ether for degreasing treatment, and then filtered and dried to obtain a defatted material; Among them, the drying temperature is 120℃ and the time is 20min; The defatted material was mixed with the extractant, stirred for 4 hours, then subjected to ultrasonic treatment for 30 minutes, and finally subjected to high-speed centrifugation for 20 minutes, and the supernatant was collected; The supernatant is spray-dried to obtain the desired anthocyanins.

7. A method for improving the survival rate of bifidobacterium fermentation broth according to claim 6, Features: The mass ratio of grape skin to grape seed is 1:2-3; The average particle size of the mixed crushed material is 80 mesh; The mixing ratio of the mixed crushed material and petroleum ether is 15-20g:200mL.

8. A method for improving the survival rate of bifidobacterium fermentation broth according to claim 6, Features: The ultrasonic frequency is 50kHz; The high-speed centrifugal rotation speed is 8000r / min.

9. A method for improving the survival rate of bifidobacterium fermentation broth according to claim 6, Features: The mixing mass ratio of the degreasing material to the extractant is 1:5; The extractant is an ethanol solution; The mass fraction of the ethanol solution is 80%.

10. A method for improving the storage stability of the bifidobacterium fermentation liquid obtained by the method according to claim 1, Features: The following steps are involved: (1) Centrifuging the obtained fermentation broth at 5000 g for 15-18 min to collect the bacterial sludge; (2) adding the obtained bacterial sludge to the lyophilization protective agent in a mass ratio of 1:1, mixing evenly, standing for 20 min, pre-freezing at -78°C overnight, and then placing in a vacuum freeze dryer for freeze drying at a freezing temperature of -55°C, a vacuum degree of 12-15 Pa, and a time of 24 h to obtain a lyophilized powder; The freeze-drying protective agent includes, by weight percentage, 4.5% glycerol, 1.2% ascorbic acid, 0.8% trehalose, 0.2% D-mannitol, 1.5% glycine, 3% leucine, 10% skim milk powder, and the rest is pure water.

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