Composite protective agent for preserving strains and method for preparing strain freeze-dried powder

Abstract

The invention discloses a protective agent composite formula for freeze-drying preserved strains and a method for freeze-drying preserved strains by using the formula. The composite formula of the protective agent disclosed by the invention is suitable for protecting various mixed bacteria, especially bifidobacteria, lactobacillus and bacillus. Compared with the prior art, the method for freeze-drying the preserved strain by using the protective agent disclosed by the invention has the advantages that the survival rate of the freeze-dried mixed bacteria can reach 78% on the premise of ensuring the beautiful form of the mixed bacteria.

Description

Technical Field

[0001] The present invention discloses a composite protective agent for freeze - drying and preserving strains, and a method for preparing probiotic freeze - dried powder using the composite protective agent. Specifically, the composite protective agent disclosed in the present invention is applicable to preserving mixed bacteria. Background Art

[0002] Freeze drying is a drying method in which a water - containing substance is first cooled and frozen into a solid, and then under low - temperature and reduced - pressure conditions, the sublimation property of water is utilized to dehydrate the substance at low temperature to achieve the purpose of drying.

[0003] During the entire freeze - drying process, various stresses generated can cause damage (passivation of some protein molecules) or even death of some microbial cells. In order to reduce the damage caused by the freeze - drying process to microbial cells and improve the solubility and stability of freeze - dried products, some additional substances need to be added to the freeze - drying system, and such substances are collectively referred to as freeze - drying protective agents. When freeze - drying is carried out without the presence of a protective agent, the survival rate of microbial cells is very low; the survival rate after freeze - drying depends on the protective agent used. The protective effect of a single protective agent component (hereinafter referred to as a single factor) is limited and cannot provide due protection to live bacterial preparations. Therefore, several single factors with different protective effects need to be formulated into a composite formula in a certain proportion. Thus, finding a suitable freeze - drying protective agent formula to improve the survival rate of strains after freeze - drying has become an important issue in the production of live bacterial vaccines and the process of strain preservation. Summary of the Invention

[0004] Aiming at the shortcomings in the prior art, the present invention provides a composite formula of a strain - preserving protective agent.

[0005] According to one aspect of the present invention, a protective agent solution is disclosed, and the protective agent solution contains one or more of mannitol 3 - 9%, starch 3 - 9%, skim milk powder 4 - 10%, xylose 1 - 4%, galactose 1 - 4%, lactose 1 - 4%, sucrose 1 - 4%, glucose 1 - 4%, trehalose 1 - 4%.

[0006] According to certain embodiments of the present application, the protective agent solution contains one or more of mannitol 5%, starch 5%, skim milk powder 8%, xylose 2%, galactose 2%, lactose 2%, sucrose 2%, glucose 2%, trehalose 2%.

[0007] According to certain embodiments of the present application, the protective agent solution contains 2% glucose, 2% xylose, 2% trehalose, 2% galactose, 5% mannitol, 5% starch, and 8% skim milk powder; 2% glucose, 2% lactose, 2% trehalose, 2% sucrose, 5% starch, 5% mannitol, and 8% skim milk powder; 2% glucose, 2% lactose, 2% xylose, 2% trehalose, 5% starch, 5% mannitol, and 8% skim milk powder; or 2% lactose, 2% trehalose, 2% galactose, 2% sucrose, 5% starch, 5% mannitol, and 8% skim milk powder.

[0008] According to one aspect of the present invention, the present invention discloses a method for preparing a mixed probiotic freeze-dried powder using the protective agent solution, and the method includes:

[0009] 1) Place the fermentation broth containing probiotics in an environment of 2 - 6°C, centrifuge at a speed of 5000 - 8000 r / min for 15 - 20 minutes, pour off the supernatant, and collect the cell precipitate; wash the cell precipitate with sterile normal saline 2 - 3 times,

[0010] After each washing, centrifuge at 2 - 6°C and 5000 - 8000 r / min for 15 - 20 minutes, and discard the supernatant;

[0011] 2) Resuspend the washed cells with a small amount of sterile normal saline, adjust the concentration of the cell suspension so that the concentration of various probiotics after mixing reaches 10^9 - 10^11 CFU / mL;

[0012] 3) Mix the cell suspension with a concentration of 10^9 - 10^11 CFU / mL evenly with 2 - 5 mL of the sterilized protective agent solution;

[0013] 4) Aliquot the mixed cell solution into a freeze-drying tray, control the thickness to 1 - 2 cm, and then place it in a freeze-dryer for pre-freezing,

[0014] The pre-freezing condition is to cool down to -30 - -50°C at a cooling rate of 1 - 2°C / min and maintain for 2 - 3 hours

[0015] 5) Start the vacuum pump to evacuate. When the vacuum degree reaches 10 - 20 Pa, start heating. The heating process is as follows: first heat up to -20°C at a rate of 0.5 - 1°C / min and maintain for 3 - 4 hours to promote ice crystal sublimation; then heat up to 0°C at a rate of 1 - 2°C / min and maintain for 2 - 3 hours;

[0016] 6) Continue to heat up to 25 - 30°C and maintain for 3 - 4 hours to remove residual moisture and reduce the water content of the product to 3%

[0017] Thereafter, obtain the probiotic freeze-dried powder.

[0018] According to certain embodiments of the present application, the protective agent is autoclaved at 121 °C for 15 - 20 minutes.

[0019] According to certain embodiments of the present application, the mixed probiotics include Bifidobacterium, Lactobacillus, and Bacillus.

[0020] According to certain embodiments of the present application, the mixed probiotics contain Bifidobacterium, Lactobacillus, and Bacillus mixed in any proportion.

[0021] The technical solution of the present invention has the following beneficial effects:

[0022] 1) The present invention discloses a freeze-dried excipient formula for mixed strains, not a single strain.

[0023] 2) For the excipient formula of freeze-dried single strains in the prior art, the survival rate after freeze-drying can reach more than 80%; however, for the excipient formula of freeze-dried mixed bacteria in the prior art, the survival rate of the mixed bacteria after freeze-drying cannot reach 60%. The freeze-dried mixed bacteria formula disclosed by the present invention can simultaneously freeze-dry 3 types of mixed bacteria, and the survival rate of the mixed bacteria after freeze-drying can reach 78% while ensuring a good appearance.

[0024] 3) The formula disclosed by the present invention is applicable to the mixed bacteria of 3 types: Bifidobacterium, Lactobacillus, and Bacillus.

[0025] 4) The formula ABCD disclosed by the present invention is significantly better than the freeze-dried excipient formula disclosed in the prior art. Among them, formula B has particularly good effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shows the freeze-drying effects of different concentrations of glucose. Each vial contains 3 ml of liquid bacteria for freeze-drying. From left to right, the glucose concentrations are 1%, 2%, 3%, and 4% respectively. Figure 1 The results show that none of the vials at the four concentrations were freeze-dried within 24 hours, and the freeze-drying effects of the 1% and 2% concentrations were better than those of the other two groups of concentrations.

[0027] Figure 2 Shows the freeze-drying effects of different concentrations of lactose. Each vial contains 3 ml of liquid bacteria for freeze-drying. From left to right, the lactose concentrations are 1%, 2%, 3%, and 4% respectively. Figure 2 The results show that none of the vials at the four concentrations were freeze-dried within 24 hours, and the freeze-drying effects of the 2% and 3% concentrations were better than those of the other two groups of concentrations.

[0028] Figure 3 Shows the freeze-drying effects of different concentrations of xylose. Each vial contains 3 ml of liquid bacteria for freeze-drying. From left to right, the xylose concentrations are 1%, 2%, 3%, and 4% respectively. Figure 3The results showed that none of the vials at the four concentrations were freeze-dried within 24 hours, and the freeze-drying effects of the 1% and 2% concentrations were better than those of the other two groups of concentrations.

[0029] Figure 4 The freeze-drying effects of different concentrations of trehalose are shown. Each vial was filled with 3 ml of liquid bacteria for freeze-drying, and the trehalose concentrations from left to right were 1%, 2%, 3%, and 4%. Figure 4 The results showed that none of the vials at the four concentrations were freeze-dried within 24 hours, and the freeze-drying effects of the 1%, 2%, and 3% concentrations were better than those of the other two groups of concentrations.

[0030] Figure 5 The freeze-drying effects of different concentrations of galactose are shown. Each vial was filled with 3 ml of liquid bacteria for freeze-drying, and the galactose concentrations from left to right were 1%, 2%, 3%, and 4%. Figure 5 The results showed that none of the vials at the four concentrations were freeze-dried within 24 hours, and the freeze-drying effects of the 1% and 2% concentrations were better than those of the other two groups of concentrations.

[0031] Figure 6 The freeze-drying effects of different concentrations of sucrose are shown. Each vial was filled with 3 ml of liquid bacteria for freeze-drying, and the sucrose concentrations from left to right were 1%, 2%, 3%, and 4%. Figure 6 The results showed that none of the vials at the four concentrations were freeze-dried within 24 hours, and the freeze-drying effects of the 1%, 2%, and 3% concentrations were better than those of the other two groups of concentrations.

[0032] Figure 7 The freeze-drying effects of different concentrations of starch are shown. Each vial was filled with 3 ml of liquid bacteria for freeze-drying, and the starch concentrations from left to right were 3%, 5%, 7%, and 9%. Figure 7 The results showed that all vials at the four concentrations could be freeze-dried within 24 hours, and the freeze-drying effects of the four groups of concentrations were comparable.

[0033] Figure 8 The freeze-drying effects of different concentrations of mannitol are shown. Each vial was filled with 3 ml of liquid bacteria for freeze-drying, and the mannitol concentrations from left to right were 3%, 5%, 7%, and 9%. Figure 8 The results showed that all vials at the four concentrations could be freeze-dried within 24 hours. Except for the poor freeze-drying morphology of the 3% concentration, the freeze-drying effects of the other three groups of concentrations were better.

[0034] Figure 9 The freeze-drying effects of different concentrations of skim milk powder are shown. Each vial was filled with 3 ml of liquid bacteria for freeze-drying, and the skim milk powder concentrations from left to right were 4%, 6%, 8%, and 10%. Figure 9 The results showed that all vials at the four concentrations could be freeze-dried within 24 hours. Except for the poor freeze-drying morphology of the 4% concentration, the freeze-drying effects of the other three groups of concentrations were better.

[0035] Figure 10 Shows the freeze-dried state of pure bacteria without adding excipients. From left to right, there are 1 portion of bacteria (1*10 9 CFU), 2 portions of bacteria (2*10 9 CFU), and 3 portions of bacteria (3*10 9 CFU). Figure 10 The results show that without adding cryoprotectant and directly freeze-drying different amounts of bacteria, there is no fixed form.

[0036] Figure 11 Shows the freeze-dried state of composite formula A, with 4 replicates. Figure 11 The results show that after adding the cryoprotectant of composite formula A, there is a fixed form after freeze-drying.

[0037] Figure 12 Shows the freeze-dried state of composite formula B, with 4 replicates. Figure 12 The results show that after adding the cryoprotectant of composite formula B, there is a fixed form after freeze-drying.

[0038] Figure 13 Shows the freeze-dried state of composite formula C, with 4 replicates. Figure 13 The results show that after adding the cryoprotectant of composite formula C, there is a fixed form after freeze-drying.

[0039] Figure 14 Shows the freeze-dried state of composite formula D, with 4 replicates. Figure 14 The results show that after adding the cryoprotectant of composite formula D, there is a fixed form after freeze-drying. Detailed description of the invention

[0041] Definition

[0042] Freeze-drying technology

[0043] Freeze drying is a drying method in which a water-containing substance is first cooled and frozen into a solid, and then under low-temperature and reduced-pressure conditions, the sublimation property of water is used to dehydrate the substance at low temperature to achieve the purpose of drying.

[0044] The outstanding advantages of freeze-drying technology compared with other drying methods are as follows:

[0045] 1) The product is dried at low temperature, and the activities of various components are less lost;

[0046] 2) When frozen, the product can form a certain shape and basically remains unchanged after drying;

[0047] 3) It has good rehydration performance and can quickly return to the state before freeze-drying after absorbing water;

[0048] 4) Thorough dehydration, suitable for long-distance transportation and storage.

[0049] Due to the above advantages, freeze-drying technology is widely used in many fields such as biopharmaceuticals.

[0050] The complete freeze-drying process includes three stages: pre-freezing, primary drying (sublimation drying), and secondary drying (desorption drying). Various stresses generated during the entire freeze-drying process can cause damage (passivation of certain protein molecules) or even death of some microbial cells.

[0051] Lyoprotectant

[0052] In order to reduce the damage caused by the freeze-drying process to microbial cells and improve the solubility and stability of freeze-dried products, some additional substances need to be added to the freeze-drying system, and such substances are collectively referred to as lyoprotectants.

[0053] Currently, the commonly used protective agent components can be divided into low-molecular compounds, such as sodium glutamate and arginine hydrochloride; high-molecular substances, such as dextran and dextrin; and some natural mixtures, such as skim milk and serum. According to whether they can penetrate into the interior of cells, lyoprotectants can also be divided into permeable and non-permeable types. Permeable lyoprotectants are considered to be more ideal protective agents. Permeable protective agents are further subdivided into those that can penetrate both the cell wall and the cell membrane, such as glycerol; and those that can only pass through the cell wall but not the cell membrane, such as glucose. Non-permeable protective agents cannot penetrate both the cell wall and the cell membrane, such as polysaccharides. According to the chemical properties of the protective agents, they can be classified into several categories such as sugars, alcohols, inorganic salts, polymers, etc.

[0054] According to certain embodiments of the present invention, glucose, lactose, xylose, trehalose, galactose, sucrose, and starch are sugars; mannitol is an alcohol; VC is an antioxidant; glutamic acid, glycine, and lysine are amino acids; and skim milk powder is a complex.

[0055] Probiotics

[0056] Probiotics, namely "probiotics" or "microecological regulators". The latest definition of probiotics by the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO / WHO) in 2001 is: the general term for live microorganisms that can produce certain beneficial effects on the host when ingested in sufficient amounts. Probiotics play a significant role in preventing and regulating intestinal diseases. A sufficient number of probiotics can effectively colonize the intestine, form a dominant flora, and improve the balance of the intestinal flora.

[0057] Single strain and mixed bacteria

[0058] According to certain embodiments of the present invention, a single strain refers to one type of probiotic.

[0059] According to certain embodiments of the present invention, a probiotic mixed bacteria refers to a probiotic preparation formed by combining two or more probiotic strains together.

[0060] According to certain embodiments of the present invention, the mixed bacteria are a mixture of three types of bacteria: Bifidobacterium, Lactobacillus, and Bacillus

[0061] According to certain embodiments of the present invention, the mixed bacteria are an equi-proportion mixture of Bifidobacterium, Lactobacillus, and Bacillus

[0062] According to certain embodiments of the present invention, the mixed bacteria are a mixture of Bifidobacterium, Lactobacillus, and Bacillus in any proportion

[0063] According to certain embodiments of the present invention, in the following various situations, it is usually necessary to use mixed bacteria:

[0064] Complex intestinal problems

[0065] Constipation: Slow intestinal peristalsis and imbalance of intestinal flora are often the causes of constipation. Lactobacillus bifidus in the mixed bacteria can produce short-chain fatty acids to stimulate intestinal peristalsis; Lactobacillus acidophilus and others can increase the number of beneficial bacteria in the intestine and optimize the intestinal environment. The cooperation of multiple strains is more effective in solving the problem of constipation than a single strain

[0066] Inflammatory bowel disease: Inflammatory bowel diseases represented by ulcerative colitis and Crohn's disease have the intestine in an inflammatory state for a long time, and the mucosal barrier is damaged. Bifidobacterium, Lactobacillus acidophilus, and Bacillus in the mixed bacteria can play a comprehensive therapeutic advantage through multiple mechanisms such as regulating immunity, inhibiting inflammatory reactions, and repairing the intestinal mucosa, and may achieve more significant effects than a single strain

[0067] Immune enhancement needs

[0068] The elderly: As they age, the immune system function of the elderly gradually declines, and the intestinal flora also changes. Using a mixed bacteria preparation containing multiple probiotics such as Bifidobacterium, Lactobacillus acidophilus, and Enterococcus faecalis can more comprehensively regulate intestinal immune function, enhance the resistance of the elderly, and help prevent diseases such as infections

[0069] Children: The immune system of children is not yet fully developed, and there is an urgent need to enhance immunity during disease-susceptible stages such as seasonal changes and starting daycare or school. The mixed bacteria can enhance the immune function of children and resist the invasion of external pathogens by promoting the development and maturation of the intestinal immune system and generating immune regulatory factors

[0070] People with low immunity: Such as those who stay up late for a long time, are overworked, have chronic diseases, or receive radiotherapy and chemotherapy, whose immune systems are damaged to varying degrees. Various strains in the mixed bacteria can regulate immunity from different dimensions, some activate immune cells, and some promote the generation of immune factors, jointly enhancing the immune ability of the body

[0071] Specific nutritional needs

[0072] Lactose-intolerant people: Such people lack lactase and cannot fully digest lactose. Lactobacillus bulgaricus and Streptococcus thermophilus in the mixed bacteria can produce lactase during the fermentation process, helping to decompose lactose and providing other nutrients at the same time, improving the nutritional absorption status of lactose-intolerant people.

[0073] People in need of specific vitamin supplementation: Some probiotics, such as Bifidobacterium and Lactobacillus acidophilus, have the ability to synthesize nutrients such as vitamin B complex. Using the mixed bacteria can increase the amount of vitamin synthesis, better meet the human body's demand for these vitamins, which is particularly important for people with unbalanced diets or vegetarians.

[0074] Other situations

[0075] Long-term use of antibiotics: While antibiotics kill pathogenic bacteria, they also affect beneficial bacteria in the intestine. The mixed bacteria can more quickly and comprehensively supplement various beneficial bacteria damaged by antibiotics, restore the balance of the intestinal flora, and reduce the incidence of adverse reactions such as antibiotic-associated diarrhea.

[0076] Optimizing the diversity of the intestinal microecology: A healthy intestinal microecology depends on rich flora diversity. The improvement of a single strain on the diversity of the intestinal flora is relatively limited, while the mixed bacteria can introduce multiple probiotic strains, increase the types and numbers of beneficial bacteria in the intestine, optimize the intestinal microecological structure, and enhance the intestinal stability and function.

[0077] Protective agent formulation

[0078] The concentration of the protective agent formulation is calculated in g / 100ml. 1% means there is 1 gram of solute in 100ml of solution. In this scheme, the solute is different protective agent excipients, and the solution is pure water, and different concentration excipient solutions are prepared.

[0079] According to a certain embodiment of the present invention, the protective agent solution comprises one or more of 3-9% (preferably 5%) of mannitol, 3-9% (preferably 5%) of starch, 4-10% (preferably 8%) of skim milk powder, 1-4% (preferably 2%) of xylose, 1-4% (preferably 2%) of galactose, 1-4% (preferably 2%) of lactose, 1-4% (preferably 2%) of sucrose, 1-4% (preferably 2%) of glucose, and 1-4% (preferably 2%) of trehalose.

[0080] According to a certain embodiment of the present invention, the protective agent solution comprises 3-9% of mannitol, such as 3%, %, 4%, 5%, 6%, 7%, 8% or 9% mannitol.

[0081] According to a certain embodiment of the present invention, the protective agent solution comprises 3-9% of starch, such as 3%, %, 4%, 5%, 6%, 7%, 8% or 9% starch.

[0082] According to certain embodiments of the present invention, the protective agent solution comprises 4-10% skim milk powder, such as 4%, 5%, 6%, 7%, 8%, 9% or 10% skim milk powder.

[0083] According to certain embodiments of the present invention, the protective agent solution comprises 1-4% xylose, such as 1%, 2%, 3% or 4% xylose.

[0084] According to certain embodiments of the present invention, the protective agent solution comprises 1-4% galactose, such as 1%, 2%, 3% or 4% galactose.

[0085] According to certain embodiments of the present invention, the protective agent solution comprises 1-4% lactose, such as 1%, 2%, 3% or 4% lactose.

[0086] According to certain embodiments of the present invention, the protective agent solution comprises 1-4% sucrose, such as 1%, 2%, 3% or 4% sucrose.

[0087] According to certain embodiments of the present invention, the protective agent solution comprises 1-4% glucose, such as 1%, 2%, 3% or 4% glucose.

[0088] According to certain embodiments of the present invention, the protective agent solution comprises 1-4% trehalose, such as 1%, 2%, 3% or 4% trehalose.

[0089] According to certain embodiments of the present invention, the protective agent solution comprises one or more of 5% mannitol, 5% starch, 8% skim milk powder, 2% xylose, 2% galactose, 2% lactose, 2% sucrose, 2% glucose, 2% trehalose.

[0090] According to certain embodiments of the present invention, the protective agent solution comprises 2% glucose, 2% xylose, 2% trehalose, 2% galactose, 5% mannitol, 5% starch, 8% skim milk powder.

[0091] According to certain embodiments of the present invention, the protective agent solution comprises 2% glucose, 2% lactose, 2% trehalose, 2% sucrose, 5% starch, 5% mannitol, 8% skim milk powder.

[0092] According to certain embodiments of the present invention, the protective agent solution comprises 2% glucose, 2% lactose, 2% xylose, 2% trehalose, 5% starch, 5% mannitol, 8% skim milk powder.

[0093] According to certain embodiments of the present invention, the protective agent solution comprises 2% lactose, 2% trehalose, 2% galactose, 2% sucrose, 5% starch, 5% mannitol, 8% skim milk powder.

[0094] According to a certain embodiment of the present invention, the protective agent solution is composed of one or more of 3-9% (preferably 5%) mannitol, 3-9% (preferably 5%) starch, 4-10% (preferably 8%) skim milk powder, 1-4% (preferably 2%) xylose, 1-4% (preferably 2%) galactose, 1-4% (preferably 2%) lactose, 1-4% (preferably 2%) sucrose, 1-4% (preferably 2%) glucose, and 1-4% (preferably 2%) trehalose.

[0095] According to a certain embodiment of the present invention, the protective agent solution is composed of 3-9% mannitol, such as 3%, 4%, 5%, 6%, 7%, 8%, or 9% mannitol.

[0096] According to a certain embodiment of the present invention, the protective agent solution is composed of 3-9% starch, such as 3%, 4%, 5%, 6%, 7%, 8%, or 9% starch.

[0097] According to a certain embodiment of the present invention, the protective agent solution is composed of 4-10% skim milk powder, such as 4%, 5%, 6%, 7%, 8%, 9%, or 10% skim milk powder.

[0098] According to a certain embodiment of the present invention, the protective agent solution is composed of 1-4% xylose, such as 1%, 2%, 3%, or 4% xylose.

[0099] According to a certain embodiment of the present invention, the protective agent solution is composed of 1-4% galactose, such as 1%, 2%, 3%, or 4% galactose.

[0100] According to a certain embodiment of the present invention, the protective agent solution is composed of 1-4% lactose, such as 1%, 2%, 3%, or 4% lactose.

[0101] According to a certain embodiment of the present invention, the protective agent solution is composed of 1-4% sucrose, such as 1%, 2%, 3%, or 4% sucrose.

[0102] According to a certain embodiment of the present invention, the protective agent solution is composed of 1-4% glucose, such as 1%, 2%, 3%, or 4% glucose.

[0103] According to a certain embodiment of the present invention, the protective agent solution is composed of 1-4% trehalose, such as 1%, 2%, 3%, or 4% trehalose.

[0104] According to a certain embodiment of the present invention, the protective agent solution is composed of one or more of 5% mannitol, 5% starch, 8% skim milk powder, 2% xylose, 2% galactose, 2% lactose, 2% sucrose, 2% glucose, and 2% trehalose.

[0105] According to a certain embodiment of the present invention, the protective agent solution is composed of 2% glucose, 2% xylose, 2% trehalose, 2% galactose, 5% mannitol, 5% starch, and 8% skim milk powder.

[0106] According to a certain embodiment of the present invention, the protective agent solution is composed of 2% glucose, 2% lactose, 2% trehalose, 2% sucrose, 5% starch, 5% mannitol, and 8% skim milk powder.

[0107] According to a certain embodiment of the present invention, the protective agent solution is composed of 2% glucose, 2% lactose, 2% xylose, 2% trehalose, 5% starch, 5% mannitol, and 8% skim milk powder.

[0108] According to a certain embodiment of the present invention, the protective agent solution is composed of 2% lactose, 2% trehalose, 2% galactose, 2% sucrose, 5% starch, 5% mannitol, and 8% skim milk powder.

[0109] Method for preparing mixed probiotic freeze-dried powder

[0110] According to a certain embodiment of the present invention, the present invention discloses a method for preparing mixed probiotic freeze-dried powder using the protective agent solution, and the method includes:

[0111] 1) Place the fermentation broth containing probiotics in an environment of 2-6°C, centrifuge at a speed of 5000-8000 r / min for 15-20 minutes, pour off the supernatant, and collect the cell precipitate; wash the cell precipitate with sterile physiological saline 2-3 times, and centrifuge at 2-6°C and 5000-8000 r / min for 15-20 minutes after each washing, and discard the supernatant;

[0112] 2) Resuspend the washed cells with a small amount of sterile physiological saline, adjust the concentration of the cell suspension so that the concentration of various probiotics after mixing reaches 10^9-10^11 CFU / mL;

[0113] 3) Mix the cell suspension with a concentration of 10^9-10^11 CFU / mL evenly with 2-5 mL of the sterilized protective agent solution;

[0114] 4) Aliquot the mixed cell solution into a freeze-drying tray, control the thickness to 1-2 cm, and then place it in a freeze-dryer for pre-freezing. The pre-freezing conditions are to cool down at a rate of 1-2°C / min to -30 - -50°C and maintain for 2-3 hours;

[0115] 5) Start the vacuum pump to evacuate. When the vacuum degree reaches 10-20 Pa, start heating. The heating procedure is as follows: first heat at a rate of 0.5-1°C / min to -20°C and maintain for 3-4 hours to promote ice crystal sublimation; then heat at a rate of 1-2°C / min to 0°C and maintain for 2-3 hours;

[0116] 6) Continue to raise the temperature to 25 - 30 °C and maintain for 3 - 4 hours to remove residual moisture and reduce the water content of the product to 3%.

[0117] Next, obtain the freeze-dried probiotic powder.

[0118] According to a certain embodiment of the present invention, place the fermentation broth containing probiotics in an environment of 2 - 6 °C, such as 2 °C, 3 °C, 4 °C, 5 °C, or 6 °C.

[0119] According to a certain embodiment of the present invention, centrifuge at a rotational speed of 5000 - 8000 r / min, such as 5000 r / min, 6000 r / min, 7000 r / min, or 8000 r / min.

[0120] According to a certain embodiment of the present invention, centrifuge for 15 - 20 minutes, such as 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, or 20 minutes.

[0121] According to a certain embodiment of the present invention, the probiotic concentration reaches 10^9 - 10^11 CFU / mL, such as 10^9 CFU / mL, 10^10 CFU / mL, or 10^11 CFU / mL.

[0122] According to a certain embodiment of the present invention, 2 - 5 mL of the protective agent is, for example, 2 mL, 3 mL, 4 mL, or 5 mL.

[0123] According to a certain embodiment of the present invention, the thickness is controlled at 1 - 2 cm, such as 1.0 cm, 1.2 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.8 cm, or 2.0 cm.

[0124] According to a certain embodiment of the present invention, the rate of 1 - 2 °C / min is 1.0 °C / min, 1.2 °C / min, 1.4 °C / min, 1.5 °C / min, 1.6 °C / min, 1.8 °C / min, or 2.0 °C / min.

[0125] According to a certain embodiment of the present invention, cool down to -30 - -50 °C, such as -30 °C, -35 °C, -40 °C, -45 °C, or -50 °C.

[0126] According to a certain embodiment of the present invention, maintain for 2 - 3 hours, such as 2 hours, 2.5 hours, or 3 hours.

[0127] According to a certain embodiment of the present invention, raise the temperature at a rate of 0.5 - 1 °C / min, such as 0.5 °C / min, 0.6 °C / min, 0.7 °C / min, 0.8 °C / min, 0.9 °C / min, or 1.0 °C / min.

[0128] According to a certain embodiment of the present invention, the holding is for 3 - 4 hours, such as 3 hours, 3.5 hours, or 4 hours.

[0129] According to a certain embodiment of the present invention, the temperature is raised to 25 - 30 °C, such as 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C.

[0130] According to a certain embodiment of the present invention, the water content of the product is reduced to less than 3%, such as less than 33%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, or less than 0.01%.

[0131] According to a certain embodiment of the present invention, the protective agent is autoclaved at 121 °C for 15 - 20 minutes, such as 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, or 20 minutes.

[0132] Total number of colonies

[0133] The total number of colonies refers to the total number of bacterial colonies growing in each gram (each milliliter) of the sample.

[0134] The unit of colonies is CFU (colony - forming unit). The number of colonies represented by the total number of colonies is not the number of bacteria in the colonies, nor does it distinguish the types of bacteria. Detailed implementation methods

[0135] Example 1: Formulation and effects in the prior art

[0136] The preparation process of the mixed probiotic freeze - dried powder generally includes steps such as strain resuscitation, seed cultivation, fermentation culture, bacterial cell collection and concentration, protective agent addition and mixing, freeze - drying, etc.:

[0137] I. Strain resuscitation

[0138] (I) Preparation of resuscitation medium

[0139] Lactobacillus acidophilus: Use MRS medium, which contains 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate. Then add 15 g of agar and make up to 1000 mL with distilled water, and adjust the pH to 6.2 - 6.6.

[0140] Bifidobacterium: Use modified MRS medium, that is, add 0.05% cysteine hydrochloride to the ordinary MRS medium and adjust the pH to 6.8 - 7.0.

[0141] Lactobacillus rhamnosus: For the MRS medium used, the pH also needs to be adjusted to 6.2 - 6.6.

[0142] (II) Strain resuscitation operation

[0143] Inoculate the preserved strain onto the corresponding resuscitation medium slant. Lactobacillus acidophilus and Lactobacillus rhamnosus need to be cultured in an anaerobic environment at 37°C for 24 - 48 hours; while Bifidobacterium needs to be cultured under strict anaerobic conditions at 37°C for 48 - 72 hours. Then pick a single colony and inoculate it again into a new resuscitation medium, repeating the culture 1 - 2 times.

[0144] II. Seed cultivation

[0145] (I) Preparation of seed medium

[0146] The formula of the seed medium is the same as that of the resuscitation medium, but agar can be not added to make a liquid medium.

[0147] (II) Inoculation and culture process

[0148] Use an inoculation loop to pick a resuscitated single colony and inoculate it into the seed medium. Among them, the inoculation amounts of Lactobacillus acidophilus and Lactobacillus rhamnosus are 2% - 3% by volume ratio, and the inoculation amount of Bifidobacterium is 3% - 5%.

[0149] Under an anaerobic environment at 37°C, Lactobacillus acidophilus and Lactobacillus rhamnosus are cultured with shaking at a speed of 120 - 150 r / min for 18 - 24 hours; while Bifidobacterium is statically cultured under strict anaerobic conditions for 24 - 36 hours.

[0150] After the culture is completed, detect the bacterial concentration in the seed liquid. Generally, it is required that the bacterial concentration of Lactobacillus acidophilus and Lactobacillus rhamnosus reaches 10^8 - 10^9 CFU / mL, and the bacterial concentration of Bifidobacterium reaches 10^7 - 10^8 CFU / mL.

[0151] III. Fermentation culture stage

[0152] (I) Optimization of fermentation medium

[0153] Based on the basic MRS medium, optimize the proportions of carbon sources (such as glucose, lactose, maltose, etc.), nitrogen sources (such as soy peptone, beef extract, yeast powder, etc.) and other nutritional components through experiments. For example, the optimized fermentation medium formula for Lactobacillus acidophilus may be 25 g of glucose, 12 g of soy peptone, 8 g of yeast powder, 5 g of beef extract, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, made up to 1000 mL with distilled water, and the pH adjusted to 6.5.

[0154] The optimized Bifidobacterium fermentation medium is as follows: 20 g of glucose, 10 g of soy peptone, 6 g of yeast powder, 4 g of beef extract, 0.5 g of cysteine hydrochloride, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate. It is made up to 1000 mL with distilled water and the pH is 6.8.

[0155] For the fermentation medium of Lactobacillus rhamnosus, after optimization, it is: 22 g of glucose, 11 g of soy peptone, 7 g of yeast powder, 5 g of beef extract, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate. It is made up to 1000 mL with distilled water and the pH is set at 6.4.

[0156] (II) Inoculation and fermentation operations

[0157] The seed liquid is inoculated into the fermenter at an inoculation amount of 5%-10% (volume ratio).

[0158] Control the fermentation conditions: The fermentation temperature of Lactobacillus acidophilus and Lactobacillus rhamnosus is maintained at 37°C, the stirring speed is 100 - 150 r / min, an anaerobic environment is created by introducing nitrogen, and the fermentation duration is 24 - 36 hours; the fermentation temperature of Bifidobacterium is also 37°C, in a strictly anaerobic environment without stirring, and the fermentation time is 36 - 48 hours.

[0159] During the fermentation process, parameters such as pH value and bacterial concentration are detected regularly. When the pH of the fermentation broth of Lactobacillus acidophilus and Lactobacillus rhamnosus drops to 4.0 - 4.5 and the bacterial concentration reaches 10^9 - 10^10 CFU / mL; when the pH of the Bifidobacterium fermentation broth drops to 4.5 - 5.0 and the bacterial concentration reaches 10^8 - 10^9 CFU / mL, the fermentation ends.

[0160] IV. Steps for cell collection and concentration

[0161] (I) Centrifugation to collect cells

[0162] The fermentation broth is placed in an environment at 4°C and centrifuged at a speed of 5000 - 8000 r / min for 15 - 20 minutes. The supernatant is discarded and the cell precipitate is collected.

[0163] (II) Cell washing operation

[0164] The cell precipitate is washed 2 - 3 times with sterile normal saline. After each washing, it is centrifuged at 4°C and 5000 - 8000 r / min for 15 - 20 minutes, and then the supernatant is discarded.

[0165] (III) Cell concentration treatment

[0166] Resuspend the washed bacterial cells with a small amount of sterile normal saline, and adjust the concentration of the bacterial suspension so that the concentration of various probiotics after mixing reaches 10^10 - 10^11 CFU / mL.

[0167] V. Protective Agent Addition and Mixing Process

[0168] (I) Selection and Preparation of Protective Agent

[0169] After the protective agent is stirred evenly, autoclave it at 121°C for 15 - 20 minutes.

[0170] Add the corresponding protective agent according to different strains in Table 1.

[0171] (II) Operation of Mixing Protective Agent and Bacterial Cells

[0172] Mix the concentrated bacterial suspension (total concentration 1*10^9) evenly with 3 mL of the protective agent solution.

[0173] VI. Freeze-Drying Process

[0174] (I) Pre-Freezing Stage

[0175] Dispense the mixed bacterial solution into the freeze-drying trays, with the thickness controlled at 1 - 2 cm, and then place it in the freeze-dryer for pre-freezing. The pre-freezing program is: cool down at a rate of 1 - 2°C / min to -40°C and maintain for 2 - 3 hours.

[0176] (II) Sublimation Drying Stage

[0177] After pre-freezing is completed, start the vacuum pump to evacuate. When the vacuum degree reaches 10 - 20 Pa, start heating. The heating program is: first heat up at a rate of 0.5 - 1°C / min to -20°C and maintain for 3 - 4 hours to promote ice crystal sublimation; then heat up at a rate of 1 - 2°C / min to 0°C and maintain for 2 - 3 hours.

[0178] (III) Desorption Drying Stage

[0179] Continue to heat up to 25 - 30°C and maintain for 3 - 4 hours to remove residual moisture, reduce the water content of the product to less than 3%, and obtain the freeze-dried powder.

[0180] VII. Detection of Bacterial Survival Rate

[0181] Take 1 tube of freeze-dried probiotics with 3 mL of protective agent, add 3 mL of normal saline for gradient dilution, plate count each gradient, and place it in the corresponding culture medium and culture conditions of the corresponding strain in the above "Step I. Strain Resuscitation" for 48 hours.

[0182] The viable count is based on the national standard "National Food Safety Standard Microbiological Examination of Foods - Examination of Lactic Acid Bacteria" (GB 4789.35—2016).

[0183] Table 1: Lyoprotectants for freeze-drying single strains in the prior art

[0184]

[0185] Example 2: Survival rate and morphology of freeze-dried strains using single protective agent excipients at different concentrations

[0186] Carry out strain resuscitation, seed cultivation, fermentation culture, cell collection and concentration, addition and mixing of lyoprotectant, and freeze-drying according to the steps of Example 1. The components of the lyoprotectant are added with the components in Tables 2-4 respectively.

[0187] After freeze-drying the equal-ratio mixed bacteria of Bifidobacterium, Lactobacillus, Bacillus and other single lyoprotectant excipients, carry out viable count and calculate the survival rate.

[0188] The morphology after freeze-drying of glucose at different concentrations is as Figure 1 shown. The freeze-drying morphology of the 1% and 2% concentrations is better than that of the other two groups of concentrations. Among them, the survival rate of the bacteria at the 2% concentration is the highest (Table 2). Finally, 2% glucose is selected.

[0189] The morphology after freeze-drying of lactose at different concentrations is as Figure 2 shown. The freeze-drying morphology of the 2% and 3% concentrations is better than that of the other two groups of concentrations. Among them, the survival rate of the bacteria at the 2% concentration is the highest (Table 2). Finally, 2% lactose is selected.

[0190] The morphology after freeze-drying of xylose at different concentrations is as Figure 3 shown. The freeze-drying morphology of the 1% and 2% concentrations is better than that of the other two groups of concentrations. Among them, the survival rate of the bacteria at the 2% concentration is the highest (Table 2). Finally, 2% xylose is selected.

[0191] The morphology after freeze-drying of trehalose at different concentrations is as Figure 4 shown. The freeze-drying morphology of the 1%, 2% and 3% concentrations is better than that of the other two groups of concentrations. Among them, the survival rate of the bacteria at the 2% concentration is the highest (Table 2). Finally, 2% trehalose is selected.

[0192] The morphology after freeze-drying of galactose at different concentrations is as Figure 5 shown. The freeze-drying morphology of the 1% and 2% concentrations is better than that of the other two groups of concentrations. Among them, the survival rate of the bacteria at the 2% concentration is the highest (Table 2). Finally, 2% galactose is selected.

[0193] The morphology after freeze-drying of sucrose at different concentrations is as Figure 6 shown. The freeze-drying morphology of the 1%, 2% and 3% concentrations is better than that of the other two groups of concentrations. Among them, the survival rate of the bacteria at the 2% concentration is the highest (Table 2). Finally, 2% sucrose is selected.

[0194] The morphology after freeze-drying of starch at different concentrations is asFigure 7 As shown, the freeze-drying effects of the 4 groups with different concentrations are equivalent, and the survival rate of bacteria at 5% concentration is the highest (Table 3). Finally, 5% starch is selected.

[0195] The morphology of starches with different concentrations after freeze-drying is as Figure 8 shown. The freeze-dried morphologies of 5%, 7%, and 9% concentrations are good, and the survival rate of bacteria at 5% concentration is the highest (Table 3). Finally, 5% starch is selected.

[0196] The morphology of skim milk powder with different concentrations after freeze-drying is as Figure 9 shown. The freeze-dried morphologies of 6%, 8%, and 10% concentrations are good, and the survival rate of bacteria at 8% concentration is the highest (Table 4). Finally, 8% skim milk powder is selected.

[0197] Table 2

[0198] 1％ 2％ 3％ 4％ Glucose 39.35％ 85.92％ 31.70％ 65.28％ Figure 1 Lactose 8.38％ 38.46％ 13.85％ 23.89％ Figure 2 Xylose 41.69％ 74.67％ 41.30％ 42.27％ Figure 3 Trehalose 40.29％ 86.44％ 75.63％ 63.93％ Figure 4 Galactose 52.86％ 77.64％ 61.61％ 61.20％ Figure 5 Sucrose 52.87％ 74.43％ 60.22％ 58.04％ Figure 6

[0199] Table 3

[0200] 3％ 5％ 7％ 9％ Starch 26.84％ 60.74％ 17.76％ 35.64％ Figure 7 Mannitol 0.02％ 16.84％ 1.05％ 10.64％ Figure 8

[0201] Table 4

[0202] 4％ 6％ 8％ 10％ Skim milk powder 52.18％ 35.17％ 85.65％ 72.93％ Figure 9

[0203] Example 3: Survival rate and morphology of freeze-dried composite strains using composite formulation protective agent excipients at different concentrations

[0204] Carry out bacterial strain resuscitation, seed cultivation, fermentation culture, bacterial cell collection and concentration, protective agent addition and mixing, and freeze-drying according to the steps of Example 1. The components of the protective agent are added with the components in Table 5 respectively.

[0205] Figure 10 Shows the freeze-dried state of pure bacteria without adding protective agent excipients. From left to right, they are 1 portion of bacteria (1*10 9 CFU), 2 portions of bacteria (2*10 9 CFU), 3 portions of bacteria (3*10 9 CFU), all without a fixed morphology. The survival rate results show that without adding a protective agent, the direct freeze-drying survival rate of different amounts of bacteria is 0.

[0206] Figure 11 Shows the freeze-dried state of compound protective agent formula A. There are 4 replicates. After adding the compound formula A protective agent, it has a fixed morphology after freeze-drying, and the survival rate of bacteria after freeze-drying is 62.10%.

[0207] Figure 12 Shows the freeze-dried state of compound protective agent formula B. There are 4 replicates. After adding the compound formula B protective agent, it has a fixed morphology after freeze-drying, and the survival rate of bacteria after freeze-drying is 78.14%.

[0208] Figure 13Shows the freeze-dried state of the composite protectant formulation C, with 4 replicates. After adding the composite formulation C protectant, it has a fixed form after freeze-drying, and the survival rate of bacteria after freeze-drying is 35.59%.

[0209] Figure 14 Shows the freeze-dried state of the composite protectant formulation D, with 4 replicates. After adding the composite formulation D protectant, it has a fixed form after freeze-drying, and the survival rate of bacteria after freeze-drying is 53.18%.

[0210] Table 5

[0211]

[0212] The results of this example show that by using the composite protectant formulations A, B, C, and D disclosed in the present invention to freeze-dry and preserve the mixed strains, a very high survival rate can be obtained, and the effect of formulation B is particularly good.

[0213] Example 4: Comparison of the survival rate of freeze-dried mixed bacteria between excipients of Formulation B and the prior art freeze-drying protective agent formulation

[0214] In this example, the formulation B disclosed in Example 3 and the freeze-drying protectant adjuvant formulations of 5 kinds of prior arts disclosed in Table 1 were used to prepare the freeze-dried powder of the mixed strains.

[0215] According to the experimental steps in Example 1, the strain resuscitation, seed cultivation, fermentation culture, bacterial cell collection and concentration, protectant addition and mixing, and freeze-drying were carried out. The components of the protectant were added with the components in Table 6 respectively.

[0216] Table 6

[0217]

[0218] The results of this example show that by using the composite protectant formulation B disclosed in the present invention to freeze-dry and preserve the mixed strains, the survival rate is significantly better than the freeze-drying protectant adjuvant formulations disclosed in the prior art.

Claims

1. A protective agent solution, which contains one or more of mannitol at 3-9%, starch at 3-9%, skim milk powder at 4-10%, xylose at 1-4%, galactose at 1-4%, lactose at 1-4%, sucrose at 1-4%, glucose at 1-4%, and trehalose at 1-4%.

2. The method according to claim 1, wherein, The protective agent solution contains one or more of mannitol at 5%, starch at 5%, skim milk powder at 8%, xylose at 2%, galactose at 2%, lactose at 2%, sucrose at 2%, glucose at 2%, and trehalose at 2%.

3. The method according to claim 2, wherein The protective agent solution contains glucose at 2%, xylose at 2%, trehalose at 2%, galactose at 2%, mannitol at 5%, starch at 5%, and skim milk powder at 8%; glucose at 2%, lactose at 2%, trehalose at 2%, sucrose at 2%, starch at 5%, mannitol at 5%, and skim milk powder at 8%; glucose at 2%, lactose at 2%, xylose at 2%, trehalose at 2%, starch at 5%, mannitol at 5%, and skim milk powder at 8%; or lactose at 2%, trehalose at 2%, galactose at 2%, sucrose at 2%, starch at 5%, mannitol at 5%, and skim milk powder at 8%.

4. A method for preparing a freeze-dried powder of mixed probiotics using the protective agent solution according to any one of claims 1-3, the method comprising: 1) Placing the fermentation broth containing probiotics in an environment at 2-6°C, centrifuging at a speed of 5000-8000 r / min for 15-20 minutes, pouring off the supernatant, and collecting the cell precipitate; washing the cell precipitate with sterile normal saline 2-3 times, and centrifuging at 2-6°C and 5000-8000 r / min for 15-20 minutes after each washing, and discarding the supernatant; 2) Resuspending the washed cells with a small amount of sterile normal saline, adjusting the concentration of the cell suspension to make the concentration of various probiotics after mixing reach 10^9-10^11 CFU / mL; 3) Mixing the cell suspension with a concentration of 10^9-10^11 CFU / mL evenly with 2-5 mL of the sterilized protective agent solution; 4) Aliquoting the mixed cell solution into a freeze-drying tray, controlling the thickness to 1-2 cm, and then placing it in a freeze dryer for pre-freezing. The pre-freezing condition is to cool down at a rate of 1-2°C / min to -30 - -50°C and hold for 2-3 hours 5) Starting the vacuum pump to evacuate, when the vacuum degree reaches 10-20 Pa, start heating up. The heating process is as follows: first heat up at a rate of 0.5-1°C / min to -20°C and hold for 3-4 hours to promote the sublimation of ice crystals; then heat up at a rate of 1-2°C / min to 0°C and hold for 2-3 hours; 6) Continue to heat up to 25-30°C and hold for 3-4 hours to remove the residual moisture, reducing the water content of the product to less than 3%, to obtain the freeze-dried powder of probiotics.

5. The method according to claim 4, wherein, The protective agent is autoclaved at 121°C for 15-20 minutes.

6. The method according to claim 4, wherein The mixed probiotics contain Bifidobacterium, Lactobacillus, and Bacillus.

7. The method according to claim 6, wherein The mixed probiotics contain Bifidobacterium, Lactobacillus, and Bacillus mixed in any proportion.

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