Preparation method of bifidobacterium breve powder with high viable count

Abstract

The invention relates to the technical field of microorganisms, and provides a preparation method of bifidobacterium breve powder with high viable count, which comprises the following steps: S1, inoculating a bifidobacterium breve strain into a seed culture medium for culture to obtain a bifidobacterium breve seed solution, inoculating the bifidobacterium breve seed solution into a high-density fermentation culture medium for fermentation culture to obtain a bifidobacterium breve seed solution; fermentation liquid is obtained; s2, centrifuging the fermentation liquid to obtain first bacterial sludge, mixing the first bacterial sludge with a washing liquid, resuspending, and centrifuging again to obtain second bacterial sludge; s3, adding a freeze-drying protective agent into the second bacterial sludge, and mixing to obtain an emulsion; and S4, dropwise adding liquid nitrogen into the emulsion, pre-freezing, and performing vacuum freeze drying to obtain bifidobacterium breve powder. According to the technical scheme, the problems of low viable count and low freeze-drying survival rate of the bifidobacterium breve fermentation liquor in related technologies are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology. Specifically, it relates to a method for preparing short Bifidobacterium powder with a high viable count. Background Art

[0002] In recent years, the food industry related to lactic acid bacteria fermentation in China has developed rapidly. Statistical data shows that from 2015 to 2019, the growth rate of the national lactic acid bacteria market was higher than 10%. In 2020, the lactic acid bacteria market exceeded 35 billion, and it is expected to reach a scale of about 50 billion in 2024. Bifidobacterium ( Bifidobacterium ) is a Gram-positive bacterium without spores, non-motile, strictly anaerobic, and presenting various forms such as V or Y. It is the dominant flora naturally present in the colon and is one of the first microorganisms to colonize the human intestine, playing an important role in regulating the balance of intestinal flora and promoting the normal development of the intestine.

[0003] Bifidobacterium breve is isolated from infant feces, breast milk, and vaginal environment. It has various probiotic functions such as relieving seasonal allergic rhinitis and intermittent asthma, changing fat metabolism, immune regulation, and inhibiting pathogenic bacteria. Its application prospects are broad, and it can be used as both a dietary supplement and a functional ingredient in health foods.

[0004] In addition to having high nutritional requirements, Bifidobacterium breve also has strict requirements for the surrounding living environment. For example, in the literature "Wang Shunyu, Zhang Hairong, Li Yuan, etc. Simulation analysis of the death law of Bifidobacterium at different stages before freeze-drying in industrial production [J]. Food and Industrial Fermentation, 2019, 45(12): 55-61.", Wang Shunyu et al. found that the exposure time to air and temperature during the industrial preparation process have a greater impact on the viable count of Bifidobacterium; in the literature "Li Debin, Zhao Min. Research on cryoprotectants during the preparation of freeze-dried Bifidobacterium powder [J]. China Dairy Industry, 2011, 39(11): 20-23.", Li Debin et al. found that the highest freeze-dried survival rate of Bifidobacterium is 71.11% after adding a compound cryoprotectant; in the literature "Jiang Wenxin, Cui Shumao, Mao Bingyong, etc. Optimization of cryoprotectants for Bifidobacterium breve and high-density freeze-drying process [J]. Food and Fermentation Industries, 2020, 46(9): 31-36.", Jiang Wenxin et al. found that the cryoprotectant consists of sorbitol, raffinose, and collagen in a mass ratio of 3:3:2, and the freeze-dried survival rate of Bifidobacterium breve reaches 80%. Therefore, it is necessary to study a high-density fermentation medium and its fermentation method to increase the viable count of Bifidobacterium breve fermentation broth. In addition, as a probiotic, Bifidobacterium breve is very easy to inactivate during production, preparation, and storage, and improving the freeze-dried survival rate of Bifidobacterium breve is also an urgent problem to be solved. Summary of the Invention

[0005] The present invention provides a method for preparing a short Bifidobacterium powder with a high viable cell count, which solves the problems of low viable cell count in the fermentation broth of Bifidobacterium breve and low freeze-drying survival rate in the related art.

[0006] The technical solution of the present invention is as follows: The present invention provides a method for preparing a short Bifidobacterium powder with a high viable cell count, comprising the following steps: S1. Inoculate a Bifidobacterium breve strain into a seed medium for cultivation to obtain a Bifidobacterium breve seed liquid, and inoculate the Bifidobacterium breve seed liquid into a high-density fermentation medium for fermentation cultivation to obtain a fermentation broth; S2. Centrifuge the fermentation broth to obtain a first bacterial sludge, mix and resuspend the first bacterial sludge with a washing solution, and then centrifuge again to obtain a second bacterial sludge; S3. Add a freeze-drying protectant to the second bacterial sludge and mix to obtain an emulsion; S4. Drop the emulsion into liquid nitrogen, pre-freeze it, and then perform vacuum freeze-drying to obtain a Bifidobacterium breve powder; The high-density fermentation medium comprises the following components: 25 - 50 g of lactose, 15 - 30 g of yeast nutrient, 10 - 20 g of soy peptone, 2 - 8 g of sodium acetate, 0.5 - 2 g of citric acid, 0.5 - 2 g of diammonium hydrogen phosphate, 0.2 - 0.5 g of magnesium sulfate, 0.1 - 0.5 g of emulsifier, 0.5 - 2 g of sodium ascorbate, 0.5 - 2 g of calcium carbonate, and 1000 mL of water.

[0007] As a further technical solution, the weight ratio of citric acid to calcium carbonate is 3 - 4:2.

[0008] In the present invention, when the weight ratio of citric acid to calcium carbonate is 3 - 4:2, the viable cell count in the fermentation broth of Bifidobacterium breve can be further increased.

[0009] As a further technical solution, in step S1, the addition amount of the Bifidobacterium breve seed liquid is 3% - 8% of the volume of the high-density fermentation medium.

[0010] As a further technical solution, in step S1, the specific steps of the fermentation cultivation are as follows: anaerobic fermentation is carried out for 10 - 15 h under the conditions of a temperature of 35 - 40 °C, a rotation speed of 60 - 150 rpm, and an initial pH of 6.2 - 6.5. During the anaerobic fermentation, the pH is adjusted to 5.5 - 6.0 with an alkaline solution, and the tank pressure is maintained at 0.05 MPa; The alkaline solution is one of a sodium carbonate solution and an ammonia water solution; The mass fraction of the alkaline solution is 20% - 30%.

[0011] As a further technical solution, after anaerobic fermentation, the temperature is adjusted to 30-35°C and cultivation is continued for 2-4 h.

[0012] As a further technical solution, the washing liquid is composed of the following components by weight percentage: Emulsifier 0.3%-2.5%, peptone 0.5%-1.5%, and the balance is water; The mass ratio of the first bacterial sludge to the washing liquid is 1:7-12; In the components of the high-density fermentation medium and the washing liquid of the present invention, the emulsifier is one or both of Tween 80 and Tween 20, and preferably Tween 80.

[0013] As a further technical solution, the lyoprotectant is composed of the following components by weight percentage: Trehalose 10%-12%, skim milk powder 8%-12%, lactose 4%-6%, glycerol 1%-3%, inulin 1%-3%, sodium ascorbate 0.5%-2%, sodium alginate 3%-5%, yeast peptone 0.5%-2%, and the balance is water; The mass ratio of the second bacterial sludge to the lyoprotectant is 1:0.5-2.

[0014] As a further technical solution, when adding the lyoprotectant, it is divided into the first addition and the second addition. The first addition is 45%-70% of the mass of the lyoprotectant, and the second addition is the remaining mass of the lyoprotectant.

[0015] In the present invention, when adding the lyoprotectant, it is divided into the first addition and the second addition. Adding a part of the lyoprotectant for the first time can make the protectant and the second bacterial sludge mix evenly initially, can fully contact with Bifidobacterium breve, and form a preliminary protection barrier on the surface of Bifidobacterium breve, providing basic protection for the subsequent freeze-drying process; adding the remaining lyoprotectant for the second time can further enhance the protection effect. Adding in two times can continuously play a protective role at different stages, form a stable emulsion, so as to ensure that Bifidobacterium breve can be well protected during the whole freeze-drying process, thereby increasing the viable count of the final emulsion and the viable count of the bacterial powder.

[0016] By regulating the mass of the lyoprotectant added in two times, when the first addition is 45%-70% of the mass of the lyoprotectant and the second addition is the remaining mass of the lyoprotectant, the viable count of the emulsion and the viable count of the bacterial powder can be further increased.

[0017] As a further technical solution, the skim milk powder is skim milk powder treated with protease for 3-5 h before use.

[0018] As a further technical solution, the specific preparation method of the Bifidobacterium breve seed liquid is as follows: inoculate the Bifidobacterium breve strain into 10 mL of the first seed medium at an inoculation amount of 8-12%, perform the first static culture to obtain the primary seed, and inoculate the primary seed into 200 mL of the second seed medium at an inoculation amount of 4-6%, perform the second static culture to obtain the secondary seed, which is the Bifidobacterium breve seed liquid.

[0019] As a further technical solution, the temperatures of the first static culture and the second static culture are each independently 35-37 °C; The time of the first static culture is 20-24 h; The time of the second static culture is 16-20 h.

[0020] As a further technical solution, the first seed medium and the second seed medium are each independently composed of the following components by weight percentage: Lactose 18-22 g, yeast nutrient 13-16 g, soy peptone 8-11 g, sodium acetate 1.5-2.2 g, citric acid 0.2-0.4 g, diammonium hydrogen phosphate 0.15-0.24 g, magnesium sulfate 0.14-0.24 g, Tween 80 0.05-0.15 g, sodium ascorbate 0.4-0.6 g, calcium carbonate 0.2-0.32 g, water 1000 mL.

[0021] As a further technical solution, the dropping into liquid nitrogen is to drop the emulsifier into liquid nitrogen through a nozzle.

[0022] As a further technical solution, the temperature of the liquid nitrogen is -195 to -198 °C.

[0023] As a further technical solution, during the vacuum freeze-drying, the primary sublimation temperature is 8-12 °C, the first vacuum degree is 0.013-0.016 mbar, the analytical drying temperature is 30-33 °C, the second vacuum degree is 0.004-0.006 mbar, and freeze-dry until the water content is 2.6-3.3%.

[0024] As a further technical solution, after the vacuum freeze-drying, it further includes pulverization.

[0025] The working principle and beneficial effects of the present invention are as follows: 1. In the present invention, a high-density fermentation medium is formed with lactose, yeast nutrient, soy peptone, sodium acetate, citric acid, diammonium hydrogen phosphate, magnesium sulfate, Tween 80, sodium ascorbate, calcium carbonate and water as raw materials. After high-density culture of Bifidobacterium breve, the viable bacteria count in the fermentation broth can reach 1.89×10 10 ~2.38×10 10 cfu / mL; 2. When the high-density fermentation medium in the present invention contains citric acid and calcium carbonate, the viable cell count of the fermentation broth of Bifidobacterium breve can be significantly increased by the combined use of the two.

[0026] 3. After the Bifidobacterium breve of the present invention is protected by a lyophilization protectant and quickly frozen in liquid nitrogen, the lyophilization survival rate reaches 85%, and the viable cell count is as high as 1.5×10 12 cfu / g. At the same time, the appearance of the product is uniform, and the rehydration property is better, avoiding problems such as incomplete dissolution during use. Detailed implementation manners

[0027] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0028] In the following examples and comparative examples, the Bifidobacterium breve strain was isolated from the feces of infants in the mother and infant cohort in the southwestern region.

[0029] Example 1 The first seed medium is composed of the following components by weight percentage: Lactose 20 g, yeast nutrient 15 g, soy peptone 10 g, sodium acetate 2 g, citric acid 0.3 g, diammonium hydrogen phosphate 0.2 g, magnesium sulfate 0.2 g, Tween 80 0.1 g, sodium ascorbate 0.5 g, calcium carbonate 0.3 g, water 1000 mL; The second seed medium is composed of the following components by weight percentage: Lactose 20 g, yeast nutrient 15 g, soy peptone 10 g, sodium acetate 2 g, citric acid 0.3 g, diammonium hydrogen phosphate 0.2 g, magnesium sulfate 0.2 g, Tween 80 0.1 g, sodium ascorbate 0.5 g, calcium carbonate 0.3 g, water 1000 mL; The high-density fermentation medium includes the following components by weight: Lactose 25 g, yeast nutrient 15 g, soy peptone 10 g, sodium acetate 2 g, citric acid 0.5 g, diammonium hydrogen phosphate 0.5 g, magnesium sulfate 0.2 g, Tween 80 0.1 g, sodium ascorbate 0.5 g, calcium carbonate 0.5 g, water 1000 mL; The washing solution is composed of the following components by weight percentage: Tween 80 0.3%, peptone 0.5%, and the balance is water; The lyophilization protectant is composed of the following components by weight percentage: 10% trehalose, 8% skim milk powder, 4% lactose, 1% glycerol, 1% inulin, 0.5% sodium ascorbate, 3% sodium alginate, 1% yeast peptone, the balance being water; The skim milk powder is the skim milk powder treated with protease for 3 h before use; A method for preparing a short Bifidobacterium powder with a high viable bacteria count, comprising the following steps: S1. Inoculate the Bifidobacterium breve strain into 10 mL of the first seed culture medium at an inoculation amount of 8%, and statically culture at 35 °C for 24 h to obtain the first-stage seeds. Inoculate the first-stage seeds into 200 mL of the second seed culture medium at an inoculation amount of 4%, and statically culture at 35 °C for 20 h to obtain the second-stage seeds, which are the Bifidobacterium breve seed liquid. Inoculate the Bifidobacterium breve seed liquid into a fermenter filled with the sterilized high-density fermentation medium at an inoculation amount of 5%, and anaerobically ferment at a temperature of 35 °C, a rotation speed of 60 rpm, and an initial pH of 6.2 for 10 h. During the anaerobic fermentation, adjust the pH to 5.5 with a 20% sodium carbonate solution by mass fraction, maintain the tank pressure at 0.05 MPa, adjust the temperature to 30 °C after anaerobic fermentation, and continue to culture for 4 h to obtain the fermentation broth; S2. Centrifuge the above fermentation broth to obtain the first bacterial sludge, mix and resuspend the first bacterial sludge with the washing liquid at a mass ratio of 1:7, and centrifuge again to obtain the second bacterial sludge; S3. Add a freeze-drying protectant to the second bacterial sludge (the mass ratio of the second bacterial sludge to the freeze-drying protectant is 1:0.5). Among them, the freeze-drying protectant is added in batches. First, add 30% of the mass of the freeze-drying protectant, mix evenly, and then add the remaining mass of the freeze-drying protectant and mix evenly to obtain an emulsion; S4. Drop the emulsion into liquid nitrogen at a temperature of -195 °C through a nozzle. After pre-freezing, carry out vacuum freeze-drying under the conditions of a primary sublimation temperature of 8 °C, a first vacuum degree of 0.013 mbar, an analytical drying temperature of 30 °C, and a second vacuum degree of 0.004 mbar. After freeze-drying until the water content of the emulsion is 2.6%, backfill with nitrogen and pulverize to obtain the Bifidobacterium breve powder.

[0030] Example 2 The first seed culture medium is composed of the following components by weight percentage: 20 g of lactose, 15 g of yeast nutrient, 10 g of soy peptone, 2 g of sodium acetate, 0.3 g of citric acid, 0.2 g of diammonium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.1 g of Tween 80, 0.5 g of sodium ascorbate, 0.3 g of calcium carbonate, 1000 mL of water; The second seed culture medium is composed of the following components by weight percentage: 20 g of lactose, 15 g of yeast nutrient, 10 g of soy peptone, 2 g of sodium acetate, 0.3 g of citric acid, 0.2 g of diammonium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.1 g of Tween 80, 0.5 g of sodium ascorbate, 0.3 g of calcium carbonate, 1000 mL of water; The high-density fermentation medium comprises the following components in parts by weight: 40 g of lactose, 20 g of yeast nutrient, 15 g of soy peptone, 5 g of sodium acetate, 1.15 g of citric acid, 1 g of diammonium hydrogen phosphate, 0.35 g of magnesium sulfate, 0.3 g of Tween 80, 1.1 g of sodium ascorbate, 0.95 g of calcium carbonate, 1000 mL of water; The washing solution consists of the following components by weight percentage: 1.4% of Tween 80, 1% of peptone, and the balance is water; The lyophilization protectant consists of the following components by weight percentage: 11% of trehalose, 10% of skim milk powder, 5% of lactose, 2% of glycerol, 2% of inulin, 1.2% of sodium ascorbate, 4% of sodium alginate, 0.8% of yeast peptone, and the balance is water; The skim milk powder is the skim milk powder treated with protease for 4 h before use; A preparation method of a short Bifidobacterium infantis bacterial powder with a high viable bacteria count comprises the following steps: S1. Inoculate the short Bifidobacterium infantis strain into 10 mL of a first seed culture medium at an inoculation amount of 10%, and statically culture at 37 °C for 22 h to obtain a first-stage seed. Inoculate the first-stage seed into 200 mL of a second seed culture medium at an inoculation amount of 5%, and statically culture at 37 °C for 18 h to obtain a second-stage seed, which is the short Bifidobacterium infantis seed liquid. Inoculate the short Bifidobacterium infantis seed liquid into a fermentation tank filled with the sterilized high-density fermentation medium at an inoculation amount of 5%, and anaerobically ferment at a temperature of 37 °C, a rotation speed of 100 rpm, and an initial pH of 6.3 for 13 h. During the anaerobic fermentation, adjust the pH to 6.0 with a 23% sodium carbonate solution by mass fraction, maintain the tank pressure at 0.05 MPa, adjust the temperature to 34 °C after anaerobic fermentation, and continue to culture for 3 h to obtain a fermentation broth; S2. Centrifuge the above fermentation broth to obtain a first bacterial sludge, mix and resuspend the first bacterial sludge with the washing solution at a mass ratio of 1:10, and centrifuge again to obtain a second bacterial sludge; S3. Add the lyophilization protectant to the second bacterial sludge (the mass ratio of the second bacterial sludge to the lyophilization protectant is 1:1.3). Among them, the lyophilization protectant is added in batches. Add 30% of the mass of the lyophilization protectant for the first time, mix evenly, then add the remaining mass of the lyophilization protectant, mix evenly to obtain an emulsion; S4. Drop the emulsion into liquid nitrogen at -196°C through a nozzle. After pre-freezing, conduct vacuum freeze-drying under the conditions of a primary sublimation temperature of 10°C, a first vacuum degree of 0.015 mbar, an analytical drying temperature of 32°C, and a second vacuum degree of 0.005 mbar. After freeze-drying until the water content of the emulsion is 3%, backfill with nitrogen and pulverize to obtain the Bifidobacterium breve powder.

[0031] Example 3 The first seed culture medium consists of the following components by weight percentage: 20 g of lactose, 15 g of yeast nutrient, 10 g of soy peptone, 2 g of sodium acetate, 0.3 g of citric acid, 0.2 g of diammonium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.1 g of Tween 80, 0.5 g of sodium ascorbate, 0.3 g of calcium carbonate, 1000 mL of water; The second seed culture medium consists of the following components by weight percentage: 20 g of lactose, 15 g of yeast nutrient, 10 g of soy peptone, 2 g of sodium acetate, 0.3 g of citric acid, 0.2 g of diammonium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.1 g of Tween 80, 0.5 g of sodium ascorbate, 0.3 g of calcium carbonate, 1000 mL of water; The high-density fermentation medium includes the following components by weight: 50 g of lactose, 30 g of yeast nutrient, 20 g of soy peptone, 8 g of sodium acetate, 2 g of citric acid, 2 g of diammonium hydrogen phosphate, 0.5 g of magnesium sulfate, 0.5 g of Tween 80, 2 g of sodium ascorbate, 2 g of calcium carbonate, 1000 mL of water; The washing solution consists of the following components by weight percentage: 2.5% of Tween 80, 1.5% of peptone, and the balance is water; The lyoprotectant consists of the following components by weight percentage: 12% of trehalose, 12% of skim milk powder, 6% of lactose, 3% of glycerol, 3% of inulin, 2% of sodium ascorbate, 5% of sodium alginate, 1.5% of yeast peptone, and the balance is water; The skim milk powder is the skim milk powder treated with protease for 5 h before use; A method for preparing a Bifidobacterium breve powder with a high viable count includes the following steps: S1. Inoculate the Bifidobacterium breve strain into 10 mL of the first seed medium at an inoculation amount of 12%, and statically culture it at 37 °C for 20 h to obtain the first-stage seeds. Then inoculate the first-stage seeds into 200 mL of the second seed medium at an inoculation amount of 6%, and statically culture it at 37 °C for 16 h to obtain the second-stage seeds, which are the Bifidobacterium breve seed liquid. Inoculate the Bifidobacterium breve seed liquid into a fermenter filled with sterilized high-density fermentation medium at an inoculation amount of 5%, and carry out anaerobic fermentation at a temperature of 40 °C, a rotation speed of 150 rpm, and an initial pH of 6.5 for 15 h. During the anaerobic fermentation, adjust the pH to 6.0 with a 30% sodium carbonate solution by mass, maintain the tank pressure at 0.05 MPa, adjust the temperature to 35 °C after anaerobic fermentation, and continue to culture for 2 h to obtain the fermentation broth; S2. Centrifuge the above fermentation broth to obtain the first bacterial sludge, mix and resuspend the first bacterial sludge with the washing liquid at a mass ratio of 1:12, and centrifuge again to obtain the second bacterial sludge; S3. Add a freeze-drying protectant to the second bacterial sludge (the mass ratio of the second bacterial sludge to the freeze-drying protectant is 1:2). Among them, the freeze-drying protectant is added in batches. First, add 30% of the mass of the freeze-drying protectant, mix evenly, and then add the remaining mass of the freeze-drying protectant and mix evenly to obtain an emulsion; S4. Drop the emulsion into liquid nitrogen at a temperature of -198 °C through a nozzle. After pre-freezing, carry out vacuum freeze-drying under the conditions of a primary sublimation temperature of 12 °C, a first vacuum degree of 0.016 mbar, an analytical drying temperature of 33 °C, and a second vacuum degree of 0.006 mbar. After freeze-drying until the water content of the emulsion is 3.3%, backfill with nitrogen and pulverize to obtain the Bifidobacterium breve powder.

[0032] Example 4 The difference between this example and Example 2 is only that in this example, 1.5 g of citric acid and 0.6 g of calcium carbonate are added to the high-density fermentation medium.

[0033] Example 5 The difference between this example and Example 2 is only that in this example, 1.26 g of citric acid and 0.84 g of calcium carbonate are added to the high-density fermentation medium.

[0034] Example 6 The difference between this example and Example 2 is only that in this example, 1.4 g of citric acid and 0.7 g of calcium carbonate are added to the high-density fermentation medium.

[0035] Example 7 The difference between this embodiment and Embodiment 6 is only that in this embodiment, step S3 is as follows: adding a lyoprotectant to the second bacterial sludge (the mass ratio of the second bacterial sludge to the lyoprotectant is 1:1.3), wherein the lyoprotectant is added in batches. 80% of the mass of the lyoprotectant is added for the first time. After mixing evenly, the remaining mass of the lyoprotectant is added and mixed evenly to obtain an emulsion.

[0036] Embodiment 8 The difference between this embodiment and Embodiment 7 is only that in this embodiment, 45% of the mass of the lyoprotectant is added for the first time. After mixing evenly, the remaining mass of the lyoprotectant is added.

[0037] Embodiment 9 The difference between this embodiment and Embodiment 7 is only that in this embodiment, 70% of the mass of the lyoprotectant is added for the first time. After mixing evenly, the remaining mass of the lyoprotectant is added.

[0038] Embodiment 10 The difference between this embodiment and Embodiment 7 is only that in this embodiment, the lyoprotectant is not added in batches.

[0039] Embodiment 11 The difference between this embodiment and Embodiment 2 is only that in this embodiment, the sodium carbonate solution with a mass fraction of 23% is replaced by a sodium hydroxide solution with a mass fraction of 23%.

[0040] Comparative Example 1 The difference between this comparative example and Embodiment 2 is only that in this comparative example, citric acid is replaced by an equal amount of dipotassium hydrogen phosphate.

[0041] Comparative Example 2 The difference between this comparative example and Embodiment 2 is only that in this comparative example, citric acid is replaced by an equal amount of ammonium hydrogen citrate.

[0042] Comparative Example 3 The difference between this comparative example and Embodiment 2 is only that in this comparative example, calcium carbonate is replaced by an equal amount of sodium carbonate.

[0043] Comparative Example 4 The difference between this comparative example and Embodiment 2 is only that in this comparative example, citric acid is not added.

[0044] Comparative Example 5 The difference between this comparative example and Embodiment 2 is only that in this comparative example, calcium carbonate is not added.

[0045] Comparative Example 6 The difference between this comparative example and Embodiment 2 is only that in this comparative example, neither citric acid nor calcium carbonate is added.

[0046] Comparative Example 7 The difference between this comparative example and Example 2 is only that in this comparative example, in step S4, the emulsion is pre-frozen by a common pre-freezing method without liquid nitrogen freezing.

[0047] Comparative Example 8 The difference between this comparative example and Example 2 is only that in this comparative example: Step S2 is: centrifuging the above fermentation broth to obtain the first bacterial sludge; Step S3 is: adding a freeze-drying protectant to the first bacterial sludge (the mass ratio of the first bacterial sludge to the freeze-drying protectant is 1:1.3), wherein the freeze-drying protectant is added in batches. 30% of the mass of the freeze-drying protectant is added for the first time. After mixing evenly, the remaining mass of the freeze-drying protectant is added and mixed evenly to obtain an emulsion; Step S4 is: dropping the emulsion into liquid nitrogen at a temperature of -196°C through a nozzle. After pre-freezing, vacuum freeze-drying is carried out under the conditions of a primary sublimation temperature of 10°C, a first vacuum degree of 0.015 mbar, an analytical drying temperature of 32°C, and a second vacuum degree of 0.005 mbar. After freeze-drying until the water content of the emulsion is 3%, nitrogen backfilling is carried out and pulverized to obtain the Bifidobacterium breve powder.

[0048] Comparative Example 9 The difference between this comparative example and Example 2 is only that in this comparative example, in step S3, no freeze-drying protectant is added, and the freeze-drying protectant is replaced with an equal mass of water.

[0049] Comparative Example 10 The difference between this comparative example and Example 2 is only that in this comparative example, citric acid is replaced with an equal amount of potassium citrate.

[0050] During the preparation of the Bifidobacterium breve powder in Examples 1 to 11 and Comparative Examples 1 to 10, the viable bacteria counts in the fermentation broth, emulsion, and powder in Examples 1 to 11 and Comparative Examples 1 to 10 were measured, and the freeze-drying survival rate was calculated according to the following formula: freeze-drying survival rate = (the value of the viable bacteria count after freeze-drying / the total number of bacteria) / (the viable bacteria count before freeze-drying / the total number of bacteria) × 100%. The test results are shown in Table 1: Table 1 Test results of viable bacteria counts and freeze-drying survival rates in Examples 1 to 11 and Comparative Examples 1 to 10

[0051] As can be seen from the table, compared with Comparative Examples 1-6 and Comparative Example 10, the viable count of the fermentation broth in Example 2 is significantly increased, indicating that when citric acid and calcium carbonate are contained in the high-density fermentation medium and used in combination, the viable count of the fermentation broth of Bifidobacterium breve can be significantly increased. Among them, the addition of citric acid will change the metabolic pathway of the bacteria, resulting in more carbon dioxide production in the changed pathway, which plays a role in reducing dissolved oxygen. Calcium carbonate may play two roles. On the one hand, it is used as an internal neutralizer to play a buffering role when the bacteria rapidly produce acid. On the other hand, the free Ca formed after the reaction of calcium carbonate with acid 2+ will promote the compartmentalization of the cell wall during bacterial division, making the bacteria shorter, which is beneficial to the high-density culture of the bacteria. By using citric acid and calcium carbonate in combination, the viable count of the fermentation broth of Bifidobacterium breve can be significantly increased. Among them, compared with Examples 2 and 4, the viable count of the fermentation broth in Examples 5-6 is increased, indicating that when the weight ratio of citric acid to calcium carbonate is 3-4:2, the viable count of the fermentation broth of Bifidobacterium breve can be further increased.

[0052] In addition, compared with Examples 6-7 and Example 10, the viable count of the emulsion and the viable count of the bacterial powder in Examples 8-9 are increased, indicating that when the lyoprotectant is added in batches and 45%-70% of the mass of the lyoprotectant is added for the first time and the remaining mass of the lyoprotectant is added for the second time, the viable count of the emulsion and the viable count of the bacterial powder of Bifidobacterium breve can be increased.

[0053] Compared with Example 11, the viable count of the fermentation broth in Example 2 is increased, indicating that when the pH is adjusted with a sodium carbonate solution, carbon dioxide will be produced after the sodium carbonate neutralizes the acid produced by the bacteria, which will play a role in reducing dissolved oxygen, thereby increasing the viable count of the fermentation broth of Bifidobacterium breve.

[0054] Compared with Comparative Example 7, the freeze-drying survival rate of Example 2 is significantly increased, indicating that rapid freezing in liquid nitrogen will increase the freeze-drying survival rate. Compared with Comparative Example 8, the freeze-drying survival rate of Example 2 is increased, indicating that in step S2, the washing of the bacterial sludge with the washing solution will increase the freeze-drying survival rate.

[0055] Compared with Comparative Example 9, the freeze-drying survival rate of Example 2 is significantly increased, indicating that the addition of the lyoprotectant can increase the freeze-drying survival rate.

[0056] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a high viable count Bifidobacterium breve powder, characterized in that: The following steps are involved: S1, inoculating a Bifidobacterium breve strain into a seed culture medium for culturing to obtain a Bifidobacterium breve seed solution, inoculating the Bifidobacterium breve seed solution into a high-density fermentation culture medium for fermentation culture to obtain a fermentation solution; S2, centrifuging the fermentation liquid to obtain a first bacterial sludge, mixing the first bacterial sludge with a washing solution, resuspending the mixture, and centrifuging the mixture again to obtain a second bacterial sludge; S3, adding a lyophilization protective agent to the second bacterial sludge, mixing, and obtaining an emulsion; S4, dropping the emulsion into liquid nitrogen, pre-freezing, and vacuum freeze-drying to obtain Bifidobacterium breve powder; The high-density fermentation medium comprises the following components: Lactose 25~50g, yeast nutrient 15~30g, soy peptone 10~20g, sodium acetate 2~8g, citric acid 0.5~2g, diammonium phosphate 0.5~2g, magnesium sulfate 0.2~0.5g, emulsifier 0.1~0.5g, sodium ascorbate 0.5~2g, calcium carbonate 0.5~2g, water 1000mL.

2. The method for preparing a high viable count Bifidobacterium breve powder according to claim 1, characterized in that: The weight ratio of the citric acid to calcium carbonate is 3-4:

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3. The method for preparing a high viable count Bifidobacterium breve powder according to claim 1, characterized in that: In step S1, the specific steps of the fermentation culture are: anaerobic fermentation for 10 to 15 hours at a temperature of 35 to 40°C, a rotation speed of 60 to 150 rpm, and an initial pH of 6.2 to 6.5, during the anaerobic fermentation, an alkaline solution is used to adjust the pH to 5.5 to 6.0, and the tank pressure is maintained at 0.05 MPa; The alkaline solution is one of a sodium carbonate solution and an ammonia solution; The mass fraction of the alkaline solution is 20% to 30%.

4. The method for preparing a high viable count Bifidobacterium breve powder according to claim 1, characterized in that: The washing liquid is composed of the following components in percentage by weight: Emulsifier 0.3%~2.5%, peptone 0.5%~1.5%, the balance is water; The mass ratio of the first bacterial sludge to the washing liquid is 1:7-12.

5. The method for preparing a high viable count Bifidobacterium breve powder according to claim 1, characterized in that: The freeze-drying protective agent is composed of the following components in weight percentage: Trehalose 10%~12%, skim milk powder 8%~12%, lactose 4%~6%, glycerol 1%~3%, inulin 1%~3%, sodium ascorbate 0.5%~2%, sodium alginate 3%~5%, yeast peptone 0.5%~2%, and the balance is water; The mass ratio of the second bacterial sludge to the freeze-drying protective agent is 1:0.5~2.

6. The method for preparing a high viable count Bifidobacterium breve powder according to claim 1, characterized in that: The adding of the freeze-dried protective agent is divided into a first adding and a second adding, wherein the first adding is 45% to 70% of the mass of the freeze-dried protective agent, and the second adding is the remaining mass of the freeze-dried protective agent.

7. The method for preparing a high viable count Bifidobacterium breve powder according to claim 5, characterized in that: The skimmed milk powder is skimmed milk powder that has been treated with protease for 3 to 5 hours before use.

8. The method for preparing a high viable count Bifidobacterium breve powder according to claim 1, characterized in that: The specific preparation method of the Bifidobacterium breve seed liquid is as follows: the Bifidobacterium breve strain is inoculated into 10 mL of a first seed culture medium at an inoculation rate of 8% to 12%, and a first static culture is performed to obtain primary seeds; the primary seeds are inoculated into 200 mL of a second seed culture medium at an inoculation rate of 4% to 6%, and a second static culture is performed to obtain secondary seeds, namely, the Bifidobacterium breve seed liquid.

9. The method for preparing a high viable count Bifidobacterium breve powder according to claim 8, characterized in that: The temperature of the first static culture and the second static culture are independently 35-37° C.; The first static culture time is 20 to 24 hours; The second static culture time is 16 to 20 hours.

10. The method for preparing a high viable count Bifidobacterium breve powder according to claim 1, characterized in that: During the vacuum freeze drying, the primary sublimation temperature is 8-12° C., the first vacuum degree is 0.013-0.016 mbar, the analytical drying temperature is 30-33° C., the second vacuum degree is 0.004-0.006 mbar, and the freeze drying is performed until the moisture content is 2.6%-3.3%.

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