Bifidobacterium preparation capable of being preserved at normal temperature and preparation method of bifidobacterium preparation

Abstract

The invention discloses a bifidobacterium preparation capable of being preserved at normal temperature and a preparation method thereof, and the preparation method comprises the following steps: firstly, culturing bifidobacterium to obtain bifidobacterium liquid; wherein the bifidobacterium is cultured in a liquid culture medium; carrying out centrifugal treatment on the bifidobacterium liquid, and collecting bifidobacterium sludge; mixing the obtained bifidobacterium sludge with a composite freeze-drying protective additive, and then performing freeze-drying treatment to obtain freeze-dried powder; carrying out top spray granulation on the obtained freeze-dried powder by using maltodextrin as an adhesive through a fluidized bed coating machine to obtain granules; and respectively loading the obtained granules into catechol-gelatin composite capsule shells to obtain the catechol-gelatin composite capsule. According to the method, in the process of preparing the bifidobacterium preparation, the bifidobacterium is subjected to freeze-drying treatment, in order to reduce the cell activity loss of the bifidobacterium to the maximum extent, the specially-made composite freeze-drying protective agent is adopted, the excellent anti-freezing performance is achieved, and the integrity and functions of the bifidobacterium cells are kept at the low temperature.

Description

Technical Field

[0001] The invention belongs to the technical field of bifidobacterium cultivation, in particular to a bifidobacterium preparation which can be stored at room temperature and a preparation method thereof. Background Art

[0002] Bifidobacterium is an important probiotic belonging to the kingdom of bacteria, phylum Actinomycetes, class Actinomycetes, orders Bifidobacteriales, family Bifidobacteriaceae, and genus Bifidobacterium.

[0003] Bifidobacterium is an obligate anaerobe and is extremely sensitive to oxygen. Their optimal growth temperature is usually between 37°C and 41°C, but they can also grow within a certain range (such as 25°C to 28°C or 43°C to 45°C). Bifidobacterium's glucose metabolism belongs to heterofermentation. They do not have aldolase and glucose-6-phosphate dehydrogenase in their bodies, but have 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] There are some problems with storing bifidobacteria at room temperature: as a probiotic, the activity of bifidobacteria is sensitive to temperature. Usually at room temperature, the activity of bifidobacteria may gradually weaken, resulting in reduced drug efficacy. This is because high temperature accelerates the metabolism of microorganisms, thereby shortening their survival time and affecting their efficacy, causing their storage period to be significantly shortened.

[0005] Therefore, the present invention solves the corresponding problems by providing a method for preparing a bifidobacterium preparation that can be stored at room temperature. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a bifidobacterium preparation that can be stored at room temperature, so as to solve the deficiencies in the prior art.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A method for preparing a bifidobacterium preparation that can be stored at room temperature comprises: firstly, culturing bifidobacteria to obtain a bifidobacterium bacterial liquid;

[0009] Wherein, the bifidobacterium is cultured in a liquid culture medium, wherein the liquid culture medium is:

[0010] 20.0 g glucose, 10.0 g tryptone, 6.0 g beef extract, 5 g sodium acetate, 5.0 g yeast extract powder, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 2.0 g potassium hydrogen phosphate, 0.2 g cysteine ​​hydrochloride, 0.58 g magnesium sulfate, 1.0 mL Tween-80, 1000 mL distilled water; adjust pH to 6.5, sterilize at 121°C for 25 min to obtain a liquid culture medium;

[0011] The culture conditions were as follows: culture in a shaker at 37°C and 220 rpm for 24 h, transfer to a 40°C incubator and continue culture for 24 h;

[0012] The bifidobacterium bacterial liquid is centrifuged to collect the bifidobacterium bacterial mud;

[0013] The obtained bifidobacterium sludge is mixed with a composite freeze-drying protective agent, and then freeze-dried to obtain freeze-dried powder;

[0014] The obtained freeze-dried powder is granulated by top spraying in a fluidized bed coater with maltodextrin as a binder to obtain granules;

[0015] The granules obtained above are respectively filled into catechol-gelatin composite capsule shells to obtain a bifidobacterium preparation that can be stored at room temperature.

[0016] As a further technical solution: the bifidobacterium is Bifidobacterium longum;

[0017] The number of viable bacteria in the bifidobacterium liquid is greater than 5.5×10 8 Pieces / mL.

[0018] As a further technical solution: the centrifugal treatment speed is 5500r / min;

[0019] The centrifugation time was 10 min.

[0020] As a further technical solution: the mass ratio of the bifidobacterium sludge to the freeze-drying protective agent is 6:1-1.5.

[0021] As a further technical solution: the preparation method of the composite freeze-drying protective agent is:

[0022] First, weigh 20-25g of hydrogenated lecithin and 2-3g of mannitol;

[0023] Add hydrogenated lecithin to chloroform, stir and mix until dissolved, to obtain a hydrogenated lecithin solution;

[0024] Adding mannitol to methanol, stirring and mixing until dissolved, to obtain a mannitol solution;

[0025] The hydrogenated lecithin solution and the mannitol solution were mixed, and chloroform and methanol were removed by concentrating using a rotary evaporator at 44°C, and then 1-1.5 g of alginate and 3-4 g of bovine serum albumin were added, and the volume was adjusted to 100 mL with water for injection to obtain a mixed solution;

[0026] The obtained mixed solution was subjected to one ultrasonic treatment by a cell disruptor for 10 min.

[0027] As a further technical solution: the freeze-drying process is:

[0028] First, pre-freeze for 20 min at -60 °C, then lower to -45 °C, keep warm for 30 min, and then evacuate to 0.18 mbar for 2 h.

[0029] The analytical drying vacuum was reduced to 0.1 mbar, the temperature was raised to 23°C and maintained for 2 hours to complete the freeze-drying process.

[0030] As a further technical solution: the mass ratio of the freeze-dried powder to maltodextrin is 1:5-6.

[0031] As a further technical solution: the parameters of the fluidized bed coating machine top spray granulation are an inlet air temperature of 60°C and an air flow rate of 35m 3 / h, atomization pressure is 1.8bar, material temperature is 55℃, and spray rate is 8.5mL / min.

[0032] As a further technical solution: the preparation method of the catechol-gelatin composite capsule shell is:

[0033] First, gelatin is added into deionized water and fully dissolved to obtain a gelatin solution;

[0034] Adding catechol to deionized water, stirring and mixing evenly to obtain a catechol solution;

[0035] The gelatin solution and the catechol solution are mixed evenly, and then mixed with glycerol, the temperature is adjusted to 65°C, and the mixture is kept warm and homogenized for 10 minutes to obtain a composite glue solution;

[0036] After the capsule mold is immersed in the composite glue solution, it is taken out at a uniform speed and turned evenly, and dried at 45°C for 2.5 hours, and the formed capsules are peeled and cut one by one from the mold to finally obtain the catechol-gelatin composite capsule shell;

[0037] The capacity of the catechol-gelatin composite capsule shell is 0.25 g.

[0038] A method for preparing a bifidobacterium preparation that can be stored at room temperature is disclosed. The method comprises the steps of preparing a bifidobacterium preparation that can be stored at room temperature.

[0039] As a further technical solution: the bifidobacterium preparation that can be stored at room temperature contains more than 3.2×10 11 Pieces / g.

[0040] Beneficial effects:

[0041] In the process of preparing the bifidobacterium preparation, the method of the present invention performs freeze-drying treatment on the bifidobacterium, in order to minimize the loss of bifidobacterium cell activity, this is mainly through the use of the special composite freeze-drying protective agent of the present invention, which has excellent antifreeze performance, can maintain the integrity and function of bifidobacterium cells at low temperatures, can produce a strong protective effect on bifidobacterium cells during freeze-drying, and can avoid the damage of bifidobacterium cells caused by the formation of ice crystals during freeze-drying. The composite freeze-drying protective agent prepared by the present invention can form a thin protective film on the surface of bifidobacterium cells, can greatly reduce the water evaporation rate on the surface of bifidobacterium during freeze-drying to remove water, thereby enhancing its drying resistance, protecting bifidobacterium, reducing damage to bifidobacterium, and improving the activity of bifidobacterium, thereby effectively improving the survival rate of bifidobacterium.

[0042] In addition, the composite lyophilization protective agent prepared by the present invention can reduce the metabolism and energy loss of bifidobacteria, and can help prevent apoptosis and inactivation of bifidobacteria during storage.

[0043] The high quality and high activity of bifidobacterium freeze-dried powder during storage and transportation can be improved by using the combined freeze-drying protective agent of the present invention and the freeze-drying method.

[0044] The present invention can isolate the bifidobacterium from the external environment by using the catechol-gelatin composite capsule shell, mainly effectively isolating factors such as air, moisture and light that may have an adverse effect on the bifidobacterium, thereby maintaining its activity and stability. The catechol-gelatin composite capsule shell can also prevent the possibility of mechanical damage to the bifidobacterium cells, mainly preventing the bifidobacterium from being damaged by mechanical impact or friction. The catechol-gelatin composite capsule shell can provide a certain buffer and protection, reducing the damage caused to the bifidobacterium by these external factors.

[0045] The catechol-gelatin composite capsule shell used in the present invention also has certain anti-oxidation performance, can remove free radicals and inhibit the occurrence of oxidation reactions, which plays an important role in protecting bifidobacteria from damage by oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The figure is a statistical chart of the survival rate of Bifidobacterium species in each group. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] The following are specific embodiments

[0049] Example 1

[0050] A method for preparing a bifidobacterium preparation that can be stored at room temperature comprises: first, culturing bifidobacteria to obtain a bifidobacterium bacterial liquid; the number of viable bacteria in the bifidobacterium bacterial liquid is greater than 5.5×10 8 Pieces / mL.

[0051] Wherein, the culturing of Bifidobacterium longum is culturing in a liquid culture medium, wherein the liquid culture medium is:

[0052] 20.0 g glucose, 10.0 g tryptone, 6.0 g beef extract, 5 g sodium acetate, 5.0 g yeast extract powder, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 2.0 g potassium hydrogen phosphate, 0.2 g cysteine ​​hydrochloride, 0.58 g magnesium sulfate, 1.0 mL Tween-80, 1000 mL distilled water; adjust pH to 6.5, sterilize at 121°C for 25 min to obtain a liquid culture medium;

[0053] The culture conditions were as follows: culture in a shaker at 37°C and 220 rpm for 24 h, transfer to a 40°C incubator and continue culture for 24 h;

[0054] The bifidobacterium bacterial liquid was centrifuged to collect the bifidobacterium bacterial sludge; the centrifugal speed was 5500r / min;

[0055] The centrifugation time was 10 min.

[0056] The obtained bifidobacterium sludge is mixed with a composite freeze-drying protective agent, and then freeze-dried to obtain freeze-dried powder; the mass ratio of the bifidobacterium sludge to the freeze-drying protective agent is 6:1.

[0057] The obtained freeze-dried powder is granulated by top spraying in a fluidized bed coater with maltodextrin as a binder to obtain granules;

[0058] The granules obtained above are respectively filled into catechol-gelatin composite capsule shells to obtain a bifidobacterium preparation that can be stored at room temperature.

[0059] The preparation method of the composite freeze-drying protective agent is as follows:

[0060] First, weigh 20g of hydrogenated lecithin and 2g of mannitol;

[0061] Add hydrogenated lecithin to chloroform, stir and mix until dissolved, to obtain a hydrogenated lecithin solution;

[0062] Adding mannitol to methanol, stirring and mixing until dissolved, to obtain a mannitol solution;

[0063] The hydrogenated lecithin solution and the mannitol solution were mixed, and chloroform and methanol were removed by concentrating using a rotary evaporator at 44°C, and then 1 g of alginate and 3 g of bovine serum albumin were added, and the volume was adjusted to 100 mL using water for injection to obtain a mixed solution;

[0064] The obtained mixed solution was subjected to one ultrasonic treatment by a cell disruptor for 10 min.

[0065] Freeze-dried:

[0066] First, pre-freeze for 20 min at -60 °C, then lower to -45 °C, keep warm for 30 min, and then evacuate to 0.18 mbar for 2 h.

[0067] The analytical drying vacuum was reduced to 0.1 mbar, the temperature was raised to 23°C and maintained for 2 hours to complete the freeze-drying process.

[0068] The mass ratio of freeze-dried powder to maltodextrin is 1:5.

[0069] The parameters of the top spray granulation of the fluidized bed coating machine are as follows: the inlet air temperature is 60°C, the air flow rate is 35m 3 / h, atomization pressure is 1.8bar, material temperature is 55℃, and spray rate is 8.5mL / min.

[0070] The preparation method of catechol-gelatin composite capsule shell is as follows:

[0071] First, gelatin is added into deionized water and fully dissolved to obtain a gelatin solution;

[0072] Adding catechol to deionized water, stirring and mixing evenly to obtain a catechol solution;

[0073] The gelatin solution and the catechol solution are mixed evenly, and then mixed with glycerol, the temperature is adjusted to 65°C, and the mixture is kept warm and homogenized for 10 minutes to obtain a composite glue solution;

[0074] After the capsule mold is immersed in the composite glue solution, it is taken out at a uniform speed and turned evenly, and dried at 45°C for 2.5 hours, and the formed capsules are peeled and cut one by one from the mold to finally obtain the catechol-gelatin composite capsule shell;

[0075] The capacity of the catechol-gelatin composite capsule shell is 0.25 g.

[0076] Example 2

[0077] A method for preparing a bifidobacterium preparation that can be stored at room temperature comprises: first, culturing bifidobacteria to obtain a bifidobacterium bacterial liquid; the number of viable bacteria in the bifidobacterium bacterial liquid is greater than 5.5×10 8 Pieces / mL.

[0078] Wherein, the culturing of Bifidobacterium longum is culturing in a liquid culture medium, wherein the liquid culture medium is:

[0079] 20.0 g glucose, 10.0 g tryptone, 6.0 g beef extract, 5 g sodium acetate, 5.0 g yeast extract powder, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 2.0 g potassium hydrogen phosphate, 0.2 g cysteine ​​hydrochloride, 0.58 g magnesium sulfate, 1.0 mL Tween-80, 1000 mL distilled water; adjust pH to 6.5, sterilize at 121°C for 25 min to obtain a liquid culture medium;

[0080] The culture conditions were as follows: culture in a shaker at 37°C and 220 rpm for 24 h, transfer to a 40°C incubator and continue culture for 24 h;

[0081] The bifidobacterium bacterial liquid was centrifuged to collect the bifidobacterium bacterial sludge; the centrifugal speed was 5500r / min;

[0082] The centrifugation time was 10 min.

[0083] The obtained bifidobacterium sludge is mixed with a composite freeze-drying protective agent, and then freeze-dried to obtain freeze-dried powder; the mass ratio of the bifidobacterium sludge to the freeze-drying protective agent is 6:1.2.

[0084] The obtained freeze-dried powder is granulated by top spraying in a fluidized bed coater with maltodextrin as a binder to obtain granules;

[0085] The granules obtained above are respectively filled into catechol-gelatin composite capsule shells to obtain a bifidobacterium preparation that can be stored at room temperature.

[0086] The preparation method of the composite freeze-drying protective agent is as follows:

[0087] First, weigh 21 g of hydrogenated lecithin and 2.5 g of mannitol;

[0088] Add hydrogenated lecithin to chloroform, stir and mix until dissolved, to obtain a hydrogenated lecithin solution;

[0089] Adding mannitol to methanol, stirring and mixing until dissolved, to obtain a mannitol solution;

[0090] The hydrogenated lecithin solution and the mannitol solution were mixed, and chloroform and methanol were removed by concentrating using a rotary evaporator at 44°C, and then 1.2 g of alginate and 3.1 g of bovine serum albumin were added, and the volume was adjusted to 100 mL using water for injection to obtain a mixed solution;

[0091] The obtained mixed solution was subjected to one ultrasonic treatment by a cell disruptor for 10 min.

[0092] Freeze-dried:

[0093] First, pre-freeze for 20 min at -60 °C, then lower to -45 °C, keep warm for 30 min, and then evacuate to 0.18 mbar for 2 h.

[0094] The analytical drying vacuum was reduced to 0.1 mbar, the temperature was raised to 23°C and maintained for 2 hours to complete the freeze-drying process.

[0095] The mass ratio of freeze-dried powder to maltodextrin is 1:5.3.

[0096] The parameters of the top spray granulation of the fluidized bed coating machine are as follows: the inlet air temperature is 60°C, the air flow rate is 35m 3 / h, atomization pressure is 1.8bar, material temperature is 55℃, and spray rate is 8.5mL / min.

[0097] The preparation method of catechol-gelatin composite capsule shell is as follows:

[0098] First, gelatin is added into deionized water and fully dissolved to obtain a gelatin solution;

[0099] Adding catechol to deionized water, stirring and mixing evenly to obtain a catechol solution;

[0100] The gelatin solution and the catechol solution are mixed evenly, and then mixed with glycerol, the temperature is adjusted to 65°C, and the mixture is kept warm and homogenized for 10 minutes to obtain a composite glue solution;

[0101] After the capsule mold is immersed in the composite glue solution, it is taken out at a uniform speed and turned evenly, and dried at 45°C for 2.5 hours, and the formed capsules are peeled and cut one by one from the mold to finally obtain the catechol-gelatin composite capsule shell;

[0102] The capacity of the catechol-gelatin composite capsule shell is 0.25 g.

[0103] Example 3

[0104] A method for preparing a bifidobacterium preparation that can be stored at room temperature comprises: first, culturing bifidobacteria to obtain a bifidobacterium bacterial liquid; the number of viable bacteria in the bifidobacterium bacterial liquid is greater than 5.5×10 8Pieces / mL.

[0105] Wherein, the culturing of Bifidobacterium longum is culturing in a liquid culture medium, wherein the liquid culture medium is:

[0106] 20.0 g glucose, 10.0 g tryptone, 6.0 g beef extract, 5 g sodium acetate, 5.0 g yeast extract powder, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 2.0 g potassium hydrogen phosphate, 0.2 g cysteine ​​hydrochloride, 0.58 g magnesium sulfate, 1.0 mL Tween-80, 1000 mL distilled water; adjust pH to 6.5, sterilize at 121°C for 25 min to obtain a liquid culture medium;

[0107] The culture conditions were as follows: culture in a shaker at 37°C and 220 rpm for 24 h, transfer to a 40°C incubator and continue culture for 24 h;

[0108] The bifidobacterium bacterial liquid was centrifuged to collect the bifidobacterium bacterial sludge; the centrifugal speed was 5500r / min;

[0109] The centrifugation time was 10 min.

[0110] The obtained bifidobacterium sludge is mixed with a composite freeze-drying protective agent, and then freeze-dried to obtain freeze-dried powder; the mass ratio of the bifidobacterium sludge to the freeze-drying protective agent is 6:1.4.

[0111] The obtained freeze-dried powder is granulated by top spraying in a fluidized bed coater with maltodextrin as a binder to obtain granules;

[0112] The granules obtained above are respectively filled into catechol-gelatin composite capsule shells to obtain a bifidobacterium preparation that can be stored at room temperature.

[0113] The preparation method of the composite freeze-drying protective agent is as follows:

[0114] First, weigh 24 g of hydrogenated lecithin and 2.6 g of mannitol;

[0115] Add hydrogenated lecithin to chloroform, stir and mix until dissolved, to obtain a hydrogenated lecithin solution;

[0116] Adding mannitol to methanol, stirring and mixing until dissolved, to obtain a mannitol solution;

[0117] The hydrogenated lecithin solution and the mannitol solution were mixed, and chloroform and methanol were removed by concentrating at 44° C. using a rotary evaporator, and then 1.2 g of alginate and 3.8 g of bovine serum albumin were added, and the volume was adjusted to 100 mL using water for injection to obtain a mixed solution;

[0118] The obtained mixed solution was subjected to one ultrasonic treatment by a cell disruptor for 10 min.

[0119] Freeze-dried:

[0120] First, pre-freeze for 20 min at -60 °C, then lower to -45 °C, keep warm for 30 min, and then evacuate to 0.18 mbar for 2 h.

[0121] The analytical drying vacuum was reduced to 0.1 mbar, the temperature was raised to 23°C and maintained for 2 hours to complete the freeze-drying process.

[0122] The mass ratio of freeze-dried powder to maltodextrin is 1:5.2.

[0123] The parameters of the top spray granulation of the fluidized bed coating machine are as follows: the inlet air temperature is 60°C, the air flow rate is 35m 3 / h, atomization pressure is 1.8bar, material temperature is 55℃, and spray rate is 8.5mL / min.

[0124] The preparation method of catechol-gelatin composite capsule shell is as follows:

[0125] First, gelatin is added into deionized water and fully dissolved to obtain a gelatin solution;

[0126] Adding catechol to deionized water, stirring and mixing evenly to obtain a catechol solution;

[0127] The gelatin solution and the catechol solution are mixed evenly, and then mixed with glycerol, the temperature is adjusted to 65°C, and the mixture is kept warm and homogenized for 10 minutes to obtain a composite glue solution;

[0128] After the capsule mold is immersed in the composite glue solution, it is taken out at a uniform speed and turned evenly, and dried at 45°C for 2.5 hours, and the formed capsules are peeled and cut one by one from the mold to finally obtain the catechol-gelatin composite capsule shell;

[0129] The capacity of the catechol-gelatin composite capsule shell is 0.25 g.

[0130] Example 4

[0131] A method for preparing a bifidobacterium preparation that can be stored at room temperature comprises: first, culturing bifidobacteria to obtain a bifidobacterium bacterial liquid; the number of viable bacteria in the bifidobacterium bacterial liquid is greater than 5.5×10 8 Pieces / mL.

[0132] Wherein, the culturing of Bifidobacterium longum is culturing in a liquid culture medium, wherein the liquid culture medium is:

[0133] 20.0 g glucose, 10.0 g tryptone, 6.0 g beef extract, 5 g sodium acetate, 5.0 g yeast extract powder, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 2.0 g potassium hydrogen phosphate, 0.2 g cysteine ​​hydrochloride, 0.58 g magnesium sulfate, 1.0 mL Tween-80, 1000 mL distilled water; adjust pH to 6.5, sterilize at 121°C for 25 min to obtain a liquid culture medium;

[0134] The culture conditions were as follows: culture in a shaker at 37°C and 220 rpm for 24 h, transfer to a 40°C incubator and continue culture for 24 h;

[0135] The bifidobacterium bacterial liquid was centrifuged to collect the bifidobacterium bacterial sludge; the centrifugal speed was 5500r / min;

[0136] The centrifugation time was 10 min.

[0137] The obtained bifidobacterium sludge is mixed with a composite freeze-drying protective agent, and then freeze-dried to obtain freeze-dried powder; the mass ratio of the bifidobacterium sludge to the freeze-drying protective agent is 6:1.4.

[0138] The obtained freeze-dried powder is granulated by top spraying in a fluidized bed coater with maltodextrin as a binder to obtain granules;

[0139] The granules obtained above are respectively filled into catechol-gelatin composite capsule shells to obtain a bifidobacterium preparation that can be stored at room temperature.

[0140] The preparation method of the composite freeze-drying protective agent is as follows:

[0141] First, weigh 23 g of hydrogenated lecithin and 2.8 g of mannitol;

[0142] Add hydrogenated lecithin to chloroform, stir and mix until dissolved, to obtain a hydrogenated lecithin solution;

[0143] Adding mannitol to methanol, stirring and mixing until dissolved, to obtain a mannitol solution;

[0144] The hydrogenated lecithin solution and the mannitol solution were mixed, and chloroform and methanol were removed by concentrating at 44° C. using a rotary evaporator, and then 1.2 g of alginate and 3-4 g of bovine serum albumin were added, and the volume was adjusted to 100 mL with water for injection to obtain a mixed solution;

[0145] The obtained mixed solution was subjected to one ultrasonic treatment by a cell disruptor for 10 min.

[0146] Freeze-dried:

[0147] First, pre-freeze for 20 min at -60 °C, then lower to -45 °C, keep warm for 30 min, and then evacuate to 0.18 mbar for 2 h.

[0148] The analytical drying vacuum was reduced to 0.1 mbar, the temperature was raised to 23°C and maintained for 2 hours to complete the freeze-drying process.

[0149] The mass ratio of freeze-dried powder to maltodextrin is 1:5.8.

[0150] The parameters of the top spray granulation of the fluidized bed coating machine are as follows: the inlet air temperature is 60°C, the air flow rate is 35m 3 / h, atomization pressure is 1.8bar, material temperature is 55℃, and spray rate is 8.5mL / min.

[0151] The preparation method of catechol-gelatin composite capsule shell is as follows:

[0152] First, gelatin is added into deionized water and fully dissolved to obtain a gelatin solution;

[0153] Adding catechol to deionized water, stirring and mixing evenly to obtain a catechol solution;

[0154] The gelatin solution and the catechol solution are mixed evenly, and then mixed with glycerol, the temperature is adjusted to 65°C, and the mixture is kept warm and homogenized for 10 minutes to obtain a composite glue solution;

[0155] After the capsule mold is immersed in the composite glue solution, it is taken out at a uniform speed and turned evenly, and dried at 45°C for 2.5 hours, and the formed capsules are peeled and cut one by one from the mold to finally obtain the catechol-gelatin composite capsule shell;

[0156] The capacity of the catechol-gelatin composite capsule shell is 0.25 g.

[0157] Example 5

[0158] A method for preparing a bifidobacterium preparation that can be stored at room temperature comprises: first, culturing bifidobacteria to obtain a bifidobacterium bacterial liquid; the number of viable bacteria in the bifidobacterium bacterial liquid is greater than 5.5×10 8 Pieces / mL.

[0159] Wherein, the culturing of Bifidobacterium longum is culturing in a liquid culture medium, wherein the liquid culture medium is:

[0160] 20.0 g glucose, 10.0 g tryptone, 6.0 g beef extract, 5 g sodium acetate, 5.0 g yeast extract powder, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 2.0 g potassium hydrogen phosphate, 0.2 g cysteine ​​hydrochloride, 0.58 g magnesium sulfate, 1.0 mL Tween-80, 1000 mL distilled water; adjust pH to 6.5, sterilize at 121°C for 25 min to obtain a liquid culture medium;

[0161] The culture conditions were as follows: culture in a shaker at 37°C and 220 rpm for 24 h, transfer to a 40°C incubator and continue culture for 24 h;

[0162] The bifidobacterium bacterial liquid was centrifuged to collect the bifidobacterium bacterial sludge; the centrifugal speed was 5500r / min;

[0163] The centrifugation time was 10 min.

[0164] The obtained bifidobacterium sludge is mixed with a composite freeze-drying protective agent, and then freeze-dried to obtain freeze-dried powder; the mass ratio of the bifidobacterium sludge to the freeze-drying protective agent is 6:1.5.

[0165] The obtained freeze-dried powder is granulated by top spraying in a fluidized bed coater with maltodextrin as a binder to obtain granules;

[0166] The granules obtained above are respectively filled into catechol-gelatin composite capsule shells to obtain a bifidobacterium preparation that can be stored at room temperature.

[0167] The preparation method of the composite freeze-drying protective agent is as follows:

[0168] First, weigh 25g of hydrogenated lecithin and 2g of mannitol;

[0169] Add hydrogenated lecithin to chloroform, stir and mix until dissolved, to obtain a hydrogenated lecithin solution;

[0170] Adding mannitol to methanol, stirring and mixing until dissolved, to obtain a mannitol solution;

[0171] The hydrogenated lecithin solution and the mannitol solution were mixed, and chloroform and methanol were removed by concentrating using a rotary evaporator at 44°C, and then 1.5 g of alginate and 4 g of bovine serum albumin were added, and the volume was adjusted to 100 mL with water for injection to obtain a mixed solution;

[0172] The obtained mixed solution was subjected to one ultrasonic treatment by a cell disruptor for 10 min.

[0173] Freeze-dried:

[0174] First, pre-freeze for 20 min at -60 °C, then lower to -45 °C, keep warm for 30 min, and then evacuate to 0.18 mbar for 2 h.

[0175] The analytical drying vacuum was reduced to 0.1 mbar, the temperature was raised to 23°C and maintained for 2 hours to complete the freeze-drying process.

[0176] The mass ratio of freeze-dried powder to maltodextrin is 1:6.

[0177] The parameters of the top spray granulation of the fluidized bed coating machine are as follows: the inlet air temperature is 60°C, the air flow rate is 35m 3 / h, atomization pressure is 1.8bar, material temperature is 55℃, and spray rate is 8.5mL / min.

[0178] The preparation method of catechol-gelatin composite capsule shell is as follows:

[0179] First, gelatin is added into deionized water and fully dissolved to obtain a gelatin solution;

[0180] Adding catechol to deionized water, stirring and mixing evenly to obtain a catechol solution;

[0181] The gelatin solution and the catechol solution are mixed evenly, and then mixed with glycerol, the temperature is adjusted to 65°C, and the mixture is kept warm and homogenized for 10 minutes to obtain a composite glue solution;

[0182] After the capsule mold is immersed in the composite glue solution, it is taken out at a uniform speed and turned evenly, and dried at 45°C for 2.5 hours, and the formed capsules are peeled and cut one by one from the mold to finally obtain the catechol-gelatin composite capsule shell;

[0183] The capacity of the catechol-gelatin composite capsule shell is 0.25 g.

[0184] Comparative Example 1:

[0185] Based on the technical solution of Example 1, the product is stored only in the form of freeze-dried powder.

[0186] Comparative Example 2:

[0187] Based on the technical solution of Example 1, it is stored only in the form of pellets.

[0188] Comparative Example 3:

[0189] On the basis of the technical solution of Example 1, catechol was not added in the preparation of the catechol-gelatin composite capsule shell, and the rest of the technical solutions remained unchanged.

[0190] test:

[0191] The test results of the viable bacteria counts in the examples and comparative examples are shown in Table 1:

[0192] Table 1

[0193]

[0194]

[0195] It can be seen from Table 1 that the bifidobacterium preparation prepared by the method of the present invention and which can be stored at room temperature contains a relatively high content of live bifidobacteria.

[0196] The samples of the embodiment and the comparative example were stored at room temperature (25°C, air humidity 40%) for 4 weeks, and the results of the comparison were as follows:

[0197] Table 2

[0198] Viable bacteria count cfu / mL Example 1 <![CDATA[3.06×10 11 ]]> Example 2 <![CDATA[3.01×10 11 ]]> Example 3 <![CDATA[3.10×10 11 ]]> Example 4 <![CDATA[3.03×10 11 ]]> Example 5 <![CDATA[3.02×10 11 ]]> Comparative Example 1 <![CDATA[2.18×10 9 ]]> Comparative Example 2 <![CDATA[5.04×10 10 ]]> Comparative Example 3 <![CDATA[2.87×10 11 ]]>

[0199] It can be seen from Table 2 that different treatments have different effects on the storage time of bifidobacteria, and the bifidobacterium preparation prepared by the present invention can significantly prolong the survival number of bifidobacteria.

[0200] Taking Example 1 as the basic sample, the number of viable bacteria was detected after storage at different temperatures for 24 hours, and the temperature resistance was compared, while other parameters were the same;

[0201] Table 3

[0202]

[0203]

[0204] It can be seen from Table 3 that the bifidobacterium preparation prepared by the present invention has excellent temperature resistance and can still maintain a high content of live bifidobacteria when stored at a relatively high temperature.

[0205] Gastric acid resistance test:

[0206] Take 2 g of each sample of the embodiment and the comparative example, and then add them to 120 mL of hydrochloric acid solution with a pH of 1.2, and keep them in a water bath at 37° C. for 1 hour. Then, determine the number of viable bifidobacteria, and calculate the survival rate of bifidobacteria in each group:

[0207] Table 4

[0208] Bifidobacterium survival rate % Example 1 88.13 Example 2 88.37 Example 3 87.12 Example 4 87.68 Example 5 88.05 Comparative Example 1 0.12 Comparative Example 2 10.24 Comparative Example 3 75.69

[0209] It can be seen from Table 4 that the bifidobacterium preparation prepared in the present invention has excellent gastric acid resistance.

[0210] The above description is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which still do not exceed the spirit covered by the specification, should be within the protection scope of the present invention.

Claims

1. A method for preparing a bifidobacterium preparation that can be stored at room temperature, characterized in that: First, bifidobacteria are cultured to obtain bifidobacterium bacterial liquid; Wherein, the bifidobacterium is cultured in a liquid culture medium, wherein the liquid culture medium is: 20.0 g glucose, 10.0 g tryptone, 6.0 g beef extract, 5 g sodium acetate, 5.0 g yeast extract powder, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 2.0 g potassium hydrogen phosphate, 0.2 g cysteine ​​hydrochloride, 0.58 g magnesium sulfate, 1.0 mL Tween-80, 1000 mL distilled water; adjust pH to 6.5, sterilize at 121°C for 25 min to obtain a liquid culture medium; The culture conditions were as follows: culture in a shaker at 37°C and 220 rpm for 24 h, transfer to a 40°C incubator and continue culture for 24 h; The bifidobacterium bacterial liquid is centrifuged to collect the bifidobacterium bacterial mud; The obtained bifidobacterium sludge is mixed with a composite freeze-drying protective agent, and then freeze-dried to obtain freeze-dried powder; The obtained freeze-dried powder is granulated by top spraying in a fluidized bed coater with maltodextrin as a binder to obtain granules; The granules obtained above are respectively filled into catechol-gelatin composite capsule shells to obtain a bifidobacterium preparation that can be stored at room temperature.

2. The method for preparing a bifidobacterium preparation that can be stored at room temperature according to claim 1, characterized in that: The bifidobacterium is Bifidobacterium longum; The number of viable bacteria in the bifidobacterium liquid is greater than 5.5×10 8 Pieces / mL.

3. The method for preparing a bifidobacterium preparation that can be stored at room temperature according to claim 1, characterized in that: The centrifugal treatment speed is 5500r / min; The centrifugation time was 10 min.

4. The method for preparing a bifidobacterium preparation that can be stored at room temperature according to claim 1, characterized in that: The mass ratio of the bifidobacterium sludge to the freeze-drying protective agent is 6:1-1.

5.

5. The method for preparing a bifidobacterium preparation that can be stored at room temperature according to claim 4, characterized in that: The preparation method of the composite freeze-drying protective agent is as follows: First, weigh 20-25g of hydrogenated lecithin and 2-3g of mannitol; Add hydrogenated lecithin to chloroform, stir and mix until dissolved, to obtain a hydrogenated lecithin solution; Adding mannitol to methanol, stirring and mixing until dissolved, to obtain a mannitol solution; The hydrogenated lecithin solution and the mannitol solution were mixed, and chloroform and methanol were removed by concentrating using a rotary evaporator at 44°C, and then 1-1.5 g of alginate and 3-4 g of bovine serum albumin were added, and the volume was adjusted to 100 mL with water for injection to obtain a mixed solution; The obtained mixed solution was subjected to one ultrasonic treatment by a cell disruptor for 10 min.

6. The method for preparing a bifidobacterium preparation that can be stored at room temperature according to claim 5, characterized in that: The freeze-drying process is: First, pre-freeze for 20 min at -60 °C, then lower to -45 °C, keep warm for 30 min, and then evacuate to 0.18 mbar for 2 h. The analytical drying vacuum was reduced to 0.1 mbar, the temperature was raised to 23°C, and maintained for 2 hours to complete the freeze-drying process; The mass ratio of freeze-dried powder to maltodextrin is 1:5-6.

7. The method for preparing a bifidobacterium preparation that can be stored at room temperature according to claim 6, characterized in that: The parameters of the fluidized bed coating machine top spray granulation are as follows: the inlet air temperature is 60°C, the air flow rate is 35m 3 / h, atomization pressure is 1.8bar, material temperature is 55℃, and spray rate is 8.5mL / min.

8. The method for preparing a bifidobacterium preparation that can be stored at room temperature according to claim 1, characterized in that: The preparation method of the catechol-gelatin composite capsule shell is as follows: First, gelatin is added into deionized water and fully dissolved to obtain a gelatin solution; Adding catechol to deionized water, stirring and mixing evenly to obtain a catechol solution; The gelatin solution and the catechol solution are mixed evenly, and then mixed with glycerol, the temperature is adjusted to 65°C, and the mixture is kept warm and homogenized for 10 minutes to obtain a composite glue solution; After the capsule mold is immersed in the composite glue solution, it is taken out at a uniform speed and turned evenly, and dried at 45°C for 2.5 hours, and the formed capsules are peeled and cut one by one from the mold to finally obtain the catechol-gelatin composite capsule shell; The capacity of the catechol-gelatin composite capsule shell is 0.25 g.

9. A bifidobacterium preparation that can be stored at room temperature is prepared according to the method for preparing a bifidobacterium preparation that can be stored at room temperature according to any one of claims 1 to 8.

10. The bifidobacterium preparation that can be stored at room temperature according to claim 9, characterized in that: The bifidobacterium preparation that can be stored at room temperature contains more than 3.2×10 11 Pieces / g.

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