Preparation method of freeze-dried powder of bifidobacterium bifidum and freeze-dried powder of freeze-dried powder of bifidobacterium bifidum

Abstract

The invention belongs to the technical field of food processing, and provides a preparation method of freeze-dried bifidobacterium bifidum powder and freeze-dried bifidobacterium bifidum powder.The preparation method comprises the steps that a bifidobacterium bifidum strain is subjected to enlarged culture to obtain fermentation liquor, centrifugation is conducted to obtain bacterial sludge, the bacterial sludge and a protective agent are evenly mixed to obtain bacterial suspension, and the bacterial suspension is dried to obtain the freeze-dried bifidobacterium bifidum powder. And carrying out pre-freezing and staged variable-temperature vacuum freeze-drying to obtain the freeze-dried powder of the bifidobacterium bifidum. According to the preparation method of the freeze-dried bacterial powder, the efficiency of a freeze dryer is fully exerted, and the time consumed by the freeze-drying process can be as low as 25.25 h; the freeze-drying survival rate of the prepared freeze-dried bifidobacterium powder can reach 100%, the total number of viable bacteria is larger than or equal to 1.4 * 10 < 11 > CFU / g, and the freeze-dried bifidobacterium powder can efficiently act on intestines and stomach and is beneficial to providing a stronger probiotic effect; the water content of the prepared freeze-dried bifidobacterium powder is appropriate (3.30%-3.54%), so that the freeze-drying survival rate of the freeze-dried bifidobacterium powder is improved, and the freeze-dried bifidobacterium powder has excellent storage stability; and the method has remarkable energy-saving and emission-reducing significance and economic benefits.

Description

Technical Field

[0001] The invention relates to a preparation method of freeze-dried bacterial powder of Bifidobacterium bifidum and the freeze-dried bacterial powder, belonging to the technical field of food processing. Background Art

[0002] Bifidobacterium bifidum is a Gram-positive, non-spore-forming, non-motile, obligate anaerobic bacterium with a variety of morphological characteristics including long rods, bifurcated Y-shaped rods, V-shaped rods, etc., and there are differences under different nutritional environments. Bacteria of the genus Bifidobacterium are one of the important members of the intestinal flora of humans and animals. Some strains of Bifidobacterium can be used as probiotics in food, medicine and feed. Studies have shown that Bifidobacterium bifidum can produce neuroserine protease inhibitors to protect the adhesion ability of extracellular proteins and pili, thereby achieving its colonization and adhesion in the host. Bifidobacterium bifidum has been studied for decades at home and abroad, and its application prospects are broad. It can be used as a dietary supplement and as a functional ingredient in health foods. A large number of animal and clinical trial studies have shown that Bifidobacterium bifidum has the characteristics of improving digestive system metabolism, enhancing immune system regulation, improving nervous system regulation, and being safe and without side effects.

[0003] Bifidobacterium bifidum is a probiotic strain that can be used in health foods. Health foods and probiotic preparations with added probiotics on the market are mainly in the form of ordinary foods, including cheese, beverages, yogurt, etc. Probiotic preparations health products mainly in the form of medicines include powders, hard capsules, soft capsules, granules, oral liquids, microcapsules and other dosage forms.

[0004] In order to provide stronger probiotic effects and meet dosage requirements, it is necessary to improve the powder preparation process to obtain high-viability powder. Vacuum freeze drying is the most mature method for preparing probiotic freeze-dried powder. It reduces the water activity of bacteria to below 0.2, so that it can be stored for a long time in an environment above zero degrees, reducing its transportation and storage costs, and preserving the vitality of bacterial cells to the maximum extent. However, this method has the disadvantages of being time-consuming and energy-intensive. Bifidobacterium is sensitive to oxygen and has poor stress resistance, so the cells are more susceptible to damage during the freeze-drying process, making it difficult to improve the freeze-drying survival rate. For example, when sorbitol and raffinose are used as freeze-drying protectants for Bifidobacterium adolescentis CCFM3680, the freeze-drying survival rate is (56.03±1.82)%; when 10% skimmed milk powder is used as freeze-drying protectant for Bifidobacterium longum DD98, the freeze-drying survival rate is 35.7%. How to optimize the freeze-drying procedure and shorten the freeze-drying time while ensuring the survival rate has significant energy-saving and environmental protection significance. Summary of the invention

[0005]

Technical issues

[0006] In order to provide stronger probiotic effects and meet dosage requirements, it is necessary to improve the bacterial powder preparation process to obtain high-viability bacterial powder. The existing vacuum freeze-drying preparation method of bifidobacterium bacterial powder has the problems of long time consumption, high energy consumption, low freeze-dried bacterial powder survival rate, and inappropriate moisture content.

[0007]

Technical solution

[0008] In order to solve the above problems, the present invention treats Bifidobacterium bifidum through a series of activation, expansion culture, centrifugal collection of bacteria, mixing of bacterial mud with a protective agent, prefreezing, vacuum freeze drying and pulverization to obtain an activity number ≥1.4×10 11 The freeze-dried powder of Bifidobacterium bifidum with CFU / g has a high freeze-drying survival rate, suitable powder moisture content, and a short freeze-drying time, and has significant energy-saving and emission-reduction significance and economic benefits.

[0009] The first object of the present invention is to provide a method for preparing freeze-dried powder of Bifidobacterium bifidum, comprising the steps of: expanding and culturing the strain of Bifidobacterium bifidum to obtain a fermentation liquid, centrifuging to obtain bacterial mud, uniformly mixing the bacterial mud with a protective agent to obtain a bacterial suspension, prefreezing, and vacuum freeze-drying to obtain freeze-dried powder of Bifidobacterium bifidum;

[0010] Among them, the conditions for vacuum freeze drying are to adopt a stage temperature change program:

[0011] The first stage is to vacuum dry at -48 to -42°C for 0.2 to 0.6 hours; the second stage is to heat up to -13 to -10°C for 0.4 to 0.6 hours and vacuum dry for 2 to 5 hours; the third stage is to heat up to 6 to 9°C for 0.4 to 0.6 hours and vacuum dry for 6 to 8 hours; the fourth stage is to heat up to 31 to 34°C for 0.4 to 0.6 hours and vacuum dry for 10 to 13 hours;

[0012] or,

[0013] The first stage is vacuum drying at -48 to -42°C for 0.8 to 1.2 hours; the second stage is heating to -30 to -25°C for 0.4 to 0.6 hours and vacuum drying for 6 to 8 hours; the third stage is heating to -4 to -2°C for 0.4 to 0.6 hours and vacuum drying for 9 to 11 hours; the fourth stage is heating to 25 to 27°C for 0.4 to 0.6 hours and vacuum drying for 17 to 20 hours;

[0014] or,

[0015] The first stage is to vacuum dry at -48 to -42 °C for 0.8 to 1.2 hours; the second stage is to heat up to -42 to -38 °C for 0.4 to 0.6 hours and vacuum dry for 1 to 3 hours; the third stage is to heat up to -31 to -29 °C for 0.4 to 0.6 hours and vacuum dry for 7 to 9 hours; the fourth stage is to heat up to -4 to -2 °C for 0.4 to 0.6 hours and vacuum dry for 9 to 11 hours; the fifth stage is to heat up to 24 to 26 °C for 0.4 to 0.6 hours and vacuum dry for 19 to 21 hours;

[0016] or,

[0017] The first stage is to heat up to -27 to -23°C in 0.4 to 0.6 h and vacuum dry for 5 to 7 h. The second stage is to heat up to -4 to -2°C in 0.4 to 0.6 h and vacuum dry for 8 to 10 h. The third stage is to heat up to 27 to 29°C in 0.4 to 0.6 h and vacuum dry for 15 to 17 h.

[0018] In one embodiment of the present invention, the vacuum degree is 0.2-0.4 Pa at the end of the stage temperature change program.

[0019] In one embodiment of the present invention, the Bifidobacterium bifidum is Bifidobacterium bifidum F35, and the deposit number is CGMCCNO. 13632. Bifidobacterium bifidum F35 can inhibit the growth and reproduction of pathogenic bacteria, maintain liver health and normal metabolism, improve intestinal function, and enhance human immunity.

[0020] In one embodiment of the present invention, the preserved Bifidobacterium bifidum strain is activated and then inoculated into a sterilized food-grade culture medium for expansion culture.

[0021] The activation is to inoculate the preserved strain into a suitable culture medium to meet its growth requirements, so as to promote its initial growth and reproduction, thereby obtaining a vigorous strain culture with a sufficient inoculation quantity.

[0022] In one embodiment of the present invention, the Bifidobacterium bifidum strain is activated 2 to 3 times.

[0023] In one embodiment of the present invention, the conditions for activating the Bifidobacterium bifidum strain are 37° C., anaerobic culture, and each time for 16 to 20 hours.

[0024] In one embodiment of the present invention, the Bifidobacterium bifidum strain is activated using sterile MRS-cys medium.

[0025] In one embodiment of the present invention, the preparation method of sterilized MRS-cys medium is as follows: dissolving the raw materials in sterile water, mixing evenly and sterilizing at 121° C. for 15 min; the raw materials and concentrations of each raw material of the sterilized MRS-cys medium are: yeast extract 10 g / L, yeast peptone 10 g / L, glucose 20 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4 2 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.05 g / L, Tween 80 1 mL / L, and L-cysteine ​​hydrochloride 2 g / L.

[0026] In one embodiment of the present invention, the conditions for expansion culture are 37° C. and anaerobic culture.

[0027] In one embodiment of the present invention, a sterilized food-grade culture medium is used for expansion of the culture.

[0028] In one embodiment of the present invention, the Bifidobacterium bifidum strain is expanded and cultured using sterilized MRS-cys fermentation medium.

[0029] In one embodiment of the present invention, the preparation method of sterilized MRS-cys fermentation medium is to dissolve the raw materials in sterile water, mix them evenly and sterilize them at 121°C for 15 minutes; the raw materials and concentrations of each raw material of the sterilized MRS-cys fermentation medium are: yeast extract 10g / L, yeast peptone 10g / L, glucose 20g / L, MgSO4·7H2O 0.1g / L, MnSO4·H2O 0.05g / L, Tween 80 1mL / L, and L-cysteine ​​hydrochloride 2g / L.

[0030] In one embodiment of the present invention, the expanded culture is to first inoculate the seed liquid into 1 to 10 L of sterilized food-grade culture medium and culture it for 16 to 20 hours, and then transfer it to ≥100 L of sterilized food-grade culture medium for culture; by supplementing 1.5 to 2.5 mol / L of sodium hydroxide solution, the pH of the fermentation liquid is maintained at 5.5 to 5.9, and the expanded culture is terminated when the total alkali supplementation amount is 8 to 16 L.

[0031] In one embodiment of the present invention, the concentration of Bifidobacterium bifidum cells in the fermentation broth is 2 to 4.5×10 9 CFU / mL.

[0032] In one embodiment of the present invention, the centrifugal speed is 80000-12000 g, and the centrifugal temperature is 0-4°C.

[0033] In one embodiment of the present invention, the protective agent is a sterilized reconstituted skim milk aqueous solution with a concentration of 18 to 22 wt%.

[0034] In one embodiment of the present invention, the protective agent is preferably a sterilized reconstituted skim milk aqueous solution with a concentration of 20 wt%.

[0035] In one embodiment of the present invention, the mixing mass ratio of the bacterial mud to the protective agent is 1:1 to 1:3.

[0036] In one embodiment of the present invention, the mixing mass ratio of bacterial mud to protective agent is preferably 1:2.

[0037] In one embodiment of the present invention, the concentration of Bifidobacterium bifidum cells in the bacterial suspension is 3 to 7×10 10 CFU / mL.

[0038] In one embodiment of the present invention, the bacterial suspension is evenly spread and then pre-frozen and vacuum freeze-dried; the spreading thickness of the bacterial suspension is 1 to 1.2 cm.

[0039] Vacuum freeze drying mainly consists of three stages: pre-freezing, primary drying and secondary drying (desorption drying). The pre-freezing process is the first step of vacuum freeze drying, which controls the size, distribution and morphology of ice crystals. During the primary drying process, the water in the pre-frozen sample is removed by sublimation, and the bound water is further removed during the secondary drying process, and the water activity is reduced to a level that can inhibit biological activity and chemical reactions. In order to remove water from the sample, it is necessary to control the water vapor pressure gradient, which is achieved by capturing condensed water (cold trap). After the ice sublimates, the remaining concentrate still has a high content of unfrozen water (20% to 30%), which is not conducive to the storage stability of the freeze-dried product. The desorption of unfrozen water is achieved by moderately heating the product to a temperature above zero (20℃ to 30℃), thereby reducing the moisture content (<5% dry weight) and water activity (<0.2). Sublimation requires low pressure (vacuum) and heating to sublimate the ice into steam. During this process, the heat input must be strictly controlled to ensure that the temperature of the bacterial powder material is lower than the collapse temperature to reduce the adverse effects on the number of live bacteria and the structure of the bacterial community.

[0040] During the vacuum drying process, dehydration can cause cell damage. Water molecules are essential for maintaining the stability of various cell components. The removal of water molecules will affect the physiological integrity and structure of cells, increase the permeability of cell membranes, affect the fluidity and integrity of cell membranes, destroy the lipid membrane structure, affect the protein structure and function, and even cause cell death. Among them, dehydration in the early pre-freezing and primary drying processes causes less damage to cells, while dehydration in the final desorption and drying process causes greater damage to cells. Studies have shown that when the moisture content of freeze-dried bacterial powder is lower than 0.5%, it will cause cell DNA breakage and cause cell death.

[0041] The moisture content of freeze-dried bacterial powder affects the storage stability and freeze-drying survival rate of the bacterial powder. When the moisture content is high, the storage stability of the bacterial powder is poor; and when the moisture content is too low, it will also lead to the loss of bacteria and reduce the freeze-drying survival rate. Therefore, it is of great significance to ensure that the moisture content of freeze-dried bacterial powder is within an appropriate range. The moisture content of the bacterial powder is mainly affected by the freeze-drying process, including the temperature, time, and vacuum degree of sublimation drying and analytical drying.

[0042] Many factors, such as the temperature of the product during the freeze-drying process, the final moisture content, and the storage conditions (temperature, relative humidity, and air), can affect the stability of freeze-dried lactic acid bacteria. Therefore, it is necessary to comprehensively consider the above factors to determine the freeze-drying procedure so that the freeze-dried lactic acid bacteria have a high survival rate and vitality recovery rate.

[0043] In one embodiment of the present invention, the pre-freezing temperature is lower than -45°C, and the pre-freezing time is 2 to 3 hours.

[0044] In one embodiment of the present invention, the pre-freezing temperature is preferably -45°C, and the pre-freezing time is preferably 2.5 hours.

[0045] In one embodiment of the present invention, the vacuum freeze drying condition is preferably to adopt a stage temperature change program, the first stage is vacuum drying at -45°C for 0.25h; the second stage is heating to -11°C for 0.5h and vacuum drying for 3h; the third stage is heating to 8°C for 0.5h and vacuum drying for 7h; the fourth stage is heating to 33°C for 0.5h and vacuum drying for 11h. As shown in Table 1 Program 9.

[0046] In one embodiment of the present invention, the vacuum freeze drying condition is preferably a stage temperature change program, the first stage is vacuum drying at -45°C for 0.5h; the second stage is heating to -12°C for 0.5h and vacuum drying for 4h; the third stage is heating to 7°C for 0.5h and vacuum drying for 7h; the fourth stage is heating to 32°C for 0.5h and vacuum drying for 12h. As shown in Table 1 Program 8.

[0047] In one embodiment of the present invention, the vacuum freeze drying condition is preferably a stage temperature change program, the first stage is vacuum drying at -45°C for 1 hour; the second stage is heating to -25°C for 0.5 hours and vacuum drying for 6 hours; the third stage is heating to -3°C for 0.5 hours and vacuum drying for 10 hours; the fourth stage is heating to 27°C for 0.5 hours and vacuum drying for 17 hours. As shown in Table 1, Program 4.

[0048] In one embodiment of the present invention, the vacuum freeze drying condition is preferably a stage temperature change program, the first stage is vacuum drying at -45°C for 1 hour; the second stage is heating to -27°C for 0.5 hours and vacuum drying for 7 hours; the third stage is heating to -3°C for 0.5 hours and vacuum drying for 10 hours; the fourth stage is heating to 26°C for 0.5 hours and vacuum drying for 18 hours. As shown in Table 1, Program 3.

[0049] In one embodiment of the present invention, the vacuum freeze drying condition is preferably a stage temperature change program, the first stage is vacuum drying at -45°C for 1 hour; the second stage is heating to -40°C for 0.5 hours and vacuum drying for 2 hours; the third stage is heating to -30°C for 0.5 hours and vacuum drying for 8 hours; the fourth stage is heating to -3°C for 0.5 hours and vacuum drying for 10 hours; the fifth stage is heating to 25°C for 0.5 hours and vacuum drying for 20 hours. As shown in Table 1, Program 2.

[0050] In one embodiment of the present invention, freeze-dried powder of Bifidobacterium bifidum is pulverized and passed through a 40-80 mesh sieve.

[0051] The second object of the present invention is to provide freeze-dried powder of Bifidobacterium bifidum prepared by the above method.

[0052] In one embodiment of the present invention, the moisture content of the freeze-dried powder of Bifidobacterium bifidum is 3.30-3.54%, and the total number of viable bacteria of Bifidobacterium bifidum is ≥1.4×10 11 CFU / g.

[0053] The third object of the present invention is to provide the use of the freeze-dried powder of Bifidobacterium bifidum in the field of food or health care products.

[0054] In one embodiment of the present invention, the application in the food field includes application in fermented foods, fermented dairy products, functional beverages, and functional foods; the application in the health care product field includes application in probiotic capsules, tablets, and powders.

[0055] Beneficial effects:

[0056] The freeze-dried bacterial powder preparation method of the present invention adopts a stage temperature change program to give full play to the efficiency of the freeze dryer and reduce unnecessary freeze drying time. The freeze drying process can be as low as 25.25 hours. The freeze drying survival rate of the prepared freeze-dried Bifidobacterium bifidum powder can reach 100%, and the total number of viable bacteria is ≥1.4×10 11 CFU / g, can effectively act on the stomach and intestines, is conducive to providing stronger probiotic effects and meeting dosage requirements; the prepared freeze-dried powder of Bifidobacterium bifidum has an appropriate moisture content (3.30% to 3.54%), which improves the freeze-dried survival rate of the powder and has excellent storage stability; it has significant energy-saving and emission-reduction significance and economic benefits.

[0057] The total number of live bacteria of the freeze-dried Bifidobacterium bifidum powder prepared by the present invention can reach 1.4×10 11 CFU / g~3.4×10 11 CFU / g, and the freeze-dried survival rate was 70.13-100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the change curve of material, eutectic point, shelf temperature and vacuum degree during the vacuum freeze-drying process of Example 1.

[0059] Figure 2 This is a photo of freeze-dried Bifidobacterium bifidum powder. DETAILED DESCRIPTION

[0060] Raw materials and equipment

[0061] The Bifidobacterium bifidum F35 (deposit number CGMCC NO.13632) involved in the following examples and comparative examples is from the strain resource library of the Food Biotechnology Center of Jiangnan University, skim milk powder is purchased from Fonterra Group of New Zealand, yeast extract and yeast peptone are purchased from Angel Yeast Co., Ltd.; glucose is purchased from Shandong Hairong Biotechnology Co., Ltd.; sodium acetate and diammonium hydrogen citrate are purchased from Jiangsu Kolondo Food Ingredients Co., Ltd.; dipotassium hydrogen phosphate and L-cysteine ​​hydrochloride are purchased from Xuzhou Fengrui Biotechnology Co., Ltd.; magnesium sulfate is purchased from Laizhou Laiyu Chemical Co., Ltd.; manganese sulfate is purchased from Lianyungang Longtaiwei Food Ingredients Co., Ltd.

[0062] The culture medium and lyophilization protective agent involved in the following examples are as follows:

[0063] Preparation of sterilized MRS-cys medium: The raw materials and formula are yeast extract 10g / L, yeast peptone 10g / L, glucose 20g / L, sodium acetate 5g / L, diammonium hydrogen citrate 2g / L, K2HPO4 2g / L, MgSO4·7H2O 0.1g / L, MnSO4·H2O 0.05g / L, Tween 80 1mL / L, L-cysteine ​​hydrochloride 2g / L, and the rest is sterile water. Dissolve the raw materials in sterile water, mix well, and sterilize at 121℃ for 15min.

[0064] Preparation of sterilized MRS-cys agar medium: The difference from the MRS-cys medium formula is that MRS-cys agar medium also contains 10g / L agar powder. Dissolve the raw materials in sterile water, mix well, and sterilize at 121℃ for 15min.

[0065] Preparation of sterilized MRS-cys fermentation medium: The difference from the MRS-cys medium formula is that the MRS-cys fermentation medium does not contain sodium acetate, diammonium hydrogen citrate and K2HPO4. Dissolve the raw materials in sterile water, mix well and sterilize at 121℃ for 15min.

[0066] Preparation of freeze-drying protective agent (sterilized reconstituted skim milk aqueous solution): sterile water was used to prepare a 20 wt % reconstituted skim milk aqueous solution, and sterilized at 115° C. for 20 min.

[0067] The 5L, 100L, and 200L fermentation tank systems used in the following examples and comparative examples were purchased from Huisen Bio-Equipment Zhenjiang Co., Ltd.; the centrifuge model was GQ105 tubular separator, purchased from Shanghai Zhangquan Centrifuge Technology Co., Ltd.; the freeze dryer model was LGJ-100G freeze dryer (rated power 8.6kw + vacuum pump 1.1kw), purchased from Beijing Sihuan Qihang Technology Co., Ltd.

[0068] Detection Methods

[0069] Activity assessment of Bifidobacterium bifidum: Take the strain fermentation broth, concentrated bacterial broth after centrifugation and freeze-dried bacterial powder samples, perform gradient dilution treatment, use MRS-cys agar medium melted and cooled to 40-45°C for plate pouring, and anaerobic culture counting.

[0070] Freeze-dried survival rate = (number of viable bacteria per gram of powder × mass of powder) / (number of viable bacteria per milliliter of suspension × volume of suspension) × 100%

[0071] Moisture determination method: refer to GB5009.3-2016.

[0072] The pre-freezing and vacuum drying procedures used in the embodiments and comparative examples are shown in Table 1.

[0073] Table 1 Specific scheme of prefreezing and vacuum drying procedures

[0074] Procedure 1 Procedure 2 Procedure 3 Procedure 4 Procedure 5 Procedure 6 Procedure 7 Procedure 8 Procedure 9 Pre-freezing temperature (℃) -45 -45 -45 -45 -45 -45 -45 -45 -45 Pre-freezing time (h) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Shelf temperature (℃) -45 -45 -45 -45 / / / -45 -45 Drying time (h) 2 1 1 1 / / / 0.5 0.25 Heating time (h) 0.5 0.5 / / / / / / / Shelf temperature (℃) -40 -40 / / / / / / / Drying time (h) 6 2 / / / / / / / Heating time (h) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Shelf temperature (℃) -30 -30 -27 -25 -25 -25 -25 -12 -11 Drying time (h) 8 8 7 6 6 6 6 4 3 Heating time (h) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Shelf temperature (℃) -3 -3 -3 -3 -3 -3 -3 7 8 Drying time (h) 10 10 10 10 10 9 9 7 7 Heating time (h) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Shelf temperature (℃) 25 25 26 27 28 28 30 32 33 Drying time (h) 22 20 18 17 16 16 14 12 11 Vacuum degree at the end of freeze drying (Pa) 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Total time (h) 52.5 45.5 40 38 36 35 33 27.5 25.25

[0075] Example 1

[0076] A method for preparing freeze-dried powder of Bifidobacterium bifidum F35 comprises the following steps:

[0077] The preserved Bifidobacterium bifidum F35 was streaked and a single colony was picked and inoculated into a 1.5 mL EP tube filled with sterilized MRS-cys medium, and cultured anaerobically at 37°C for 18 h to obtain the first generation seed solution, and then inoculated with 50 mL sterilized MRS-cys medium at a 3% inoculation amount (i.e., the mass percentage of the seed solution relative to the liquid culture medium), and cultured anaerobically at 37°C for 18 h to obtain the second generation seed solution, thereby completing the activation;

[0078] The second generation seeds were inoculated with 1% of the inoculation amount into a fermenter filled with 5L sterilized MRS-cys medium, and cultured at 37°C for 18h to obtain the third generation seeds. The third generation seeds were transferred to a fermenter filled with 200L sterilized MRS-cys fermentation medium at a 1% inoculation amount, cultured at 37°C, and the pH of the fermentation liquid was kept constant at 5.7 by supplementing 2mol / L sodium hydroxide solution, and the total alkali supplementation amount was controlled to be 12L. The fermentation was terminated after the alkali supplementation was completed, and centrifuged (10000g, 4°C) to obtain bacterial mud;

[0079] The bacterial sludge was mixed with a 20 wt% sterilized reconstituted skim milk aqueous solution in a mass ratio of 1:2 and stirred evenly to obtain a bacterial suspension;

[0080] The bacterial suspension was evenly spread on the tray of the vacuum freeze dryer, with the material volume in each tray being 1.5 to 2 L and the material thickness being 1 to 1.2 cm. The tray was then transferred to the shelf of the freeze dryer, and pre-frozen and vacuum dried using procedure 2. The product was crushed and passed through a 60-mesh sieve to obtain freeze-dried bifidobacterium powder.

[0081] The properties of the freeze-dried powder of Bifidobacterium bifidum F35 prepared in Example 1 are shown in Table 2.

[0082] Table 2 Performance of freeze-dried powder of Bifidobacterium bifidum F35 prepared by procedure 2

[0083] Fermentation broth viable count (CFU / mL) <![CDATA[3.0×10 9 ]]> Fermentation volume (L) 200 Mushroom mass (g) 4150 Protective agent addition ratio 1:2 Number of viable bacteria in bacterial suspension (CFU / mL) <![CDATA[4.5×10 10 ]]> Bacterial suspension volume (mL) 12500 Freeze-dried bacterial powder viable count (CFU / g) <![CDATA[2.2×10 11 ]]> Freeze-dried bacterial powder mass (g) 2418 Freeze-dried survival rate (%) 94.57 Freeze-dried bacterial powder moisture (%) 3.53

[0084] Example 2

[0085] A method for preparing freeze-dried bacterial powder of Bifidobacterium bifidum F35, which differs from Example 1 in that procedure 3 is used for pre-freezing and vacuum drying.

[0086] The properties of the freeze-dried powder of Bifidobacterium bifidum F35 prepared in Example 2 are shown in Table 3.

[0087] Table 3 Performance of freeze-dried powder of Bifidobacterium bifidum F35 prepared by procedure 3

[0088] Fermentation broth viable count (CFU / mL) <![CDATA[2.5×10 9 ]]> Fermentation volume (L) 200 Mushroom mass (g) 4750 Protective agent addition ratio 1:2 Number of viable bacteria in bacterial suspension (CFU / mL) <![CDATA[3.5×10 10 ]]> Bacterial suspension volume (mL) 14250 Freeze-dried bacterial powder viable count (CFU / g) <![CDATA[1.9×10 11 ]]> Freeze-dried bacterial powder mass (g) 2520 Freeze-dried survival rate (%) 96 Freeze-dried bacterial powder moisture (%) 3.54

[0089] Example 3

[0090] A method for preparing freeze-dried bacterial powder of Bifidobacterium bifidum F35 is different from that of Example 1 in that procedure 4 is used for pre-freezing and vacuum drying.

[0091] The properties of the freeze-dried powder of Bifidobacterium bifidum F35 prepared in Example 3 are shown in Table 4.

[0092] Table 4 Performance of freeze-dried powder of Bifidobacterium bifidum F35 prepared by procedure 4

[0093]

[0094]

[0095] Example 4

[0096] A method for preparing freeze-dried powder of Bifidobacterium bifidum F35 is different from that of Example 1 in that procedure 5 is used for pre-freezing and vacuum drying.

[0097] The properties of the freeze-dried powder of Bifidobacterium bifidum F35 prepared in Example 4 are shown in Table 5.

[0098] Table 5 Performance of freeze-dried powder of Bifidobacterium bifidum F35 prepared by procedure 5

[0099] Fermentation broth viable count (CFU / mL) <![CDATA[3.0×10 9 ]]> Fermentation volume (L) 200 Mushroom mass (g) 4850 Protective agent addition ratio 1:2 Number of viable bacteria in bacterial suspension (CFU / mL) <![CDATA[4.5×10 10 ]]> Bacterial suspension volume (mL) 14550 Freeze-dried bacterial powder viable count (CFU / g) <![CDATA[1.8×10 11 ]]> Freeze-dried bacterial powder mass (g) 2551 Freeze-dried survival rate (%) 70.13 Freeze-dried bacterial powder moisture (%) 3.32

[0100] Example 5

[0101] A method for preparing freeze-dried powder of Bifidobacterium bifidum F35 is different from that of Example 1 in that procedure 6 is used for pre-freezing and vacuum drying.

[0102] The properties of the freeze-dried powder of Bifidobacterium bifidum F35 prepared in Example 5 are shown in Table 6.

[0103] Table 6 Performance of freeze-dried powder of Bifidobacterium bifidum F35 prepared by procedure 6

[0104]

[0105]

[0106] Example 6

[0107] A method for preparing freeze-dried bacterial powder of Bifidobacterium bifidum F35, which differs from Example 1 in that procedure 8 is used for pre-freezing and vacuum drying.

[0108] The properties of the freeze-dried powder of Bifidobacterium bifidum F35 prepared in Example 6 are shown in Table 7.

[0109] Table 7 Performance of freeze-dried powder of Bifidobacterium bifidum F35 prepared by procedure 8

[0110] Fermentation broth viable count (CFU / mL) <![CDATA[3.7×10 9 ]]> Fermentation volume (L) 200 Mushroom mass (g) 4750 Protective agent addition ratio 1:2 Number of viable bacteria in bacterial suspension (CFU / mL) <![CDATA[5.2×10 10 ]]> Bacterial suspension volume (mL) 14250 Freeze-dried bacterial powder viable count (CFU / g) <![CDATA[2.8×10 11 ]]> Freeze-dried bacterial powder mass (g) 2520 Freeze-dried survival rate (%) 96 Freeze-dried bacterial powder moisture (%) 3.54

[0111] Example 7

[0112] A method for preparing freeze-dried powder of Bifidobacterium bifidum F35 is different from that of Example 1 in that procedure 9 is used for pre-freezing and vacuum drying.

[0113] The properties of the freeze-dried powder of Bifidobacterium bifidum F35 prepared in Example 7 are shown in Table 8.

[0114] Table 8 Performance of freeze-dried powder of Bifidobacterium bifidum F35 prepared by procedure 9

[0115]

[0116]

[0117] Comparative Example 1

[0118] A method for preparing freeze-dried bacterial powder of Bifidobacterium bifidum F35, which is different from Example 1 in that the 200L fermentation tank is replaced by a 100L fermentation tank, the mixing mass ratio of bacterial sludge to protective agent is 1:5, and procedure 1 is used for pre-freezing and vacuum drying.

[0119] The properties of the freeze-dried powder of Bifidobacterium bifidum F35 prepared in Comparative Example 1 are shown in Table 9.

[0120] Table 9 Performance of freeze-dried powder of Bifidobacterium bifidum F35 prepared by procedure 1

[0121] Fermentation broth viable count (CFU / mL) <![CDATA[3.4×10 9 ]]> Fermentation volume (L) 100 Mushroom mass (g) 2125 Protective agent addition ratio 1:5 Number of viable bacteria in bacterial suspension (CFU / mL) <![CDATA[2.8×10 10 ]]> Bacterial suspension volume (mL) 12750 Freeze-dried bacterial powder viable count (CFU / g) <![CDATA[1.7×10 11 ]]> Freeze-dried bacterial powder mass (g) 2086 Freeze-dried survival rate (%) 99.33 Freeze-dried bacterial powder moisture (%) 3.65

[0122] Comparative Example 2

[0123] A method for preparing freeze-dried bacterial powder of Bifidobacterium bifidum F35, which differs from Example 1 in that procedure 7 is used for pre-freezing and vacuum drying.

[0124] The properties of the freeze-dried powder of Bifidobacterium bifidum F35 prepared in Comparative Example 2 are shown in Table 10.

[0125] Table 10 Performance of freeze-dried powder of Bifidobacterium bifidum F35 prepared by procedure 7

[0126]

[0127]

[0128] In the preparation process of the freeze-dried bacterial powder of the present invention, the temperature curve of the shelf, the temperature curve of the material (bacterial suspension), the temperature curve of the eutectic point of the material and the vacuum curve are as follows: Figure 1 As shown. During the freeze-drying process, the pre-frozen material is placed in a closed environment and evacuated to reduce the water vapor pressure on the surface of the material, allowing ice crystals to sublime directly under low pressure. The temperature is then raised in stages to increase the rate of sublimation of the ice crystals. During the temperature change process of the vacuum freeze-drying stage of the present invention, the material temperature is always lower than its eutectic point temperature. The eutectic point refers to the lowest melting point that different components in the material may form. If this temperature is exceeded, the ice crystals may melt or recrystallize, which will not only lead to the destruction of the material structure, but may also cause the material to further shrink, reduce the water removal efficiency, and even lead to freeze-drying failure. Figure 2 This is a photo of the prepared freeze-dried bacterial powder of Bifidobacterium bifidum F35.

[0129] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing freeze-dried powder of Bifidobacterium bifidum, characterized in that: The method comprises the following steps: expanding and culturing the Bifidobacterium bifidum strain to obtain a fermentation liquid, centrifuging to obtain bacterial mud, uniformly mixing the bacterial mud with a protective agent to obtain a bacterial suspension, prefreezing, vacuum freeze drying, and obtaining freeze-dried bacterial powder of Bifidobacterium bifidum; Among them, the conditions for vacuum freeze drying are to adopt a stage temperature change program: The first stage is to vacuum dry at -48 to -42°C for 0.2 to 0.6 hours; the second stage is to heat up to -13 to -10°C for 0.4 to 0.6 hours and vacuum dry for 2 to 5 hours; the third stage is to heat up to 6 to 9°C for 0.4 to 0.6 hours and vacuum dry for 6 to 8 hours; the fourth stage is to heat up to 31 to 34°C for 0.4 to 0.6 hours and vacuum dry for 10 to 13 hours; or, The first stage is vacuum drying at -48 to -42°C for 0.8 to 1.2 hours; the second stage is heating to -30 to -25°C for 0.4 to 0.6 hours and vacuum drying for 6 to 8 hours; the third stage is heating to -4 to -2°C for 0.4 to 0.6 hours and vacuum drying for 9 to 11 hours; the fourth stage is heating to 25 to 27°C for 0.4 to 0.6 hours and vacuum drying for 17 to 20 hours; or, The first stage is to vacuum dry at -48 to -42 °C for 0.8 to 1.2 hours; the second stage is to heat up to -42 to -38 °C for 0.4 to 0.6 hours and vacuum dry for 1 to 3 hours; the third stage is to heat up to -31 to -29 °C for 0.4 to 0.6 hours and vacuum dry for 7 to 9 hours; the fourth stage is to heat up to -4 to -2 °C for 0.4 to 0.6 hours and vacuum dry for 9 to 11 hours; the fifth stage is to heat up to 24 to 26 °C for 0.4 to 0.6 hours and vacuum dry for 19 to 21 hours; or, The first stage is to heat up to -27 to -23°C in 0.4 to 0.6 h and vacuum dry for 5 to 7 h. The second stage is to heat up to -4 to -2°C in 0.4 to 0.6 h and vacuum dry for 8 to 10 h. The third stage is to heat up to 27 to 29°C in 0.4 to 0.6 h and vacuum dry for 15 to 17 h.

2. The preparation method according to claim 1, characterized in that: At the end of the stage temperature change program, the vacuum degree is 0.2~0.4Pa.

3. The preparation method according to claim 1, characterized in that: Sterile food-grade culture medium was used for expansion culture; the conditions for expansion culture were 37°C and anaerobic culture.

4. The preparation method according to claim 1, characterized in that: The protective agent is a sterilized reconstituted skim milk aqueous solution with a concentration of 18 to 22 wt %; the mixing mass ratio of the bacterial mud to the protective agent is 1:1 to 1:3; the concentration of the bifidobacterium bifidum cells in the bacterial suspension is 3 to 7×10 10 CFU / mL.

5. The preparation method according to claim 1, characterized in that: The bacterial suspension is evenly spread and then pre-frozen and vacuum freeze-dried; the spreading thickness of the bacterial suspension is 1 to 1.2 cm; the pre-freezing temperature is lower than -45°C, and the pre-freezing time is 2 to 3 hours.

6. The preparation method according to claim 1, characterized in that: The freeze-dried powder of Bifidobacterium bifidum was crushed and passed through a 40-80 mesh sieve.

7. Freeze-dried powder of Bifidobacterium bifidum prepared by the preparation method according to any one of claims 1 to 6.

8. The freeze-dried powder of Bifidobacterium bifidum according to claim 7, characterized in that The moisture content of freeze-dried powder of Bifidobacterium bifidum is 3.30-3.54%, and the total number of viable bacteria of Bifidobacterium bifidum is ≥1.4×10 11 CFU / g.

9. Use of the freeze-dried powder of Bifidobacterium bifidum according to claim 7 in the field of food or health care products.

10. The use according to claim 9, characterized in that: The applications in the food field include applications in fermented foods, fermented dairy products, functional beverages, and functional foods; the applications in the health care product field include applications in probiotic capsules, tablets, and powders.

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