Preparation method of casein peptone for promoting growth of bifidobacterium longum and improving freeze-drying survival

Abstract

The invention discloses a preparation method of casein peptone for promoting growth of bifidobacterium longum and improving freeze-drying survival, and belongs to the technical field of microorganisms, and the preparation method comprises the following steps: sequentially hydrolyzing casein by using alkaline protease, chymotrypsin and flavourzyme under different conditions, collecting peptide liquid after passing through a ceramic membrane and ultrafiltration, and drying to obtain the casein peptone for promoting growth of bifidobacterium longum. The target product casein peptone CACF is prepared after freeze drying, the peptone is used as the sole nitrogen source to culture bifidobacterium longum, the growth of the bifidobacterium longum can be promoted, the viable count of each gram of dry bacterial powder is increased, and the method has a huge application prospect.

Description

Technical Field

[0001] The invention relates to a method for preparing casein peptone which can promote the growth of Bifidobacterium longum and improve freeze-drying survival, and belongs to the technical field of microorganisms. Background Art

[0002] Bifidobacterium longum is one of the main microorganisms in the human intestine and is widely recognized as a probiotic and used in lactic acid starter cultures or as probiotic products.

[0003] However, through the study of Bifidobacterium longum, scholars found that Bifidobacterium longum has certain limitations in the utilization of nitrogen sources. Different species of Bifidobacterium have their own specific nitrogen metabolism system, which is tantamount to increasing the difficulty of its cultivation. Bifidobacterium longum has weak osmotic pressure resistance, which limits its survival and proliferation ability in a high osmotic pressure environment. Studies have shown that high concentrations of metabolites and by-products may lead to increased osmotic pressure in the fermentation system, thereby inhibiting cell growth and affecting industrial production. In addition, during the vacuum freeze-drying process, the increase in electrolyte concentration in the unfrozen part of the solution will also produce osmotic stress and inhibit the activity of bifidobacteria. In view of the specific needs of Bifidobacterium longum for nitrogen sources and the limitations of its osmotic pressure resistance, it is particularly important to find a nitrogen source that Bifidobacterium longum prefers to use. By optimizing the nitrogen source, the culture density and freeze-drying survival rate of Bifidobacterium longum can be significantly improved, which is of great significance for improving the industrial production efficiency of Bifidobacterium longum.

[0004] Therefore, it is urgent to find a peptone suitable for the efficient proliferation of Bifidobacterium longum to increase the number of viable bacteria during the growth process of Bifidobacterium longum and the number of viable bacteria in the dry powder after freeze-drying. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for preparing casein peptone that promotes the growth of Bifidobacterium longum and improves freeze-dried survival, aiming to solve the technical problem in the prior art that there is a lack of peptone as a nitrogen source that can increase the number of viable bacteria during the growth of Bifidobacterium longum and the number of viable bacteria in the dry powder after freeze-drying.

[0006] The first technical solution provided by the present invention is a method for preparing casein peptone that promotes the growth of Bifidobacterium longum and improves freeze-dried survival. The method comprises preheating the casein, hydrolyzing it in sequence with alkaline protease, chymotrypsin and flavor protease, and then filtering it with a ceramic membrane and an ultrafiltration membrane to obtain the filtrate, which is the casein peptone.

[0007] In certain embodiments, the method comprises the steps of:

[0008] (1) casein and water are mixed thoroughly at a concentration of 5-7%, and the temperature is raised to 60°C for preheating for 10 minutes after the casein and water are mixed; then the temperature is lowered to 50°C for preservation, and after the temperature stabilizes, 1 mol / L NaOH is added to adjust the pH to 8-9, and then 2000-4000 U / g pro-alkaline protease is added to perform enzymolysis under constant temperature and constant pH conditions for 2-4 hours;

[0009] (2) When the hydrolysis in step (1) is completed, the temperature is raised to 95 degrees Celsius to inactivate the enzyme and maintained for 10 to 15 minutes. After completion, the temperature is lowered to 50 degrees Celsius, 1 mol / L HCL is added, the pH is adjusted to 7 to 8, and then 2000 to 4000 U / g pro-chymotrypsin is added. The enzymatic hydrolysis is continued for 2 to 4 hours at a constant pH of 7 to 8 and a temperature of 50 degrees Celsius.

[0010] (3) When the hydrolysis in step (2) is completed, the temperature is raised to 95 degrees Celsius to inactivate the enzyme and maintained for 10 to 15 minutes. After the hydrolysis is completed, the temperature is lowered to 50 degrees Celsius, 1 mol / L HCL is added, the pH is adjusted to 6 to 7, and then 2000 to 4000 U / g pro-flavor protease is added. The enzymatic hydrolysis is continued for 2 to 4 hours at a constant pH of 6 to 7 and a temperature of 50 degrees Celsius.

[0011] (4) After the reaction is completed, the hydrolyzate in step (3) is coarsely filtered through a 0.22 μm ceramic membrane to remove unhydrolyzed protein and enzymes in the reaction system, and the permeate is collected.

[0012] (5) The permeate collected in step (4) is ultrafiltered using an organic ultrafiltration membrane with a cutoff of 3000 Da, and the filtrate is collected.

[0013] (6) The filtrate collected in step (5) is vacuum freeze-dried to obtain casein peptone CACF dry powder.

[0014] In certain embodiments, the Bifidobacterium longum includes but is not limited to Bifidobacterium longum CCFM687 and Bifidobacterium longum CCFM1029. The strain accession number of the Bifidobacterium longum CCFM687 is GDMCC No.60387, which has been disclosed in the patent document CN109055269B. The strain accession number of the Bifidobacterium longum CCFM1029 is CGMCC NO.60461, which has been disclosed in the patent document CN1 15887504B.

[0015] The second technical solution provided by the present invention is casein peptone prepared by the method described in the first technical solution.

[0016] The third technical solution provided by the present invention is a culture medium, wherein the culture medium uses the casein peptone described in the second technical solution as the sole nitrogen source.

[0017] In certain embodiments, the concentration of casein peptone in the culture medium of the second technical solution is 1-2 g / L.

[0018] In certain embodiments, the culture medium further contains a nitrogen source, glucose, KH 2 PO 4 、NaH 2 PO 4 MgSO 4 7H 2 O、MnSO 4 ·H 2 O, Tween 80, cysteine ​​hydrochloride.

[0019] Furthermore, the culture medium also contains 10 g / L glucose, KH 2 PO 4 7g / L, NaH 2 PO 4 7g / L, MgSO 4 7H 2 O0.1g / L, MnSO 4 ·H 2 O0.05g / L, Tween 801mL / L, cysteine ​​hydrochloride 1g / L.

[0020] The fourth technical solution provided by the present invention is a culture method for promoting the growth of Bifidobacterium longum and improving the freeze-drying survival rate, wherein the method comprises inoculating Bifidobacterium longum into a culture system using casein peptone as the sole nitrogen source as described in the second technical solution for culture.

[0021] In certain embodiments, the concentration of casein peptone in the culture system of the second technical solution is 1-2 g / L.

[0022] In some embodiments, the culture system further comprises a nitrogen source, glucose, KH 2 PO 4 、NaH 2 PO 4 MgSO 4 7H 2 O、MnSO 4 ·H 2 O, Tween 80, cysteine ​​hydrochloride.

[0023] Furthermore, the culture system also contains 10 g / L glucose, KH 2 PO 4 7g / L, NaH 2 PO 4 7g / L, MgSO 4 7H 2 O 0.1g / L, MnSO4 ·H 2 O 0.05g / L, Tween 801mL / L, cysteine ​​hydrochloride 1g / L.

[0024] In certain embodiments, Bifidobacterium longum is inoculated into the culture system at an inoculation rate of 2-4%, and anaerobically cultured at 37°C for at least 12 hours.

[0025] In certain embodiments, the Bifidobacterium longum includes but is not limited to Bifidobacterium longum CCFM687 and Bifidobacterium longum CCFM1029. The strain accession number of the Bifidobacterium longum CCFM687 is GDMCC No.60387, which has been disclosed in the patent document CN109055269B. The strain accession number of the Bifidobacterium longum CCFM1029 is CGMCC NO.60461, which has been disclosed in the patent document CN1 15887504B.

[0026] The technical effects of the present invention are as follows:

[0027] Compared with the traditional peptide preparation method, after hydrolysis, compared with the traditional process: inactivate the enzyme - adjust the isoelectric point - centrifuge to remove the protein, the direct use of ceramic membrane plus ultrafiltration membrane is a simple and fast process.

[0028] The present invention adopts a composite enzyme hydrolysis method, and the small peptide content of the obtained peptone is higher through the combined use of different enzymes.

[0029] The present invention provides a casein peptone CACF for promoting the growth of Bifidobacterium longum. In a nitrogen source screening medium, Bifidobacterium longum is cultured at an addition amount of 1 g / L for 12 h, and its ΔOD 600 The values ​​of viable bacteria per gram of dry powder of Bifidobacterium longum CCFM687 and CCFM1029 cultured in CACF were (5.07±0.15)×10-1, and the values ​​of viable bacteria per gram of dry powder of Bifidobacterium longum CCFM687 and CCFM1029 were (5.07±0.15)×10-1, respectively. 11 CFU and (8.62±0.62)×10 11 CFU. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the OD of Bifidobacterium longum cultured under different nitrogen sources of the present invention 600 picture.

[0031] Figure 2 The figure is a graph showing the freeze-dried survival rate and the number of live bacteria per gram of dry bacterial powder of Bifidobacterium longum cultured under different nitrogen sources of the present invention. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0033] Test method:

[0034] Growth evaluation: Refer to the method described in patent CN109913523B and use a UV spectrophotometer to measure the OD of the strain under different nitrogen sources. 600 .

[0035] Freeze-drying evaluation: Different nitrogen sources were added to the nitrogen source screening medium at a concentration of 2g / L, and the activated bacterial solution was inoculated at a 2% inoculum to culture two strains of Bifidobacterium longum. The fermentation broth was collected at 12h, and the cells were collected by centrifugation (8000g / 20min). After the cells were washed twice with cysteine ​​buffer, 20% sorbitol was added as a protective agent and mixed with the bacterial mud at a mass ratio of 2:1. 0.5ml was taken for pre-freeze-drying counting, and then 1ml was taken and put into a cillin bottle and the weight of the wet bacterial mud was recorded, and it was placed in a freeze dryer for freeze-drying. Cysteine ​​buffer was added to the freeze-dried sample to restore it to the weight before freeze-drying, and then 0.5ml was taken for post-freeze-drying counting, and compared with the number of live bacteria before freeze-drying, and the freeze-drying survival rate was calculated.

[0036] The raw materials used in the examples are:

[0037] In the following examples, acid hydrolyzed casein was purchased from Sanmenxia Gaorui Biotechnology Co., Ltd.; tryptic peptone (sigma) was purchased from Merck Life Sciences; casein was purchased from Beijing Bailingwei Technology Co., Ltd., whey protein was purchased from Shanghai Bofei Meike Chemical Technology Co., Ltd., and gluten was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; alkaline protease (Solaibao, alkaline protease model B8361, enzyme activity definition: under the measurement conditions (40°C, pH 10.5), the absorbance of trichloroacetic acid soluble matter released by casein hydrolysis per minute at a wavelength of 275nm is equivalent to the absorbance of 1 microgram of tyrosine, the required enzyme amount is one activity unit, expressed in u / g), chymotrypsin (Solaibao, chymotrypsin model C866 0, enzyme activity definition: under the assay conditions (40°C, pH 10.5), the absorbance of the trichloroacetic acid soluble matter released by the hydrolysis of casein per minute at a wavelength of 275 nm is equivalent to the absorbance of 1 μg of tyrosine, the required amount of enzyme is one activity unit, expressed in u / g), flavor protease (Solabo, flavor protease model F8270, enzyme activity definition: under the assay conditions (40°C, pH 6.5), the absorbance of the trichloroacetic acid soluble matter released by the hydrolysis of casein per minute at a wavelength of 275 nm is equivalent to the absorbance of 1 μg of tyrosine, the required amount of enzyme is one activity unit, expressed in u / g), purchased from Beijing Solab Technology Co., Ltd.; other reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0038] The Bifidobacterium longum CCFM687 and CCFM1029 involved in the following examples are preserved by the Biotechnology Center of the School of Food Science and Engineering of Jiangnan University. The strain preservation number of the Bifidobacterium longum CCFM687 is GDMCC No.60387, which has been disclosed in the patent document CN109055269B, and the strain preservation number of the Bifidobacterium longum CCFM1029 is CGMCC NO.60461, which has been disclosed in the patent document CN1 15887504B.

[0039] The culture medium involved in the following examples is as follows:

[0040] MRS-L liquid culture medium: tryptone (LP0042B) 10g / L, yeast extract powder (LP0021B), beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, diammonium hydrogen citrate 2g / L, K 2 HPO 4 ·3H 2 O2.6g / L, MgSO 4 7H 2 O0.1g / L, MnSO 4 ·H 2 O0.05g / L, Tween 801mL / L, cysteine ​​hydrochloride 1g / L, high pressure sterilization at 115℃ for 20min.

[0041] Solid MRS-L medium: Add 1.5% agar powder to the liquid MRS-L medium and sterilize by high pressure at 115℃ for 20min. After sterilization and cooling to 50-60℃, pour 15mL of the medium into each culture dish, and after solidification, wrap it with plastic wrap and store it in a refrigerator at 4℃ for later use.

[0042] Nitrogen source screening medium: nitrogen source 2g / L, glucose 10g / L, KH 2 PO 4 7g / L, NaH 2 PO 4 7g / L, MgSO 4 7H 2 O0.1g / L, MnSO 4 ·H 2 O0.05g / L, Tween 80 1mL / L, cysteine ​​hydrochloride 1g / L, pH 6.2-6.4, sterilize at 115℃ for 20min.

[0043] Cysteine ​​buffer: cysteine ​​hydrochloride 0.5 g / L, KH 2 PO 4 6.0g / L, Na 2 HPO 4 4.5g / L, NaCl 4.0g / L, Tween 80 0.6g / L, pH 6.8~7.0, sterilization at 115℃ for 20min.

[0044] Example 1: Preparation of casein peptone CACF that can promote the growth of Bifidobacterium longum

[0045] In this embodiment, casein peptone CACF which can promote the growth of Bifidobacterium longum is prepared by enzymatic hydrolysis.

[0046] The specific operations are as follows:

[0047] Step 1: Mix casein and water thoroughly at a concentration of 7%. After the casein and water are mixed, raise the temperature to 60°C for preheating for 10 minutes; then cool to 50°C for insulation. After the temperature stabilizes, add 1 mol / L NaOH to adjust the pH to 8.5, then add 4000U / g pro-alkaline protease for enzymolysis at constant temperature and pH for 2 hours;

[0048] Step 2: When the hydrolysis in step 1 is completed, the temperature is raised to 95 degrees Celsius to inactivate the enzyme and maintained for 10 to 15 minutes. After the end, the temperature is lowered to 50°C, 1 mol / L HCL is added, the pH is adjusted to 7.5, and then 4000U / g pro-chymotrypsin is added. The enzymatic hydrolysis is continued for 2 hours at a constant pH of 7.5 and a temperature of 50°C.

[0049] Step 3: When the hydrolysis in step 2 is completed, the temperature is raised to 95 degrees Celsius to inactivate the enzyme and maintained for 10 to 15 minutes. After the end, the temperature is lowered to 50°C, 1 mol / L HCL is added, the pH is adjusted to 6.5, and 4000U / g pro flavor protease is added. The enzymatic hydrolysis is continued for 2 hours at a constant pH of 6.5 and a temperature of 50°C.

[0050] Step 4: After the reaction is completed, the hydrolyzate in step 3 is coarsely filtered through a 0.22 μm ceramic membrane to remove unhydrolyzed protein and enzymes in the reaction system, and the permeate is collected.

[0051] Step 5: Ultrafiltration is performed on the permeate collected in step 4 using an organic ultrafiltration membrane with a cutoff of 3000Da, and the filtrate is collected.

[0052] Step 6: The filtrate collected in step 5 is vacuum freeze-dried to obtain casein peptone CACF dry powder.

[0053] Step 7: Determine the molecular weight distribution of the sample, and use gel exclusion chromatography to determine the molecular weight of the peptide. The chromatographic column is TSKgel 2000SWXL (300×7.8mm), the mobile phase is acetonitrile-water-trifluoroacetic acid (45:55:0.1), the flow rate is 0.5mL / min, and the UV detection wavelength is 220nm. The relative percentage of peptides in different molecular weight ranges is obtained by peak area normalization.

[0054] In this embodiment, the oligopeptides with molecular weight of 180-3000Da account for 87.07% of the hydrolyzate after enzymatic hydrolysis, small peptides with molecular weight between 180-1000Da account for 76.06%, and free amino acids with molecular weight less than 180Da account for 10.43%.

[0055] Example 2: Determination of the growth of Bifidobacterium longum under casein peptone CACF

[0056] In this example, the growth of Bifidobacterium longum under CACF culture was determined experimentally, and the selected Bifidobacterium longum were Bifidobacterium longum CCFM687 and Bifidobacterium longum CCFM1029.

[0057] Step 1: Take out the bacteria preservation tube stored at -80℃, wait for it to thaw, use an inoculation loop to take a small amount of bacterial solution and streak it on the MRS-L solid culture medium, and culture it at 37℃ in an anaerobic workstation for 48 hours. After the colonies grow, pick a single colony and inoculate it into a 5mL MRS-L liquid tube, culture it anaerobically for 18h~24h, and repeat the above operation 2-3 times to obtain the activated bacterial solution.

[0058] Step 2: Add CACF at a concentration of 1 g / L to the nitrogen source screening medium, culture two strains of Bifidobacterium longum, inoculate the activated bacterial solution at a 2% inoculation rate, and measure the ΔOD after 12 h of culture. 600 .

[0059] Test results see Figure 1 , CCFM687 and CCFM1029 were cultured in casein peptone CACF, ΔOD 600 They are 0.52±0.02 and 0.77±0.00 respectively.

[0060] Example 3: Determination of freeze-drying of Bifidobacterium longum under casein peptone CACF

[0061] In this embodiment, the freeze-dried survival rate of Bifidobacterium longum cultured in CACF was determined by experiment. The selected Bifidobacterium longum were Bifidobacterium longum CCFM687 and Bifidobacterium longum CCFM1029.

[0062] The specific operations are as follows:

[0063] Step 1: Take out the bacteria preservation tube stored at -80℃, wait for it to thaw, use an inoculation loop to take a small amount of bacterial solution and streak it on the MRS-L solid culture medium, and culture it at 37℃ in an anaerobic workstation for 48 hours. After the colonies grow, pick a single colony and inoculate it into a 5mL MRS-L liquid tube, culture it anaerobically for 18h~24h, and repeat the above operation 2-3 times to obtain the activated bacterial solution.

[0064] Step 2: CACF was added to the nitrogen source screening medium at a concentration of 2 g / L, and the activated bacterial solution was inoculated at a 2% inoculation rate to culture two strains of Bifidobacterium longum respectively.

[0065] Step 3: Collect the fermentation broth at 12 hours, collect the bacteria by centrifugation (8000g / 20min), wash the bacteria twice with cysteine ​​buffer, add 20% sorbitol as a protective agent to the bacterial mud in a mass ratio of 2:1 and mix well, take 0.5ml for counting before lyophilization, then take 1ml and put it into a penicillin bottle and record the weight of the wet bacterial mud, and place it in a freeze dryer for lyophilization.

[0066] Step 4: Add cysteine ​​buffer to the freeze-dried sample to restore the weight to the weight before freeze-drying, then take 0.5 ml for post-freeze-drying counting, compare with the number of viable bacteria before freeze-drying, and calculate the freeze-drying survival rate.

[0067] Test results see Figure 2 The survival rates of CCFM687 and CCFM1029 in casein peptone CACF culture were 32.27±0.02% and 28.25±1.66%, respectively. The viable counts per gram weight of dry powder of the two strains were (5.07±0.15)×1011 CFU and (8.62±0.62)×10 11 CFU.

[0068] Comparative Example 1: Growth of Bifidobacterium longum under different nitrogen sources

[0069] According to the method of Example 2, the nitrogen source was replaced with imported tryptone (sigma), acid hydrolyzed casein peptone, one whey protein hydrolyzed peptone (WA), two homemade hydrolyzed casein peptones (CAF, CATF) and three homemade gluten peptones (GAF, GATF, GAPF), and the two strains were cultured with them respectively, and the ΔOD of the strains after 12 h of growth under these two nitrogen sources was measured. 600 .

[0070] Preparation method of whey protein hydrolyzate WA: Step 1: Mix whey protein and water at a concentration of 7%, raise the temperature to 60°C for preheating for 10 minutes after the protein and water are mixed; then cool to 50°C for insulation, add 1mol / LNaOH to adjust the pH to 8.5 after the temperature stabilizes, then add 4000U / g pro alkaline protease for enzymolysis under constant temperature and constant pH conditions for 2 hours; Step 2: After the reaction is completed, the hydrolyzate in step 1 is coarsely filtered through a 0.22μm ceramic membrane to remove unhydrolyzed protein and enzymes in the reaction system, and the permeate is collected. Step 3: The filtrate collected in step 2 is vacuum freeze-dried to obtain WA dry powder.

[0071] Preparation method of homemade hydrolyzed casein peptone CAF and CATF: The preparation method is the same as that of Example 1, except that the raw protein used is casein, the enzymes used for CAF are alkaline protease and flavor protease, and the enzymes used for CATF are alkaline protease and trypsin.

[0072] Preparation method of homemade hydrolyzed gluten peptone GAF, GATF, GAPF: The preparation method is the same as Example 1, except that the raw protein used is gluten, the enzymes used for GAF are alkaline protease and flavor protease, the enzymes used for GATF are alkaline protease and trypsin, and the enzymes used for GAPF are alkaline protease and papain.

[0073] Test results see Figure 1 From the results, it can be seen that under the same amount of nitrogen source added, the effects of imported trypsin (sigma), commercial acid-hydrolyzed casein and several homemade hydrolyzed peptones on the growth of Bifidobacterium longum are significantly worse than that of CACF in Example 2.

[0074] Comparative Example 2: Freeze-drying of Bifidobacterium longum under different nitrogen sources

[0075] According to the method of Example 3, the nitrogen source was replaced with imported trypsin (sigma), acid hydrolyzed casein peptone and 1 whey protein hydrolyzed peptone (WA), 2 homemade hydrolyzed casein peptones (CAF, CATF, CAPF) and 3 homemade gluten peptones (GAF, GATF, GAPF), and the two strains were cultured respectively with them, and the freeze-dried survival rate of the strains after growth for 12 hours under the two nitrogen sources and the number of viable bacteria per unit dry bacterial powder were determined.

[0076] Test results see Figure 2 For CCFM687, the survival rate of bacteria cultured in homemade casein peptone GAF and WA reached 44.93±0.94% and 50.07±2.58%, respectively, but the number of viable bacteria per gram of dry powder was (3.95±0.08)×10 11 CFU and (4.46±0.29)×10 11 The CFU was lower than that of casein peptone CACF. The freeze-dried survival rate of the strains cultured in homemade peptone CAF and GAPF was not significantly different from that in CACF, but the live bacterial count of dry powder was much lower than that of CACF.

[0077] For CCFM1029, the survival rates of freeze-dried cells were 41.67±0.34% and 51.45±1.67% under homemade casein peptone GAF and WA culture, respectively, but the number of viable bacteria per gram of dry powder was only (6.54±0.05)×10 11 CFU and (4.72±0.25)×10 11 The CFU was lower than that of casein peptone CACF.

[0078] Under the culture conditions of imported commercial tryptone (sigma) and acid-hydrolyzed casein, the freeze-drying effect and the number of live bacteria in dry powder from CCFM687 and CCFM1029 were significantly worse than those from CACF culture.

[0079] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing casein peptone that promotes the growth of Bifidobacterium longum and improves freeze-drying survival, characterized in that: The method comprises the following steps: preheating casein, hydrolyzing it with alkaline protease, chymotrypsin and flavor protease in sequence, filtering it with ceramic membrane and ultrafiltration membrane, and obtaining the filtrate, namely casein peptone.

2. The method according to claim 1, characterized in that The method comprises the following steps: (1) After casein and water are fully mixed at a concentration of 5-7%, the temperature is raised to 60°C for preheating for 10 minutes; then the temperature is lowered to 50°C for preservation, 1 mol / L NaOH is added to adjust the pH to 8-9, and then 2000-4000 U / g pro-alkaline protease is added to perform enzymolysis under constant temperature and pH conditions for 2-4 hours; (2) After the hydrolysis in step (1) is completed, the temperature is raised to 95 degrees Celsius to inactivate the enzyme and maintained for 10 to 15 minutes. After the hydrolysis is completed, the temperature is lowered to 50 degrees Celsius, 1 mol / L HCL is added, the pH is adjusted to 7 to 8, and then 2000 to 4000 U / g pro-chymotrypsin is added. The enzymatic hydrolysis is continued for 2 to 4 hours at a constant pH of 7 to 8 and a temperature of 50 degrees Celsius; (3) When the hydrolysis in step (2) is completed, the temperature is raised to 95 degrees Celsius to inactivate the enzyme and maintained for 10 to 15 minutes. After the hydrolysis is completed, the temperature is lowered to 50 degrees Celsius, 1 mol / L HCL is added, the pH is adjusted to 6 to 7, and then 2000 to 4000 U / g pro-flavor protease is added. The enzymatic hydrolysis is continued for 2 to 4 hours at a constant pH of 6 to 7 and a temperature of 50 degrees Celsius; (4) After the reaction is completed, the hydrolyzate in step (3) is coarsely filtered through a 0.22 μm ceramic membrane to remove unhydrolyzed protein and enzymes in the reaction system, and the permeate is collected; (5) ultrafiltration the permeate collected in step (4) using an organic ultrafiltration membrane with a cutoff of 3000 Da, and collecting the filtrate; (6) The filtrate collected in step (5) is vacuum freeze-dried to obtain casein peptone CACF dry powder.

3. The method according to claim 1, characterized in that The Bifidobacterium longum includes but is not limited to Bifidobacterium longum CCFM687 and Bifidobacterium longum CCFM1029. The strain collection number of the Bifidobacterium longum CCFM687 is GDMCC No.60387, and the strain collection number of the Bifidobacterium longum CCFM1029 is CGMCC NO.60461.

4. Casein peptone prepared by the method according to any one of claims 1 to 3.

5. A culture medium, characterized in that The culture medium uses the casein peptone described in claim 4 as the sole nitrogen source, and the concentration of the casein peptone in the culture medium is 1 to 2 g / L.

6. The culture medium according to claim 5, characterized in that The culture medium also contains a nitrogen source, glucose, KH2PO4, NaH2PO4, MgSO4·7H2O, MnSO4·H2O, Tween 80, and cysteine ​​hydrochloride; optionally, the culture medium also contains 10 g / L glucose, 7 g / L KH2PO4, 7 g / L NaH2PO4, 0.1 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, 1 mL / L Tween 80, and 1 g / L cysteine ​​hydrochloride.

7. A method for promoting the growth of Bifidobacterium longum and improving the freeze-drying survival rate, characterized in that: The method comprises inoculating Bifidobacterium longum into a culture system using the casein peptone described in claim 4 as the sole nitrogen source for culture, wherein the concentration of the casein peptone in the culture medium is 1 to 2 g / L.

8. The method according to claim 7, characterized in that The culture system also contains a nitrogen source, glucose, KH2PO4, NaH2PO4, MgSO4·7H2O, MnSO4·H2O, Tween 80, and cysteine ​​hydrochloride; optionally, the culture medium also contains 10 g / L glucose, 7 g / L KH2PO4, 7 g / L NaH2PO4, 0.1 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, 1 mL / L Tween 80, and 1 g / L cysteine ​​hydrochloride.

9. The method according to claim 7, characterized in that: Bifidobacterium longum was inoculated into the culture system at an inoculation rate of 2-4%, and cultured anaerobically at 37°C for at least 12 hours.

10. The method according to claim 7, characterized in that The Bifidobacterium longum includes but is not limited to Bifidobacterium longum CCFM687 and Bifidobacterium longum CCFM1029. The strain collection number of the Bifidobacterium longum CCFM687 is GDMCC No. 60387, and the strain collection number of the Bifidobacterium longum CCFM1029 is CGMCC No. 60461.

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