Preparation method of whey peptone for promoting growth of bifidobacterium breve and improving freeze-drying survival

Abstract

The invention discloses a preparation method of whey peptone for promoting growth of bifidobacterium breve and improving freeze-drying survival rate, which belongs to the technical field of microorganisms, and comprises the following steps: hydrolyzing whey protein with papain and alkaline protease in sequence, collecting peptide liquid after passing through a ceramic membrane and ultrafiltration, and freeze-drying to prepare a target product whey peptone WPP, when the peptone is used as the only nitrogen source for culturing bifidobacterium breve, the growth of the bifidobacterium breve can be promoted, the freeze-drying survival rate of the bifidobacterium breve and the viable count of each gram of dry powder after freeze-drying are improved, and the application prospect is huge.

Description

Technical Field

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

[0002] Bifidobacterium breve has been reported to have a variety of physiological functions, such as specific strains can regulate the balance of intestinal flora, promote the body's absorption of nutrients, immune regulation, anti-aging, etc. In recent years, the probiotic properties of Bifidobacterium breve have become well known, and therefore have been widely used in food, animal feed, health care and other fields.

[0003] Studies have shown that one of the key factors for the growth of lactic acid bacteria is the utilization of nitrogen sources. Lactic acid bacteria, especially bifidobacteria, as multi-amino acid auxotrophic strains, cannot directly utilize inorganic nitrogen sources and exogenous nitrogen sources. They must degrade exogenous nitrogen sources to ensure that the strains can meet the needs of amino acids in the normal growth and metabolism process. Different bifidobacteria produce their own gene mutations and gene level transfer in the evolution process to adapt to their own growth environment, so their nitrogen source utilization system has strong strain specificity. For Bifidobacterium breve, its osmotic pressure resistance is weak, so it will inhibit the final proliferation concentration of the strain. At the same time, it is also a cysteine ​​auxotrophic type, so too much inappropriate nitrogen source exists in the growth environment and is not utilized, which will increase its growth difficulty and reduce the number of live bacteria. Based on this, we need to find the nitrogen source that Bifidobacterium breve prefers to use. On the one hand, it can increase the number of live bacteria, and on the other hand, it can improve the osmotic pressure resistance of the strain and thus increase the freeze-dried survival rate and the number of live bacteria in dry powder, which is of great significance to improving the industrialization level of Bifidobacterium breve.

[0004] Therefore, there is an urgent need to find a peptone suitable for the efficient proliferation of Bifidobacterium breve to increase the number of live bacteria during the growth of Bifidobacterium breve and the survival rate during the freeze-drying process. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing whey peptone that promotes the growth of Bifidobacterium breve and improves the freeze-drying survival rate, aiming to solve the technical problem in the prior art that there is a lack of peptone that can increase the number of viable bacteria in the growth process of Bifidobacterium breve and the survival rate in the freeze-drying process.

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

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

[0008] (1) Mixing whey protein and water at a concentration of 5-7%, raising the temperature to 60°C for preheating for 10 min; then cooling to 50°C for insulation, adding 1 mol / L NaOH to adjust the pH to 6-7 after the temperature stabilizes, and then adding 2000-4000 U / g propapain for enzymolysis at constant temperature and pH for 2-4 h;

[0009] (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 NaOH is added, the pH is adjusted to 8 to 9.5, and then 2000 to 4000 U / g pro-alkaline protease is added. The enzymatic hydrolysis is continued for 2 to 4 hours at a constant pH of 8 to 9.5 and a temperature of 50 degrees Celsius;

[0010] (3) After the reaction is completed, the hydrolyzate in step (2) 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;

[0011] (4) ultrafiltration the permeate collected in step (3) using an organic ultrafiltration membrane with a cutoff of 3000 Da, and collecting the filtrate;

[0012] (5) The filtrate collected in step (4) is vacuum freeze-dried to obtain whey peptone WPP dry powder.

[0013] In certain embodiments, the Bifidobacterium breve includes but is not limited to Bifidobacterium breve CCFM1026 and Bifidobacterium breve CCFM1078. The strain accession number of the Bifidobacterium breve CCFM1026 is CGMCC NO.60459, which has been disclosed in the patent document CN109576185B. The strain accession number of the Bifidobacterium breve CCFM1078 is CGMCC NO.61011, which has been disclosed in the patent document CN112111424A.

[0014] The second technical solution provided by the present invention is a whey peptone prepared by the method described in the first technical solution.

[0015] The third technical solution provided by the present invention is a culture medium, wherein the culture medium uses the whey peptone described in the second technical solution as the sole nitrogen source.

[0016] In certain embodiments, the concentration of peptone in the culture medium of the second technical solution is 1-2 g / L.

[0017] In certain embodiments, the culture medium further contains a nitrogen source, glucose, KH2 PO 4 、NaH 2 PO 4 MgSO 4 7H 2 O、MnSO 4 ·H 2 O, Tween 80, cysteine ​​hydrochloride.

[0018] 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.

[0019] The fourth technical solution provided by the present invention is a culture method for promoting the growth of Bifidobacterium breve and improving the freeze-drying survival rate, wherein the method comprises inoculating Bifidobacterium breve into a culture system using the whey peptone described in the second technical solution as the sole nitrogen source for culture.

[0020] In certain embodiments, the concentration of whey peptone in the culture system of the second technical solution is 1-2 g / L.

[0021] 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.

[0022] 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 O0.1g / L, MnSO 4 ·H 2 O0.05g / L, Tween 801mL / L, cysteine ​​hydrochloride 1g / L.

[0023] In certain embodiments, Bifidobacterium breve is inoculated into the culture system at an inoculation rate of 2-4%, and anaerobically cultured at 37°C for at least 12 hours.

[0024] In certain embodiments, the Bifidobacterium breve includes but is not limited to Bifidobacterium breve CCFM1026 and Bifidobacterium breve CCFM1078. The strain accession number of the Bifidobacterium breve CCFM1026 is CGMCC NO.60459, which has been disclosed in the patent document CN109576185B. The strain accession number of the Bifidobacterium breve CCFM1078 is CGMCC NO.61011, which has been disclosed in the patent document CN112111424A.

[0025] The technical effects of the present invention are as follows:

[0026] Compared with the traditional peptide preparation method, after hydrolysis, compared with the traditional process: inactivate enzymes - adjust isoelectric point - centrifuge to remove proteins, the direct use of ceramic membrane plus ultrafiltration membrane is a simple and fast process.

[0027] The present invention adopts a composite enzyme hydrolysis method, and the obtained peptone oligopeptide content is higher through the combined use of different enzymes.

[0028] The invention provides a whey peptone WPP for promoting the growth of Bifidobacterium breve. In a nitrogen source screening medium, Bifidobacterium breve is cultured at an addition amount of 1 g / L for 12 h, and its ΔOD 600 The results showed that the survival rates of Bifidobacterium breve CCFM1026 and CCFM1078 cultured with WPP were 32.27% and 28.25%, respectively, and the number of viable bacteria per gram of dry powder reached (1.64±0.09)×10 12 CFU and (2.21±0.13)×10 12 CFU, significantly higher than other nitrogen sources. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the OD of Bifidobacterium breve cultured under different nitrogen sources of the present invention 600 picture.

[0030] 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 breve cultured under different nitrogen sources of the present invention.

[0031] Figure 3 is the OD of Bifidobacterium breve cultured under different whey peptones of the present invention 600 picture. 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] The raw materials used in the examples are:

[0034] In the following examples, acid-hydrolyzed casein was purchased from Sanmenxia Gaorui Biotechnology Co., Ltd.; tryptic peptone (sigma) was purchased from Merck Life Sciences; whey peptone (Oxoid LP0048) was purchased from Thermo Fisher Scientific Inc.; 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, neutral protease, flavor protease, and papain were purchased from Beijing Solebow Technology Co., Ltd.; trypsin was purchased from Sinopharm Chemical Reagent Co., Ltd.; the remaining reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0035] The Bifidobacterium breve CCFM1026 and CCFM1078 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 Bifidobacterium breve CCFM1026 is CGMCC NO.60459, which has been disclosed in the patent document CN109576185B, and the strain preservation number of Bifidobacterium breve CCFM1078 is CGMCC NO.61011, which has been disclosed in the patent document CN112111424A.

[0036] The culture medium involved in the following examples is as follows:

[0037] 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.

[0038] 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.

[0039] 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.

[0040] Cysteine ​​buffer: cysteine ​​hydrochloride 0.5 g / L, KH 2 PO 4 6.0g / L, Na2HPO44.5g / L, NaCl4.0g / L, Tween 80 0.6g / L, pH 6.8~7.0, sterilization at 115℃ for 20min.

[0041] The enzymes involved in the following examples are:

[0042] Flavor protease: Model F8270 is from Solebao. Enzyme activity definition: Under the test conditions (40℃, pH 6.5), when the absorbance of trichloroacetic acid soluble matter released by hydrolyzing casein per minute at a wavelength of 275nm is equivalent to the absorbance of 1 microgram of tyrosine, the required amount of enzyme is one activity unit, expressed as U / g.

[0043] Papain: Model G8430 is from Solebol. Enzyme activity is defined as follows: When the absorbance of trichloroacetic acid-soluble matter released by casein hydrolysis per minute at a wavelength of 275 nm is equivalent to the absorbance of 1 microgram of tyrosine under the assay conditions (37±0.2°C; pH 7.0), the amount of enzyme required is one activity unit, expressed as U / g.

[0044] Alkaline protease: Model B8361 is from Solebol. Enzyme activity is defined as follows: When the absorbance of trichloroacetic acid-soluble matter released by hydrolyzing casein per minute under the measuring conditions (40°C, pH 10.5) at a wavelength of 275 nm is equivalent to the absorbance of 1 microgram of tyrosine, the amount of enzyme required is one activity unit, expressed as U / g.

[0045] Trypsin: Model 69024138 is from Sinopharm. Enzyme activity definition: Under the measurement conditions (37°C, pH 8), when the absorbance of the trichloroacetic acid soluble matter released by hydrolyzing casein per minute at a wavelength of 275 nm is equivalent to the absorbance of 1 microgram of tyrosine, the required amount of enzyme is one activity unit, expressed as U / g.

[0046] Neutral protease model Z8032 comes from Solebo. Enzyme activity is defined as follows: when the absorbance of trichloroacetic acid soluble matter released by hydrolyzing casein per minute under the measurement conditions (40°C, pH 7) at a wavelength of 275 nm is equivalent to the absorbance of 1 microgram of tyrosine, the amount of enzyme required is one activity unit, expressed as U / g.

[0047] Example 1: Preparation of whey peptone WPP that can promote the growth of Bifidobacterium breve

[0048] In this embodiment, whey peptone WPP which can promote the growth of Bifidobacterium breve is prepared by enzymatic hydrolysis.

[0049] The specific operations are as follows:

[0050] Step 1: Mix whey protein and water at a concentration of 7%. After the protein and water are mixed, raise the temperature to 60°C and preheat for 10 minutes; then cool to 50°C and keep warm. After the temperature stabilizes, add 1 mol / L NaOH to adjust the pH to 6.5, and then add 4000U / g propapain for enzymatic hydrolysis under constant temperature and pH conditions for 2 hours.

[0051] 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 minutes. After the end, the temperature is lowered to 50°C, 1 mol / L NaOH is added, the pH is adjusted to 8.5, and then 4000U / g pro alkaline protease is added. The enzymatic hydrolysis is continued for 2 hours at a constant pH of 8.5 and a temperature of 50°C.

[0052] Step 3: After the reaction is completed, the hydrolyzate in step 2 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.

[0053] Step 4: ultrafiltration the permeate collected in step 3 using an organic ultrafiltration membrane with a cutoff of 3000Da, and collect the filtrate.

[0054] Step 5: The filtrate collected in step 4 is vacuum freeze-dried to obtain whey peptone WPP dry powder.

[0055] Step 6: Determine the molecular weight distribution of the sample. Use high performance gel chromatography to determine the molecular weight distribution of whey protein peptides, and use gel exclusion chromatography to determine the molecular weight of the peptides. 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. In this embodiment, the oligopeptides with a molecular weight of 180-3000Da in the hydrolyzate after enzymatic hydrolysis account for 96.77%, and the free amino acids with a molecular weight of less than 180Da account for 3.82%.

[0056] Example 2: Determination of the growth of Bifidobacterium breve under whey peptone WPP

[0057] In this example, the growth of Bifidobacterium breve under WPP culture was determined experimentally, and the selected Bifidobacterium breve were Bifidobacterium breve CCFM1026 and Bifidobacterium breve CCFM1078.

[0058] 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.

[0059] Step 2: Add WPP at a concentration of 1 g / L to the nitrogen source screening medium, culture two strains of Bifidobacterium breve, inoculate the activated bacterial solution at a 2% inoculation rate, and measure the ΔOD after 12 h of culture. 600 .

[0060] Test results see Figure 1 , CCFM1026 and CCFM1078 were cultured in whey peptone WPP, ΔOD 600 They are 0.487±0.024 and 0.552±0.035 respectively.

[0061] Example 3: Determination of freeze-drying of Bifidobacterium breve in whey peptone WPP

[0062] In this example, the freeze-drying survival rate of Bifidobacterium breve under WPP culture was determined experimentally. The selected Bifidobacterium breve were Bifidobacterium breve CCFM1026 and Bifidobacterium breve CCFM1078.

[0063] The specific operations are as follows:

[0064] 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.

[0065] Step 2: Add WPP at a concentration of 2 g / L to the nitrogen source screening medium, inoculate the activated bacterial solution at a 2% inoculation rate, and culture two strains of Bifidobacterium breve respectively.

[0066] Step 3: Collect the fermentation broth at 12 hours, collect the bacteria by centrifugation (8000g / 20min), wash the bacteria twice with cysteine ​​buffer, mix with 20% sorbitol as a protective agent and the bacterial mud at a mass ratio of 2:1, take 0.5ml and count it before freeze-drying, 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 freeze-drying.

[0067] 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.

[0068] Test results see Figure 2 The survival rates of CCFM1026 and CCFM1078 in whey peptone WPP 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 (1.64±0.09)×10 11 CFU / g dry powder and (1.2±0.96)×10 12 CFU / g dry bacterial powder.

[0069] Comparative Example 1: Growth of Bifidobacterium breve under different nitrogen sources

[0070] According to the method of Example 2, the nitrogen source was replaced with imported tryptone (sigma), acid hydrolyzed casein peptone, three homemade hydrolyzed casein peptones (CAF, CATF, CAPF) 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 .

[0071] 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 peptone and several homemade hydrolyzed peptones on the growth of Bifidobacterium breve are significantly worse than the effect of WPP in Example 2.

[0072] Comparative Example 2: Freeze-drying of Bifidobacterium breve under different nitrogen sources

[0073] According to the method of Example 3, the nitrogen source was replaced with imported trypsin (Sigma), acid hydrolyzed casein peptone, three homemade hydrolyzed casein peptones and three homemade gluten peptones, and the two strains were cultured respectively with them, and the freeze-dried survival rate of the strains and the number of viable bacteria per unit dry bacterial powder after growth for 12 hours under the two nitrogen sources were determined.

[0074] Test results see Figure 2The results show that, under the same nitrogen source addition amount, the freeze-dried survival rate of imported peptone tryptone (sigma) is between 20-25%, the freeze-dried survival rate of Bifidobacterium breve under acid hydrolyzed casein peptone culture is less than 15%, and the freeze-dried survival rate under WPP culture is between 25-30%. The dry bacterial powder per gram weight under WPP peptone culture can reach twice that of imported peptone tryptone (sigma) and acid hydrolyzed casein peptone.

[0075] Among the homemade peptones, the survival rate of freeze-dried Bifidobacterium breve cultured in gluten peptone GAF was not significantly different from that in WPP, but the number of viable bacteria per gram of dry bacterial powder was only (8.04±0.85)×10 10 and (8.42±0.4)×10 11 The CFU was significantly lower than that of Bifidobacterium breve cultured with WPP. The survival rate of freeze-dried and the number of viable bacteria per unit weight of bacterial powder of several other homemade peptones were significantly lower than that of Bifidobacterium breve cultured with WPP.

[0076] Comparative Example 3: Growth of Bifidobacterium breve under different whey peptone cultures

[0077] According to the method of Example 1, different whey peptones were prepared. The enzymes in step 2 were replaced with neutral protease, flavor protease, trypsin, and papain, respectively. Four kinds of peptones WPN, WPF, WPT, and WPA were prepared. The remaining preparation steps were the same.

[0078] According to the method of Example 2, the nitrogen source was replaced by the four whey peptones prepared above and a commercial whey peptone, and the two strains were cultured with them respectively, and the ΔOD of the strains after growing for 12 hours under the two nitrogen sources was measured. 600 The test results are shown in Figure 3 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 peptone and several homemade hydrolyzed peptones on the growth of Bifidobacterium breve are significantly worse than the effect of WPP in Example 2.

[0079] Test results see Figure 3 The results show that, under the same nitrogen source addition amount, the commercial whey peptone and several homemade whey peptones prepared under different enzymatic hydrolysis processes have significantly better promoting effects on the growth of Bifidobacterium breve than the effect of WPP in Example 2. 600 are 0.55 and 0.49, which are significantly higher than 0.43 and 0.32 of commercial whey peptone. However, the whey peptone prepared by other processes has a worse effect on the growth of Bifidobacterium breve than commercial whey peptone.

[0080] 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 whey peptone that promotes the growth of Bifidobacterium breve and improves the freeze-drying survival rate, characterized in that: The method comprises the following steps: preheating the whey protein, hydrolyzing it with papain and alkaline protease in sequence, filtering it with a ceramic membrane and an ultrafiltration membrane, and obtaining a filtrate, namely, whey peptone.

2. The method according to claim 1, characterized in that The steps include: (1) After the whey protein 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 insulation, 1 mol / L NaOH is added to adjust the pH to 6.-7, and then 2000-4000 U / g propapain is added to perform enzymolysis under constant temperature and constant 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 NaOH is added, the pH is adjusted to 8 to 9.5, and then 2000 to 4000 U / g pro-alkaline protease is added. The enzymatic hydrolysis is continued for 2 to 4 hours at a constant pH of 8 to 9.5 and a temperature of 50 degrees Celsius; (3) After the reaction is completed, the hydrolyzate in step (2) 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; (4) ultrafiltration the permeate collected in step (3) using an organic ultrafiltration membrane with a cutoff of 3000 Da, and collecting the filtrate; (5) The filtrate collected in step (4) is vacuum freeze-dried to obtain whey peptone WPP.

3. The method according to claim 1, characterized in that: The Bifidobacterium breve includes but is not limited to Bifidobacterium breve CCFM1026 and Bifidobacterium breve CCFM1078. The strain collection number of the Bifidobacterium breve CCFM1026 is CGMCC NO.60459, and the strain collection number of the Bifidobacterium breve CCFM1078 is CGMCC NO.61011.

4. Whey 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 whey peptone described in claim 4 as the sole nitrogen source, and the concentration of the whey 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 breve and improving the freeze-drying survival rate, characterized in that: The method comprises inoculating Bifidobacterium breve into a culture system using the whey peptone as claimed in claim 4 as the sole nitrogen source for culture, wherein the concentration of the whey peptone in the culture system 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 system also contains 10 g / L of glucose, 7 g / L of KH2PO4, 7 g / L of NaH2PO4, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4·H2O, 1 mL / L of Tween 80, and 1 g / L of cysteine ​​hydrochloride.

9. The method according to claim 7, characterized in that: Bifidobacterium breve 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 breve includes but is not limited to Bifidobacterium breve CCFM1026 and Bifidobacterium breve CCFM1078. The strain collection number of the Bifidobacterium breve CCFM1026 is CGMCC NO.60459, and the strain collection number of the Bifidobacterium breve CCFM1078 is CGMCC NO.61011.

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