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

Abstract

The invention discloses a peptone preparation method for promoting growth of bifidobacterium bifidum and improving freeze-drying survival rate, and belongs to the technical field of microorganisms, and the peptone preparation method comprises the following steps: hydrolyzing whey protein with neutral protease, passing through a ceramic membrane and ultrafiltration, collecting peptide liquid, and freeze-drying to obtain small molecule peptide WA; casein is hydrolyzed with neutral protease and alkaline protease in sequence, peptide liquid is collected after passing through a ceramic membrane and ultrafiltration, and the small molecule peptide CAF is obtained after freeze-drying; the two hydrolytic peptides are mixed in proportion to prepare peptone, and the peptone is used as the sole nitrogen source to culture bifidobacterium bifidum, so that the growth of the bifidobacterium bifidum can be promoted, the freeze-drying survival rate of the bifidobacterium bifidum can be improved, and the application prospect is huge.

Description

Technical Field

[0001] The invention relates to a method for preparing peptone for promoting the growth of Bifidobacterium bifidum and improving the freeze-drying survival rate, and belongs to the technical field of microorganisms. Background Art

[0002] Bifidobacterium bifidum belongs to the genus Bifidobacterium and is a type of strictly anaerobic Gram-positive bacteria. As a probiotic, Bifidobacterium bifidum can promote human health by anti-aging, anti-inflammatory, and enhancing immunity, and has multiple prebiotic functions such as relieving colitis, preventing Alzheimer's disease, and improving constipation. In view of its effective prebiotic functions, Bifidobacterium bifidum has become more and more the focus of research, development, and production, and it is widely used in food, health care products and other fields. However, there are currently few types of Bifidobacterium bifidum strains used commercially, but there are great problems in the cultivation of Bifidobacterium bifidum and its industrialization process. Poor osmotic pressure resistance makes the growth density of Bifidobacterium bifidum low and the freeze-dried survival rate low. Among them, nitrogen source is the key factor limiting the growth of Bifidobacterium bifidum.

[0003] At present, the optimization of nitrogen sources for Bifidobacterium bifidum at home and abroad is mainly screened from existing commercial peptones and yeast extract powders through single factor experiments and orthogonal experiments, mainly including yeast extract powder, tryptone, soy peptone, fish bone peptone, beef extract, etc. A small number of studies will add hydrolyzed protein to the screening process, but when selecting hydrolases, they are rarely combined with the nitrogen source utilization characteristics of the strain itself.

[0004] From the perspective of Bifidobacterium bifidum itself, the main factors that limit its growth are: ① Bifidobacterium bifidum generally lacks cell wall proteases, has low utilization of proteins, and can only utilize hydrolyzed peptides. ② Bifidobacterium bifidum is a cysteine ​​auxotrophic strain. ③ Bifidobacterium bifidum has fewer amino acid transporters than other bifidobacteria, and its utilization efficiency of peptides is generally higher than that of amino acids and proteins, especially for short peptides such as dipeptides and tripeptides.

[0005] Through a large number of studies on Bifidobacterium bifidum, it was also found that Bifidobacterium bifidum has a preference for peptides containing proline and leucine. Proline is often considered an important substance for cells to adapt to osmotic pressure. The accumulation of proline in cells during growth can help strains to be more tolerant to osmotic stress, thereby further improving biomass and freeze-drying resistance. Studies have found that under the cultivation of different nitrogen sources, differences in bacterial structure such as length and aspect ratio will occur during the growth of strains. These differences may cause differences in the damage to the bacteria during the freeze-drying process, which may further affect the freeze-drying survival rate.

[0006] Therefore, in order to better apply Bifidobacterium bifidum to commercial production, it is urgent to find a nitrogen source peptide that can promote the growth of Bifidobacterium bifidum and improve its freeze-dried survival rate, which is of great significance to promoting the industrialization of Bifidobacterium bifidum. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention discloses a method for preparing peptone that promotes the growth of Bifidobacterium bifidum and improves its freeze-dried survival rate, aiming to solve the technical problem in the prior art of lacking a nitrogen source peptide that can promote the growth of Bifidobacterium bifidum and improve its freeze-dried survival rate.

[0008] The first technical solution provided by the present invention is a method for preparing peptone that promotes the growth of Bifidobacterium bifidum and improves the freeze-dried survival rate, comprising the following steps:

[0009] S1, using neutral protease to hydrolyze whey protein to obtain whey peptone WA dry powder;

[0010] S2, using neutral protease and alkaline protease to hydrolyze casein to obtain casein peptone CAF dry powder;

[0011] S3, mixing the whey peptone WA dry powder in step S1 with the casein peptone CAF dry powder in step S2 in a ratio of (1-3):1 to obtain peptone.

[0012] In some embodiments, step S1 comprises the following steps:

[0013] S11, fully mixing whey protein with water at a concentration of 5-7%, raising the temperature to 60°C for preheating for 10 minutes after the protein and water are mixed; then cooling to 50°C for insulation, adding 1 mol / L NaOH to adjust the pH to 6.5-7.5 after the temperature stabilizes, and then adding 2000-4000 U / g pro neutral protease for enzymolysis under constant temperature and constant pH conditions for 2-4 hours;

[0014] S12, after the reaction is completed, the hydrolyzate in step S11 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;

[0015] S13, ultrafiltration treatment of the permeate collected in step S12 using an organic ultrafiltration membrane with a cutoff of 3000Da, and collecting the filtrate;

[0016] S14, vacuum freeze-drying the filtrate collected in step S13 to obtain whey peptone WA dry powder for standby use.

[0017] In some embodiments, step S2 comprises the following steps:

[0018] S21, casein and water are fully mixed 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 insulation, and after the temperature stabilizes, 1 mol / L NaOH is added to adjust the pH to 6.5-7.5, and then 2000-4000 U / g pro neutral protease is added to perform enzymolysis under constant temperature and constant pH conditions for 2-4 hours;

[0019] S22, until the hydrolysis in step S21 is completed, the temperature is maintained at 95°C for 10-15 minutes to inactivate the enzyme, 1 mol / L HCL is added, the pH is adjusted to 6.5-8.5, and then 2000-4000 U / g pro alkaline protease is added, and the enzymatic hydrolysis is continued for 2-4 hours at a constant pH of 6.5-8.5 and a temperature of 50°C;

[0020] S23, after the reaction is completed, the hydrolyzate in step S22 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;

[0021] S24, ultrafiltration treatment of the permeate collected in step S23 using an organic ultrafiltration membrane with a cutoff of 3000Da, and collecting the filtrate;

[0022] S25, vacuum freeze-drying the filtrate collected in step S24 to obtain CAF dry powder for standby use.

[0023] In certain embodiments, the Bifidobacterium bifidum includes but is not limited to Bifidobacterium bifidum CCFM16 and Bifidobacterium bifidum CCFM1301. The strain accession number of the Bifidobacterium bifidum CCFM16 is CGMCC NO.13632, which has been disclosed in the patent document CN106834187A. The strain accession number of the Bifidobacterium bifidum CCFM1301 is CGMCC NO.63175, which has been disclosed in the patent document CN116731904A.

[0024] The second technical solution provided by the present invention is peptone prepared by the method described in the first technical solution.

[0025] The third technical solution provided by the present invention is a culture medium, wherein the culture medium uses the peptone described in the second technical solution as the sole nitrogen source.

[0026] In certain embodiments, the concentration of peptone in the culture medium of the second technical solution is 2 to 8 g / L.

[0027] In certain embodiments, the culture medium further contains glucose, sodium acetate, diammonium hydrogen citrate, K 2 HPO 4 ·3H 2 O、MgSO4 7H 2 O、MnSO 4 ·H 2 O, Tween 80, cysteine ​​hydrochloride.

[0028] Furthermore, the culture medium also contains 20 g / L glucose, 2 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, and K 2 HPO 4 ·3H 2 O 2.6g / L, MgSO 4 7H 2 O 0.1g / L, MnSO 4 ·H 2 O 0.05g / L, Tween 80 1mL / L, cysteine ​​hydrochloride 1g / L.

[0029] The fourth technical solution provided by the present invention is a culture method for promoting the growth of Bifidobacterium bifidum and improving the freeze-drying survival rate, wherein the method comprises inoculating Bifidobacterium bifidum into a culture system using the peptone described in the second technical solution as the sole nitrogen source for culture.

[0030] In certain embodiments, the concentration of peptone in the culture system of the second technical solution is 2-8 g / L.

[0031] In some embodiments, the culture system further comprises glucose, sodium acetate, diammonium hydrogen citrate, K 2 HPO 4 ·3H 2 O、MgSO 4 7H 2 O、MnSO 4 ·H 2 O, Tween 80, cysteine ​​hydrochloride.

[0032] Furthermore, the culture system also contains 20 g / L glucose, 2 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, and K 2 HPO 4 ·3H 2 O 2.6g / L, MgSO 4 7H 2 O 0.1g / L, MnSO 4 ·H 2 O 0.05g / L, Tween 80 1mL / L, cysteine ​​hydrochloride 1g / L.

[0033] In certain embodiments, Bifidobacterium bifidum is inoculated into the culture system at an inoculation rate of 2-4%, and anaerobically cultured at 37°C for at least 12 hours.

[0034] In certain embodiments, the Bifidobacterium bifidum includes but is not limited to Bifidobacterium bifidum CCFM16 and Bifidobacterium bifidum CCFM1301. The strain accession number of the Bifidobacterium bifidum CCFM16 is CGMCC NO.13632, which has been disclosed in the patent document CN106834187A. The strain accession number of the Bifidobacterium bifidum CCFM1301 is CGMCC NO.63175, which has been disclosed in the patent document CN116731904A.

[0035] The technical effects of the present invention are as follows:

[0036] Compared with the traditional peptide preparation method, after hydrolysis, compared with the traditional process: enzyme inactivation - isoelectric point adjustment - centrifugal protein removal, the direct use of ceramic membrane plus ultrafiltration membrane is simple and fast. At the same time, the selection of enzymes and proteins in the preparation method of peptone that promotes the growth of Bifidobacterium bifidum and improves the freeze-dried survival rate combines the nitrogen source utilization characteristics of the strain itself, and is more specific than the preparation of other types of peptone.

[0037] The present invention provides a small molecule peptide WA that promotes the growth of Bifidobacterium bifidum. In a nitrogen source screening medium, Bifidobacterium bifidum is cultured at an addition amount of 2 g / L for 12 h, and its OD 600 All are greater than 1.5, which is significantly higher than other nitrogen sources. The small molecule peptide CAF provided by the present invention improves the freeze-dried survival rate of Bifidobacterium bifidum. In the nitrogen source screening medium, Bifidobacterium bifidum is cultured for 12 hours at an addition amount of 2g / L, and freeze-dried with 13% skim milk as a freeze-drying protectant. The freeze-dried survival rate is as high as 27.35%, which is significantly higher than other nitrogen sources. The peptone WA-CAF provided by the present invention replaces all nitrogen sources in MRS-L with 8g / L, and Bifidobacterium bifidum is cultured for 20 hours. The number of viable bacteria is better than that of MRS-L with a total nitrogen source of 25g / L. The number of viable bacteria produced by the two strains of bacteria per gram of nitrogen source is 278.6% and 298.5% of that in MRS-L, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a graph showing the growth of Bifidobacterium bifidum under different nitrogen sources of the present invention.

[0039] Figure 2 This is a freeze-dried survival rate graph of Bifidobacterium bifidum under different nitrogen sources of the present invention.

[0040] Figure 3 This is a graph showing the number of live bacteria of Bifidobacterium bifidum cultured in WA-CAF with different mixing ratios according to the present invention.

[0041] Figure 4 This is a freeze-dried survival rate diagram of Bifidobacterium bifidum cultured in WA-CAF with different mixing ratios according to the present invention.

[0042] Figure 5 The figure is a graph of the viable bacterial count and pH of Bifidobacterium bifidum cultured with different addition amounts of WA-CAF of the present invention.

[0043] Figure 6 The graphs are the growth curve, generation time, viable bacteria count and freeze-dried survival rate of Bifidobacterium bifidum in two different culture media of the present invention.

[0044] Figure 7 This is a graph of the number of viable bacteria produced per unit nitrogen source under two different culture media of the present invention. DETAILED DESCRIPTION

[0045] 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.

[0046] Test method:

[0047] 1. Determine the generation time

[0048] Inoculate the activated bacterial solution with 2% inoculum and measure OD every 2 hours. 600 , determine the growth curve of Bifidobacterium bifidum under the culture conditions, and calculate the growth generation time of Bifidobacterium bifidum according to formula (A) in the logarithmic phase, where G represents the growth generation time of Bifidobacterium bifidum, and A 0 Indicates the initial absorbance of the strain when it enters the logarithmic growth phase, A t represents the absorbance of the fermentation broth at time t.

[0049] Absorbance OD 600 Measured using a spectrophotometer.

[0050]

[0051] 2. Determination of freeze-dried survival rate

[0052] Collect the fermentation liquid, collect the bacteria by centrifugation, wash the bacteria twice with cysteine ​​buffer, mix with 13% skim milk as a protective agent and the bacterial mud at a mass ratio of 2:1, take 0.5ml to count before lyophilization, then take 1ml into a vial and record the weight of the wet bacterial mud, and place it in a freeze dryer for freeze drying. Add cysteine ​​buffer to the freeze-dried sample to restore it to the weight before lyophilization, then take 0.5ml for post-lyophilization counting, compare it with the number of viable bacteria before lyophilization, and calculate the lyophilization survival rate. The counting method adopts the plate pouring method specified in GB 4789.2-2022.

[0053] 3. Determine the utilization efficiency of unit nitrogen source

[0054] The number of live bacteria at different times during the growth process is measured, and the utilized nitrogen content in the fermentation supernatant at this time is measured by the Kjeldahl method. Samples are taken every 4 hours to calculate the number of live bacteria produced per gram of nitrogen source, thereby calculating the conversion efficiency per unit nitrogen source. The counting method adopts the plate pouring method specified in GB 4789.2-2022.

[0055] The raw materials used in the embodiment:

[0056] In the following examples, yeast extract FM803 was purchased from Angel Yeast Co., Ltd.; tryptone peptone (sigma) was purchased from Merck Life Sciences; and other reagents and materials, unless otherwise specified, were commercially available.

[0057] The Bifidobacterium bifidum CCFM16 and CCFM1301 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 bifidum CCFM16 is CGMCC NO.13632, which has been disclosed in the patent document CN106834187A, and the strain preservation number of Bifidobacterium bifidum CCFM1301 is CGMCC NO.63175, which has been disclosed in the patent document CN116731904A.

[0058] The culture medium involved in the following examples is as follows:

[0059] 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 O 2.6g / L, MgSO 4 7H 2 O 0.1g / L, MnSO 4 ·H 2 O 0.05g / L, Tween 80 1mL / L, cysteine ​​hydrochloride 1g / L, high pressure sterilization at 115℃ for 20min.

[0060] 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.

[0061] Nitrogen source screening medium: nitrogen source 2g / L, glucose 10g / L, KH 2 PO 4 7g / L, NaH 2PO 4 7g / L, MgSO 4 7H 2 O0.1g / L, MnSO 4 ·H 2 O 0.05g / L, Tween 80 1mL / L, cysteine ​​hydrochloride 1g / L, pH 6.2-6.4, sterilize at 115℃ for 20min.

[0062] Cysteine ​​buffer: cysteine ​​hydrochloride 0.5 g / L, KH 2 PO 4 6.0g / L, Na2HPO4 4.5g / L, NaCl4.0g / L, Tween 80 0.6g / L, pH 6.8~7.0, sterilization at 115℃ for 20min.

[0063] The enzymes involved in the following examples are:

[0064] Neutral protease: Model Z8032 is from Solebo. Enzyme activity definition: Under the measurement conditions (40°C, pH 7), when the absorbance of the 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.

[0065] 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.

[0066] Example 1: Preparation of small molecule peptide WA that can promote the growth of Bifidobacterium bifidum

[0067] In this example, a small molecule peptide WA that can promote the growth of Bifidobacterium bifidum was prepared by enzymatic hydrolysis, and then the culture effect of Bifidobacterium bifidum under WA was determined. The selected strains were Bifidobacterium bifidum CCFM16 and Bifidobacterium bifidum CCFM1301.

[0068] The specific operations are as follows:

[0069] Step 1: Mix whey protein and water thoroughly 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 7, and then add 4000U / gpro neutral protease for enzymatic hydrolysis under constant temperature and pH conditions for 2 hours.

[0070] Step 2: After the reaction is completed, the unhydrolyzed protein and enzymes in the reaction system are removed by coarse filtration through a 0.22 μm ceramic membrane, and the permeate is collected.

[0071] Step 3: Ultrafiltration the permeate using an organic ultrafiltration membrane with a cutoff of 3000Da, and collect the filtrate.

[0072] Step 4: The final filtrate is vacuum freeze-dried to obtain WA dry powder for stand-by use.

[0073] Step 5: 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 48h. After the colonies grow, pick a single colony and inoculate it into a 5mL MRS-L liquid tube, culture it anaerobically for 18h to 24h, and repeat the above operation 2-3 times to obtain the activated bacterial solution.

[0074] Step 6: Add WA at a concentration of 2 g / L to the nitrogen source screening medium, culture two strains of Bifidobacterium bifidum, inoculate the activated bacterial solution at a 2% inoculation rate, and measure the OD after 12 h of culture. 600 .

[0075] Test results are shown in Figure 1 , CCFM16 and CCFM1301 were cultured with small molecule peptide WA, OD 600 They are 1.5 and 1.59 respectively.

[0076] Example 2: Preparation of small molecule peptide CAF that can improve the freeze-dried survival rate of Bifidobacterium bifidum

[0077] In this embodiment, a small peptide CAF for improving the freeze-dried survival rate of Bifidobacterium bifidum was prepared by enzymatic hydrolysis, and then the freeze-dried survival rate of Bifidobacterium bifidum in CAF culture was determined experimentally. The selected strains were Bifidobacterium bifidum CCFM16 and Bifidobacterium bifidum CCFM1301.

[0078] The specific operations are as follows:

[0079] 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 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 7, and then add 4000U / gpro neutral protease for enzymatic hydrolysis under constant temperature and pH conditions for 2 hours.

[0080] Step 2: After the hydrolysis in step 1 is completed, maintain at 95°C for 10 min to inactivate the enzyme, add 1 mol / L NaOH, adjust the pH to 8.5, and then add 4000U / g pro alkaline protease, and continue the enzymatic hydrolysis for 2 h at a constant pH of 8.5 and a temperature of 50°C.

[0081] Step 3: After the reaction is completed, the unhydrolyzed protein and enzymes in the reaction system are removed by coarse filtration through a 0.22 μm ceramic membrane, and the permeate is collected.

[0082] Step 4: Ultrafiltration the permeate using an organic ultrafiltration membrane with a cutoff of 3000Da, and collect the filtrate.

[0083] Step 5: The final filtrate is vacuum freeze-dried to obtain CAF dry powder for standby use.

[0084] Step 6: 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 medium, and culture it at 37℃ in an anaerobic workstation for 48h. After the colonies grow, pick a single colony and inoculate it into a 5mL MRS-L liquid tube, culture it anaerobically for 18h to 24h, and repeat the above operation 2-3 times to obtain the activated bacterial solution.

[0085] Step 7: CAF was added to the nitrogen source selection medium at a concentration of 2 g / L, and the activated bacterial solution was inoculated at a 2% inoculation amount to culture two strains of Bifidobacterium bifidum respectively.

[0086] Step 8: Collect the fermentation broth at 12 hours, collect the bacteria by centrifugation (8000g / 20min), wash the bacteria twice with cysteine ​​buffer, and then mix it with 13% skim milk as a protective agent at a mass ratio of 2:1 with the bacterial mud. Take 0.5ml for counting 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.

[0087] Step 9: 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.

[0088] Test results are shown in Figure 2 , the freeze-dried survival rates of CCFFM16 and CCFM1301 were 26.33% and 19.35%, respectively, when cultured with small molecule CAFs.

[0089] Example 3: Preparation of peptone WA-CAF that can promote the growth of Bifidobacterium bifidum and improve the freeze-drying survival rate

[0090] Step 1: In this example, the small molecule peptide WA obtained in Example 1 and the small molecule peptide CAF obtained in Example 2 were mixed at a mass ratio of 2:1 to obtain the target peptone.

[0091] Step 2: 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.

[0092] Step 3: Add WA-CAF to the nitrogen source screening medium at a concentration of 2 g / L, culture two strains of Bifidobacterium bifidum respectively, inoculate the activated bacterial solution at a 2% inoculation rate, and determine the number of viable bacteria after 12 hours of culture.

[0093] Step 4: Collect the fermentation broth at 12 hours, collect the bacteria by centrifugation (8000g / 20min), wash the bacteria twice with cysteine ​​buffer, and then mix it with 13% skim milk as a protective agent in a mass ratio of 2:1 with the bacterial mud. Take 0.5ml for counting 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.

[0094] Step 5: Add cysteine ​​buffer to the freeze-dried sample to restore 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. Figure 3 The freeze-dried survival rate results are as follows Figure 4 . Figure 3 It can be seen that the viable counts of the two strains of Bifidobacterium bifidum in the culture medium of WA and CAF were significantly lower than that in the mixed peptone WA-CAF (2:1) in Example 3. Figure 4 It can be seen that the freeze-dried survival rate of the two strains of Bifidobacterium bifidum under culture was significantly lower than that of the mixed peptone WA-CAF (2:1) in Example 3. The freeze-dried survival rate obtained under the mixed peptone culture in Example 3 was similar to that of CAF, and the difference was not significant.

[0095] Example 4: Application of peptone WA-CAF to promote the growth of Bifidobacterium bifidum and improve the freeze-dried survival rate

[0096] In this example, WA-CAF peptone was added to the culture as a nitrogen source to explore its effect on the growth, generation time, viable count, and freeze-dried survival rate of Bifidobacterium bifidum. The selected strains were Bifidobacterium bifidum CCFM16 and Bifidobacterium bifidum CCFM1301.

[0097] The specific operations are as follows:

[0098] Step 1: Determination of nitrogen source addition amount:

[0099] To ensure that the nitrogen source is sufficient, the amount of nitrogen source added needs to be determined to ensure that at this amount of addition, the strain can grow to acid inhibition, rather than stop growing due to lack of nitrogen source. WA in Example 1 and CAF in Example 2 were prepared at a mass ratio of 2:1, and all nitrogen sources in MRS-L were replaced with 2, 4, 6, and 8 g / L additions, respectively, and the viable counts of Bifidobacterium bifidum in these cases were determined by culturing to the end of the logarithmic phase. MRS-L with a nitrogen source addition of 25 g / L was used as a reference for comparison.

[0100] Test results are shown in Figure 5 When the addition amount of WA:CAF (2:1) was 8g / L, the number of live bacteria in CCFM16 was higher than that in the MRS-L control group; there was no significant difference in the number of live bacteria in CCFM1301 compared with MRS-L. And from the pH of the end point of the fermentation of the two bacteria, at 4g / L, the fermentation could reach acid inhibition. Therefore, at 8g / L, the available components in the nitrogen source were already in an excess state. By comparing at this addition amount, we can better compare the potential of the culture medium with WA-CAF as the nitrogen source and the MRS-L culture medium in culturing Bifidobacterium bifidum.

[0101] Step 2: Growth curve determination, generation time determination, freeze-dried survival rate determination, and unit nitrogen source utilization efficiency determination

[0102] WA and CAF were prepared at a mass ratio of 2:1, and all nitrogen sources in MRS-L were replaced at a concentration of 8 g / L. Two strains of Bifidobacterium bifidum were inoculated into the culture medium at a 2% inoculation rate, and the OD of the bacterial solution was recorded every 2 hours. 600 , and a growth curve was prepared. In the logarithmic phase, the growth generation time of Bifidobacterium bifidum was calculated according to formula (A).

[0103] At the same time, the freeze-drying survival rate was measured and the conversion efficiency per unit nitrogen source was calculated.

[0104] Growth test results are shown in Figure 6 The highest OD measured by CCFM16 600 The maximum number of viable bacteria was (28.57±1.72)×10 8 CFU / mL, the freeze-dried survival rate was 20.48±2.23%. The highest average OD measured by CCFM1301 600 The maximum number of viable bacteria was (20.433±0.95)×10 8 CFU / mL, and the freeze-dried survival rate was 15.82±1.00%.

[0105] The results of unit nitrogen source conversion rate are shown in Figure 7With 8g / L WA-CAF peptone as the sole nitrogen source, the two strains could produce 2.399×10 12 CFU and 1.379×10 12 CFU of live bacteria.

[0106] Example 5: Differences in growth and freeze-dried survival rate when culturing Bifidobacterium bifidum at different mixing ratios of two small molecule peptides

[0107] The mixing ratio of WA to CAF in step 1 of Example 3 was changed to 3:1, 1:1, 1:2, and 1:3 to prepare new peptone for culturing Bifidobacterium bifidum. The other steps were the same as those in Example 3, and the viable count and freeze-dried survival rate of Bifidobacterium bifidum were determined.

[0108] Growth results such as Figure 3 As shown, for the growth of Bifidobacterium bifidum CCFM16, when the peptones prepared at different compounding ratios were 3:1, there was no significant difference in the number of live bacteria compared with 2:1 in Example 3, while when the ratios were 3:1, 1:1, 1:2, and 1:3, the number of live bacteria was significantly lower than 2:1 in Example 3.

[0109] With respect to the growth of Bifidobacterium bifidum CCFM1301, the peptone prepared according to the ratio in Example 3 has a significantly better effect on the growth of Bifidobacterium bifidum than the peptone prepared according to other ratios.

[0110] The results of freeze-dried survival rate are as follows Figure 4 When the two strains of Bifidobacterium bifidum were cultured under peptone prepared by mixing in other proportions, their freeze-drying survival rates were significantly lower than the freeze-drying survival rates obtained in Example 3.

[0111] In summary, the peptone obtained by mixing the two small molecule peptides in Example 3 has better growth and freeze-drying effects on Bifidobacterium bifidum than single small peptides WA and CAF.

[0112] Comparative Example 1: Growth of Bifidobacterium bifidum under different nitrogen sources

[0113] According to the method of step 5-step 6 of Example 1, the nitrogen source was replaced with MRS-L formula nitrogen source, yeast extract FM803 and imported tryptone (sigma), and the two strains were cultured separately, and the OD of the strains after growing for 12 hours under these two nitrogen sources was measured. 600 .

[0114] Test results are shown in Figure 1 The results show that, under the same nitrogen source addition amount, WA as a nitrogen source has a significant promoting effect on the growth of Bifidobacterium bifidum. The OD600 of MRS-L and yeast extract FM803 culture does not exceed 0.6, and the OD600 of imported tryptone culture is less than 0.6.600 No more than 1, while the OD 600 It can reach 1.5, far exceeding commercial yeast extract powder and peptone.

[0115] Comparative Example 2: Freeze-dried survival rate of Bifidobacterium bifidum cultured under different nitrogen sources

[0116] According to the method of step 6 to step 9 of Example 2, the nitrogen source was replaced with MRS-L formula nitrogen source, yeast extract FM803 and imported trypsin (sigma), and two strains of bacteria were cultured separately, and the freeze-dried survival rate (%) of the strains was determined.

[0117] Test results are shown in Figure 2 From the results, it can be seen that under the same amount of nitrogen source added, the freeze-dried survival rates of Bifidobacterium bifidum cultured with MRS-L formula nitrogen source, yeast extract powder FM803 and imported trypsin (sigma) as nitrogen sources were all lower than 15%, which was significantly worse than the freeze-dried survival rate obtained under CAF culture in Example 2.

[0118] Comparative Example 3: Comparison of the growth of Bifidobacterium bifidum under WA-CAF nitrogen source and MRS-L culture and freeze-drying

[0119] The nitrogen source in steps 2-5 of Example 4 was replaced with MRS-L with a nitrogen source addition amount of 25 g / L. The growth, generation time, change in viable count, freeze-drying survival rate, and unit nitrogen source conversion rate of the two strains in MRS-L were measured.

[0120] The results of growth and freeze-drying tests are shown in Figure 6 ,in Figure 6 A. Figure 6 E is the growth curve; Figure 6 B. Figure 6 F is the generation time; Figure 6 C. Figure 6 G is the number of viable bacteria; Figure 6 D. Figure 6 H is the freeze-drying survival rate.

[0121] Depend on Figure 6 A and 6E show that in Example 4, using 8 g / L WA-CAF peptone as the sole nitrogen source, its upper limit of growth is higher than that of the 25 g / L MRS-L medium in this comparative example.

[0122] Figure 6 B. 6F showed that WA-CAF was pure peptone and no nitrogen source such as yeast extract powder containing a large amount of growth factors was added, but its generation time was not significantly different from that of the MRS-L control group, and the strain grew normally.

[0123] Figure 6C and 6G show that when grown in MRS-L, the number of viable bacteria was lower than that in WA-CAF in Example 5 at all times. The maximum number of viable bacteria of the two strains in WA-CAF reached (28.57±1.72)×10 8 CFU / mL, and (20.433±0.95)×10 8 CFU / mL;

[0124] Figure 6 D, 6H show that the freeze-dried survival rates of the two strains in MRS-L were 9.96±0.81% and 8.33±1.51%, which were significantly lower than those in Example 4.

[0125] The results of unit nitrogen source conversion rate test are shown in Figure 7 The results show that, with 8 g / L WA-CAF peptone in Example 5 as the only nitrogen source, the two strains can produce 2.399×10 12 and 1.379×10 12 CFU of live bacteria, compared with 0.804×10 12 and 0.4678×10 12 The CFU increased by about 278.6% and 298.5%. Therefore, WA-CAF is a peptone that can promote the growth of Bifidobacterium bifidum and improve the freeze-dried survival rate. Compared with the nitrogen source in MRS-L, WA-CAF has a higher conversion rate as a nitrogen source.

[0126] In summary, compared with the total nitrogen source amount of 25 g / L in MRS-L, the growth indicators of WA-CAF with only 8 g / L added in Example 5 can reach or even exceed the level of MRS-L. While ensuring the growth of bacteria, the amount of nitrogen source is reduced, effectively reducing the culture cost.

[0127] 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 peptone that promotes the growth of Bifidobacterium bifidum and improves the freeze-drying survival rate, characterized in that: The steps include: S1, using neutral protease to hydrolyze whey protein to obtain whey peptone WA dry powder; S2, using neutral protease and alkaline protease to hydrolyze casein to obtain casein peptone CAF dry powder; S3, mixing the whey peptone WA dry powder in step S1 with the casein peptone CAF dry powder in step S2 in a ratio of (1-3):1 to obtain peptone.

2. The method according to claim 1, characterized in that The step S1 comprises the following steps: S11, 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 6.5-7.5, and then 2000-4000 U / g pro neutral protease is added to perform enzymolysis under constant temperature and constant pH conditions for 2-4 hours; S12, after the reaction is completed, the hydrolyzate in step S11 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; S13, ultrafiltration treatment of the permeate collected in step S12 using an organic ultrafiltration membrane with a cutoff of 3000Da, and collecting the filtrate; S14, vacuum freeze-drying the filtrate collected in step S13 to obtain whey peptone WA.

3. The method according to claim 1, characterized in that Step S2 includes the following steps: S21, after fully mixing casein and water at a concentration of 5-7%, raising the temperature to 60°C for preheating for 10 minutes; then cooling to 50°C for insulation, adding 1 mol / L NaOH to adjust the pH to 6.5-7.5, and then adding 2000-4000 U / g pro neutral protease for enzymolysis at constant temperature and pH for 2-4 hours; S22, after the hydrolysis is completed, the temperature is maintained at 95°C for 10-15 min to inactivate the enzyme, 1 mol / L HCL is added, the pH is adjusted to 6.5-8.5, and then 2000-4000 U / g pro-alkaline protease is added, and the enzymatic hydrolysis is continued for 2-4 h at a constant pH of 6.5-8.5 and a temperature of 50°C; S23, after the reaction is completed, the hydrolyzate in step S22 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; S24, ultrafiltration treatment of the permeate collected in step S23 using an organic ultrafiltration membrane with a cutoff of 3000Da, and collecting the filtrate; S25, vacuum freeze-drying the filtrate collected in step S24 to obtain CAF.

4. 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 peptone described in claim 4 as the sole nitrogen source, and the concentration of the peptone in the culture medium is 2 to 8 g / L.

6. The culture medium according to claim 5, characterized in that The culture medium also contains glucose, sodium acetate, diammonium hydrogen citrate, K2HPO4·3H2O, MgSO4·7H2O, MnSO4·H2O, Tween 80, and cysteine ​​hydrochloride; optionally, the culture medium also contains 20 g / L of glucose, 2 g / L of sodium acetate, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of K2HPO4·3H2O, 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.

7. A method for promoting the growth of Bifidobacterium bifidum and improving the freeze-drying survival rate, characterized in that: The method comprises inoculating Bifidobacterium bifidum into a culture system using the peptone described in claim 4 as the sole nitrogen source for culture, wherein the concentration of the peptone in the culture system is 2-8 g / L.

8. The method according to claim 7, characterized in that The culture system also contains glucose, sodium acetate, diammonium hydrogen citrate, K2HPO4·3H2O, MgSO4·7H2O, MnSO4·H2O, Tween 80, and cysteine ​​hydrochloride; optionally, the culture system also contains 20 g / L glucose, 2 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 2.6 g / L K2HPO4·3H2O, 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 bifidum 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 bifidum includes but is not limited to Bifidobacterium bifidum CCFM16 and Bifidobacterium bifidum CCFM1301. The strain collection number of the Bifidobacterium bifidum CCFM16 is CGMCC NO.13632, and the strain collection number of the Bifidobacterium bifidum CCFM1301 is CGMCC NO.63175.

Images