Screening method and application of freeze-dried powder protective agent

Abstract

The invention belongs to the technical field of freeze-dried powder protective agent screening, and particularly relates to a screening method and application of a freeze-dried powder protective agent. According to the screening method and application of the freeze-dried powder protective agent, a freeze-drying protective agent rapid screening technology based on cell viability can accurately and efficiently complete screening of the probiotic freeze-drying protective agent, and a brand new thought is provided for screening of the probiotic freeze-drying protective agent.

Description

Technical Field

[0001] The invention belongs to the technical field of screening freeze-dried powder protective agents, and specifically relates to a screening method and application of freeze-dried powder protective agents. Background Art

[0002] Freeze-dried powder protective agents play an important role in the preparation of microbial freeze-dried powder, and are greatly affected by freeze-dried protective agents. The addition of cryoprotectants is beneficial to reduce the formation of low-temperature ice crystals and the resulting damage to bacterial cells, and can protect the biological activity of bacterial cells to the maximum extent. Commonly used freeze-dried protective agents involve many types, such as carbohydrates, proteins, buffer salts, antioxidants and amino acids. The addition of freeze-dried protective agents helps maintain the biological stability of bacterial cells during the freeze-drying process, and reduces cell freezing damage, protein denaturation and the impact of other stress environments.

[0003] Currently, the plate coating counting method is used to screen lyophilized protective agents, which has a long cycle and low accuracy. Summary of the invention

[0004] The purpose of the present invention is to provide the application of flow cytometry in screening microbial freeze-dried powder protective agents, and flow cytometry has the advantages of being fast and accurate.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides application of flow cytometry in screening protective agents for freeze-dried powder of microorganisms.

[0007] The present invention provides a method for screening a freeze-dried powder protective agent, comprising:

[0008] The microorganisms are mixed with the protective agent to be screened and then dried to obtain a lyophilized powder;

[0009] The total number of microbial cells before drying and the number of viable microbial cells after drying were determined by flow cytometry, and the microbial survival rate was calculated; the calculation formula of the microbial survival rate was: microbial survival rate = number of viable microbial cells after drying / total number of microbial cells before drying × 100%;

[0010] Determining the freeze-dried powder protective agent according to the survival rate of the microorganisms;

[0011] When the protective agent to be screened is a type of substance, the one with the highest microbial survival rate is the lyophilized powder protective agent obtained by screening;

[0012] When the protective agents to be screened are more than two types of substances, the protective agent with the highest microbial survival rate in each type of substance is the screened protective agent in that type, and then the screened protective agents of different types are compounded to obtain the freeze-dried powder protective agent.

[0013] Preferably, the protective agent to be screened includes one or more of carbohydrate lyoprotectants, protein lyoprotectants, buffer salt lyoprotectants, antioxidant lyoprotectants and amino acid lyoprotectants.

[0014] Preferably, the carbohydrate lyoprotectant comprises one or more of glucose, fructose, galactose, ribose, xylose, trehalose, sucrose, lactose, maltose, raffinose, stachyose, mannose, sorbitol, galacto-oligosaccharides, xylo-oligosaccharides, isomalto-oligosaccharides, maltodextrin and rhamnose;

[0015] The protein freeze-drying protective agent includes one or more of yeast extract powder, yeast peptone, beef extract powder, beef liver extract powder and fish peptone;

[0016] The buffer salt freeze-drying protective agent includes one or more of sodium chloride, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate and calcium chloride;

[0017] The antioxidant freeze-drying protective agent includes one or more of glutathione, tea polyphenols, ascorbic acid, L-cysteine ​​hydrochloride, β-carotene and sodium ascorbate;

[0018] The amino acid freeze-drying protective agent includes one or more of sodium glutamate, threonine, cysteine, proline, glutamic acid, tryptophan, glutamine and glycine.

[0019] Preferably, the microorganisms include probiotics; the probiotics include bifidobacteria; one or more of the Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis.

[0020] Preferably, the drying method includes vacuum freeze drying.

[0021] Preferably, the vacuum freeze-drying includes sequentially performing first-stage drying, second-stage drying, third-stage drying, fourth-stage drying, fifth-stage drying, sixth-stage drying and seventh-stage drying; the temperature of the first-stage drying may be -40 to -45°C, and the time may be 120 to 180 min; the temperature of the second-stage drying may be -25°C to -30°C, and the time may be 180 to 240 min; the temperature of the third-stage drying may be -20°C to -25°C, and the time may be 600 to 900 min; the temperature of the fourth-stage drying may be -10 to 0°C, and the time may be 180 to 300 min; the temperature of the fifth-stage drying may be 10 to 15°C, and the time may be 180 to 300 min; the temperature of the sixth-stage drying may be 15 to 20°C, and the time may be 120 to 180 min; the temperature of the seventh-stage drying may be 25 to 30°C, and the time may be 60 to 90 min.

[0022] Preferably, the application form of the microorganism includes microbial cell concentrate.

[0023] Preferably, it is characterized in that the dyes used in the determination of the number of viable cells of microorganisms by flow cytometry include PI and SYTO9.

[0024] Beneficial effects of the present invention: The present invention provides the application of flow cytometry in screening microbial freeze-dried powder protective agents. The traditional screening of protective agents uses the plate counting method to calculate the survival rate, and the cycle is 48h to 72h, which takes a long time. The technical advantage of the present invention is that the screening of each freeze-dried protective agent can be completed in a short time (3min to 5min) by using flow cytometry, and the cycle is short. The survival rate is calculated by flow cytometry, which avoids the error of human factors and improves the accuracy of the measurement.

[0025] The present invention provides a method for screening freeze-dried powder protective agents. The screening method has the characteristics of rapidity, accuracy, high throughput, etc., and can greatly improve the accuracy of screening probiotic freeze-dried protective agents, with an accuracy rate of more than 95%. It can complete the screening of 18 carbohydrates, 23 proteins, 8 buffer salts, 6 antioxidants and 8 amino acid freeze-dried protective agents at one time, and the screening efficiency is improved by 10 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The cell membrane integrity of Bifidobacterium breve BX-18 when trehalose was used as a protective agent;

[0027] Figure 2 The cell membrane integrity of Bifidobacterium breve BX-18 when yeast extract powder FM902 was used as a protective agent;

[0028] Figure 3The cell membrane integrity of Bifidobacterium breve BX-18 when potassium dihydrogen phosphate was used as a protective agent;

[0029] Figure 4 The cell membrane integrity of Bifidobacterium breve BX-18 when glutathione was used as a protective agent;

[0030] Figure 5 The cell membrane integrity of Bifidobacterium breve BX-18 when sodium glutamate was used as a protective agent;

[0031] Figures 1 to 5 FL18-Area::488-513_26 and FL21-Area::561-614_20 are both test channels for flow cytometry.

[0032] Biological Deposit Description

[0033] Bifidobacterium breve BX-18 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on September 12, 2023, with the deposit number CGMCC No. 28446. The address of the deposit unit is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0034] The invention provides application of flow cytometry in screening protective agents for freeze-dried powder of microorganisms.

[0035] Flow cytometry screening of microbial lyophilized powder protective agents has the advantages of being fast and highly accurate. The screening of each lyophilized protective agent can be completed in (3min to 5min) with an accuracy rate of more than 80%.

[0036] The present invention provides a method for screening a protective agent for freeze-dried microbial powder, comprising:

[0037] The microorganisms are mixed with the protective agent to be screened and then dried to obtain a lyophilized powder;

[0038] The total number of microbial cells before drying and the number of viable microbial cells after drying were determined by flow cytometry, and the microbial survival rate was calculated; the calculation formula of the microbial survival rate was: microbial survival rate = number of viable microbial cells after drying / total number of microbial cells before drying × 100%;

[0039] Determining the freeze-dried powder protective agent according to the survival rate of the microorganisms;

[0040] When the protective agent to be screened is a type of substance, the one with the highest microbial survival rate is the lyophilized powder protective agent obtained by screening;

[0041] When the protective agents to be screened are more than two types of substances, the protective agent with the highest microbial survival rate in each type of substance is the screened protective agent in that type, and then the screened protective agents of different types are compounded to obtain the freeze-dried powder protective agent.

[0042] As an practicable manner, the total number of microbial cells in the present invention is the sum of the number of living cells and the number of dead cells.

[0043] As an practicable manner, the protective agent to be screened in the present invention may include one or more of carbohydrate freeze-drying protective agents, protein freeze-drying protective agents, buffer salt freeze-drying protective agents, antioxidant freeze-drying protective agents and amino acid freeze-drying protective agents. The carbohydrate freeze-drying protective agent of the present invention includes one or more of glucose, fructose, galactose, ribose, xylose, trehalose, sucrose, lactose, maltose, raffinose, stachyose, mannose, sorbose, galacto-oligosaccharides, xylo-oligosaccharides, isomalto-oligosaccharides, maltodextrin and rhamnose. The protein freeze-drying protective agent of the present invention includes one or more of yeast extract powder, yeast peptone, beef extract powder, beef liver extract powder and fish peptone. The buffer salt freeze-drying protective agent of the present invention includes one or more of sodium chloride, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate and calcium chloride. The antioxidant freeze-drying protective agent of the present invention includes one or more of glutathione, tea polyphenols, ascorbic acid, L-cysteine ​​hydrochloride, β-carotene and sodium ascorbate. The amino acid freeze-drying protective agent of the present invention includes one or more of sodium glutamate, threonine, cysteine, proline, glutamic acid, tryptophan, glutamine and glycine.

[0044] As an practicable manner, the application form of the microorganism includes a microbial cell concentrate. As an practicable manner, the present invention can mix the microbial cell concentrate with the protective agent to be screened and then dry to obtain a freeze-dried powder. During the mixing of the present invention, the mass ratio of the microbial cell concentrate to the protective agent to be screened can be (1-2): (1-2), or can be 1:1. The microorganism of the present invention can include probiotics; the probiotics can include bifidobacteria; the bifidobacteria can include one or more of Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, and Bifidobacterium longum subsp. infantis. The Bifidobacterium breve of the present invention may include Bifidobacterium breve BX-18 and / or Bifidobacterium breve B2798, the Bifidobacterium animalis lactis subspecies may include one or more of Bifidobacterium animalis lactis subspecies Probio-M8, Bifidobacterium animalis lactis subspecies V9 and Bifidobacterium animalis lactis subspecies ProSci-246, and the Bifidobacterium longum infantis subspecies may include Bifidobacterium longum infantis subspecies B8762.

[0045] As an practicable manner, the method for preparing the cell concentrate of the present invention may include: sequentially performing seed culture and expansion culture on the microorganism to obtain a fermentation liquid; and centrifuging the fermentation liquid to obtain a cell concentrate.

[0046] As an embodiment, the seed culture method of the present invention may include: inoculating the microorganism into a culture medium for three subcultures, and the obtained third-generation bacterial solution is cultured for another 18 hours to obtain a seed solution. As an embodiment, the present invention inoculates the microorganism into a culture medium for the first time for the first generation culture to obtain a first-generation bacterial solution, which is recorded as the first subculture; the first-generation bacterial solution is inoculated into a culture medium for the second generation culture to obtain a second-generation bacterial solution, which is the second subculture; the second-generation bacterial solution is inoculated into a culture medium for the third generation culture to obtain a third-generation bacterial solution, which is the third subculture; the obtained third-generation bacterial solution is inoculated into a culture medium for culture to obtain a seed solution. The present invention does not specifically limit the parameters of the first inoculation, and conventional methods can be used. The inoculation amount of the inoculation may be 1% to 3% of the volume of the culture medium. In a specific embodiment of the present invention, the inoculation amount may be 1%, 2% or 3% of the volume of the culture medium. The temperature of the first generation culture, the second generation culture, the third generation culture and the culture of the present invention can be 35-40°C respectively; in a specific embodiment of the present invention, the temperature of the first generation culture, the second generation culture, the third generation culture and the culture can be 35, 36, 37±0.2, 38, 39 or 40°C. The time of the first generation culture, the second generation culture and the third generation culture of the present invention can be 24±2h respectively; in a specific embodiment of the present invention, the time of the first generation culture, the second generation culture and the third generation culture can be 22, 23, 24, 25 or 26h respectively. The time of the culture of the present invention can be 16-19h, and in a specific embodiment of the present invention, the time of the culture can be 16, 17, 18 or 19h respectively. The culture medium of the present invention can be a RCM liquid culture medium, and the composition of the RCM liquid culture medium can be: 10g of peptone (animal source), 10g of beef powder, 3g of yeast extract powder, 5g of glucose, 1g of soluble starch, 5g of sodium chloride, 3g of sodium acetate, 0.5g of L-cysteine ​​hydrochloride and 1L of distilled water, and the pH is adjusted to 6.80±0.10; the RCM liquid culture medium is sterilized at 121°C for 15min before use.

[0047] As an practicable manner, the method for expanding culture described in the present invention may include: inoculating the seed liquid into a culture medium for fermentation culture to obtain a fermentation liquid; the temperature of the fermentation culture is 35-40°C; the fermentation culture time may be 12-24h, or 16-22h. The culture medium described in the present invention may be an RCM liquid culture medium, and the composition of the RCM liquid culture medium may be: 10g of peptone (animal source), 10g of beef powder, 3g of yeast extract powder, 5g of glucose, 1g of soluble starch, 5g of sodium chloride, 3g of sodium acetate, 0.5g of L-cysteine ​​hydrochloride and 1L of distilled water, and the pH is adjusted to 6.80±0.10; the RCM liquid culture medium is sterilized at 121°C for 15min before application.

[0048] As an implementation method, after obtaining the fermentation liquid, the present invention centrifuges the fermentation liquid to obtain a cell concentrate. The centrifugal speed can be 4000×g, the centrifugal time can be 10 minutes, and the centrifugal temperature can be 4° C. After the centrifugation, the supernatant is removed, and the obtained bacterial cells are the cell concentrate.

[0049] As an practicable method, the drying method of the present invention may include vacuum freeze drying; the parameters of the vacuum freeze drying of the present invention may include sequentially performing first stage drying, second stage drying, third stage drying, fourth stage drying, fifth stage drying, sixth stage drying and seventh stage drying. The temperature of the first stage drying of the present invention may be -45 to -40°C, or -45 to -43°C; the time may be 120 to 180 minutes, or 130 to 160 minutes. The temperature of the second stage drying of the present invention may be -30 to -25°C, or -30 to -28°C; the time may be 180 to 240 minutes, or 200 to 240 minutes. The temperature of the third stage drying of the present invention may be -25 to -20°C, or -25 to -23°C, and the time may be 600 to 900 minutes, or 800 to 900 minutes. The temperature of the fourth stage drying of the present invention can be -10 to 0 ° C, or -10 to -5 ° C, and the time can be 180 to 300 minutes, or 260 to 300 minutes; the temperature of the fifth stage drying of the present invention can be 10 to 15 ° C, or 10 to 12 ° C, and the time can be 180 to 300 minutes, or 240 to 300 minutes. The temperature of the sixth stage drying of the present invention can be 15 to 20 ° C, or 18 to 20 ° C; the time can be 120 to 180 minutes, or 150 to 180 minutes. The temperature of the seventh stage drying of the present invention can be 25 to 30 ° C, or 28 to 30 ° C; the time can be 60 to 90 minutes, or 60 to 70 minutes. The vacuum degrees of the first stage drying, the second stage drying, the third stage drying, the fourth stage drying, the fifth stage drying, the sixth stage drying, and the seventh stage drying of the present invention are respectively >0 and ≤0.3 mbar, or 0.1 to 0.2 mbar. Vacuum Freeze Drying (VFDT) is a common method for preparing probiotic powder or other microbial products. The process combines the two steps of freezing and vacuum drying to maximize the retention of probiotic bacterial activity and cell structure. The advantage of this technology is that it can be dried at a lower temperature, reducing the loss of heat-sensitive substances. In addition, the low temperature during the vacuum drying process helps to maintain the biological activity of the microorganisms.

[0050] As an practicable manner, the water content of the lyophilized powder is ≤5.0%.

[0051] Cell activity is an important detection index of cell biology experiments and the basis of cell culture and physiological research. It can be used to evaluate cell activity, metabolic activity, apoptosis, etc. The present invention proposes a rapid screening method for lyoprotectants based on cell activity. The method has the characteristics of rapidity, accuracy, and high throughput, and can screen a variety of lyoprotectants for a single strain in a short time. The rapid screening method for lyoprotectants proposed in the present invention can save a lot of time for the production of industrial probiotic freeze-dried powder, and improve the screening accuracy, and has broad application prospects. The present application can provide a rapid and accurate screening method for lyoprotectants used in the vacuum freeze-drying process of probiotics. The invention is suitable for the screening of bifidobacterium lyoprotectants and has great development and market application value.

[0052] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1 Isolation and identification of Bifidobacterium breve BX-18

[0054] The strain BX-18 was isolated from the feces of healthy elderly people in Enshi City, Hubei Province in 2018. The DNA sample of the strain BX-18 was extracted, and Shanghai Meiji Biopharmaceutical Technology Co., Ltd. was commissioned to conduct 16s rDNA molecular identification. The 16s rDNA sequence was compared with the NCBI database Blast, confirming that the strain BX-18 was Bifidobacterium breve, and its 16srDNA sequence is shown in SEQ ID NO.1.

[0055]

[0056] The morphology of Bifidobacterium breve BX-18 was observed under a microscope, and it was known that Bifidobacterium breve BX-18 was a Gram-positive bacterium, and the bacterial body was in the shape of V or Y. When Bifidobacterium breve BX-18 was cultured on RCM solid medium, the colony diameter was 2 to 3 mm, the colony was milky white or white, and the texture was soft.

[0057] Bifidobacterium breve BX-18 has passed the artificial gastrointestinal fluid simulation experiment and the acid and bile resistance test, and has good gastrointestinal fluid resistance. When prepared by high-density fermentation, the bacteria maintain a high fermentation activity. The freeze-dried bacterial powder of the strain can be used as a direct-injection starter for the preparation of fermented yogurt, which can improve the taste and nutritional value of fermented yogurt. It is proved that the Bifidobacterium breve BX-18 of the present invention has broad application prospects in maintaining the balance of intestinal microecology.

[0058] The rapid screening method for freeze-drying protective agents proposed by the present invention is applicable to the genus Bifidobacterium, and can be applied to the screening of freeze-drying protective agents of Bifidobacterium breve BX-18, Bifidobacterium animalis lactis subspecies Probio-M8, Bifidobacterium animalis lactis subspecies V9, Bifidobacterium animalis lactis subspecies ProSci-246, Bifidobacterium breve B2798 and Bifidobacterium longum infantis subspecies B8762. Taking Bifidobacterium breve BX-18 as an example to screen freeze-drying protective agents, the specific implementation is as follows.

[0059] Example 2 Screening of carbohydrate freeze-drying protective agents for Bifidobacterium breve BX-18

[0060] Carbohydrate-based protectants are the preferred protectants for freeze-drying, as they can easily replace the water molecules removed during the drying process, thereby maintaining cell integrity.

[0061] 1. Preparation of Cell Concentrate

[0062] RCM liquid culture medium: peptone (animal source) 10g, beef powder 10g, yeast extract powder 3g, glucose 5g, soluble starch 1g, sodium chloride 5g, sodium acetate 3g, L-cysteine ​​hydrochloride 0.5g and distilled water 1000mL, adjust the pH to 6.80±0.10; sterilize at 121℃ for 15min.

[0063] RCM solid culture medium: peptone (animal source) 10g, beef powder 10g, yeast extract powder 3g, glucose 5g, soluble starch 1g, sodium chloride 5g, sodium acetate 3g, L-cysteine ​​hydrochloride 0.5g, agar 0.5g and distilled water 1000mL, adjust the pH to 6.80±0.10; sterilize at 121℃ for 15min.

[0064] (1) Seed culture

[0065] The Bifidobacterium breve BX-18 frozen and stored at -80°C was inoculated into RCM liquid culture medium and anaerobically cultured at 37°C for 24±2h to obtain a first-generation bacterial solution; the first-generation bacterial solution was again inoculated into RCM liquid culture medium at an inoculum volume fraction of 2% (v / v) and anaerobically cultured at 37°C for 24±2h to obtain a second-generation bacterial solution, which was continuously passaged to the third generation, and the obtained third-generation bacterial solution was anaerobically cultured at 37°C for 18h to obtain a seed solution.

[0066] (2) Expanding training

[0067] The seed liquid was inoculated into RCM liquid culture medium at a volume ratio of 5%, and then sodium hydroxide was added to adjust the initial pH of the fermentation liquid to 6.50±0.02, the nitrogen pressure was maintained at 0.02-0.03MPa, the stirring speed was set to 100rpm, and then fermented at a temperature of 37.0±0.2°C for 12h to the end of fermentation, and ammonia water was used to maintain the pH of the fermentation liquid constant at (5.50-6.00)±0.02.

[0068] (3) Post-treatment of fermentation broth

[0069] The fermentation broth that has reached the end point of fermentation is centrifuged at high speed (4000×g, 10min) to remove the supernatant to obtain the cell concentrate, which is then stored anaerobically for later use. The fermentation end point is determined by measuring the OD value of the fermentation broth every hour. 600 Value, fermentation broth OD within 1 hour 600 If there is no significant difference between the values, it is judged as the fermentation endpoint.

[0070] 2. Preparation of freeze-dried bacterial powder

[0071] Glucose, fructose, galactose, ribose, xylose, trehalose, sucrose, lactose, maltose, raffinose, stachyose, mannose, sorbitol, galacto-oligosaccharides, xylooligosaccharides, isomalto-oligosaccharides, maltodextrin and rhamnose were selected as carbohydrate protective agents; various single protective agents were prepared into solutions with a final concentration of 100 g / L.

[0072] The cell concentrate obtained by fermentation was fully mixed with the prepared protective agent solution in a mass ratio of 1:1, and then vacuum freeze-dried to obtain freeze-dried bacterial powder. The vacuum freezing parameters are: freeze-dried at -45℃ for 180min, freeze-dried at -30℃ for 240min, freeze-dried at -25℃ for 900min, freeze-dried at -10℃ for 300min, freeze-dried at 10℃ for 300min, freeze-dried at 20℃ for 180min, and freeze-dried at 30℃ for 60min; the vacuum degree of vacuum freezing is 0.2mbar. The water content of freeze-dried bacterial powder is ≤5.0%.

[0073] 3. Detection Methods

[0074] The freeze-dried bacterial powder was stained with SYTO9 and PI combined stain for 15 minutes and then tested by flow cytometry. The specific detection process is as follows:

[0075] (1) Dyes

[0076] PI dye: Propidium iodide (PI) is a commonly used nuclear fluorescent dye that can be embedded between base pairs to bind to double-stranded DNA. PI cannot penetrate the cell membrane and is excluded from living cells, but it can pass through damaged cell membranes to stain the nucleus. The excitation wavelength of PI is 488nm and the emission wavelength is 617nm.

[0077] SYTO9 dye: fluorescent nucleic acid stain SYTO9 (SYTO TM 9GreenFluorescentNucleicAcidStain) can penetrate into prokaryotic and eukaryotic cell membranes. SYTO 9 has an excitation wavelength of 488nm and an emission wavelength of 513nm.

[0078] (2) On-line process

[0079] 1) Detection of live cell count of freeze-dried bacterial powder: Take 0.5 g of freeze-dried bacterial powder and dilute it 1000 times with sterile saline to maintain the total number of cells in the bacterial suspension at 10 4 ~10 7 Take 980 μL of the bacterial suspension, add 10 μL of PI dye and 10 μL of SYTO9 dye, and incubate in the dark for 15 min for staining.

[0080] Take 200 μL of the stained bacterial solution and mix it with an equal volume of absolute counting fluorescent microspheres, the concentration of the fluorescent microspheres is 1022 / μL, and mix thoroughly to obtain a bacterial sample.

[0081] The prepared bacterial samples were subjected to flow cytometry analysis. The light source for flow cytometry (FCM) detection was an argon ion laser, the argon ion excitation wavelength was 488nm, and the emission wavelength was greater than 600nm. 100,000 cells were collected from each freeze-dried bacterial powder sample obtained with each protective agent, and the number of live cells and the number of fluorescent microspheres were collected and recorded. The number of cells to be detected per unit volume was calculated according to the following formula:

[0082] The number of cells to be detected per unit volume = (A×B) / (C×D), where A is the number of cells observed; B is the absolute number of fluorescent microspheres; C is the number of fluorescent microspheres observed; and D is the volume of the liquid.

[0083] The test result of freeze-dried bacterial powder is the number of live cells after freeze-drying (i.e., the number of living cells), and the results are shown in Table 1.

[0084] Unstained bacterial suspension, i.e., bacterial suspension without added dye, was used as negative control.

[0085] 2) Detection of viable cell count in cell concentrate: The detection process is the same as that of freeze-dried bacterial powder, the only difference is that 0.5 g of freeze-dried bacterial powder is replaced by 0.5 g of cell concentrate.

[0086] The unstained cell concentrate, i.e., the cell concentrate without dye added, was used as a negative control. The test result of the cell concentrate is that the total cell count of the cell concentrate is the sum of the number of live cells and the number of dead cells, and the total cell count of the cell concentrate is the total cell count before lyophilization. The results are shown in Table 1.

[0087] It can be seen that among the 18 carbohydrate protective agents, when the concentration of each protective agent is 100g / L, the survival rate of Bifidobacterium breve BX-18 is from high to low: trehalose 75.93%>raffinose 41.06%>sucrose 40.82%. In summary, the present application concludes that the best carbohydrate protective effect for Bifidobacterium breve BX-18 is trehalose, and its concentration is 100g / L. When trehalose is used as a protective agent, the cell membrane integrity of Bifidobacterium breve BX-18 is tested, and the cell membrane integrity is shown in Figure 1 .according to Figure 1 It was found that the survival rate of Bifidobacterium breve BX-18 was 75.93%.

[0088] Table 1 Flow cytometry results of different carbohydrate protective agents

[0089]

[0090]

[0091] Note: Significant difference was P<0.05, different letters indicate significant difference between groups; survival rate = number of living cells / total number of cells*100%.

[0092] Example 3 Screening of Bifidobacterium breve BX-18 protein freeze-drying protective agent

[0093] Protein protectants usually reduce cell damage and protein denaturation that may be caused during the freeze-drying process by slowing down the metabolic activities of cells and biomolecules.

[0094] 1. Preparation of cell concentrate: The preparation method is the same as that in Example 2.

[0095] 2. Preparation of freeze-dried bacterial powder

[0096] Beef extract powder, beef liver extract powder, fish peptone, tryptone, soy peptone, corn steep liquor, skim milk, yeast extract powder FM502, yeast extract powder FM503, yeast extract powder FM601, yeast extract powder FM802, yeast extract powder FM803, yeast extract powder FM808, yeast extract powder FM902, yeast extract powder FM985, yeast extract powder FM986, yeast peptone FP102, yeast peptone FP103, yeast peptone FP112, yeast peptone FP220, yeast peptone FP400, yeast peptone FP501 and yeast peptone FP502 are protein protective agents; a solution with a concentration of 100 g / L of each protective agent is prepared respectively.

[0097] The cell concentrate obtained by fermentation was fully mixed with the prepared single protective agent, and then vacuum freeze-dried. The parameters of vacuum freeze-drying were the same as those in Example 2 to obtain freeze-dried bacterial powder.

[0098] 3. Detection Methods

[0099] (1) Dye. Same as Example 2.

[0100] (2) On-machine process. Same as Example 2. The results are shown in Table 2. It can be seen that among the 23 protein protective agents, when the protective agent concentration is 100g / L, the survival rate of Bifidobacterium breve BX-18 is from high to low: yeast extract powder FM902> yeast peptone FP103> yeast extract powder FM502. In summary, the present application concludes that the best protein protective effect for Bifidobacterium breve BX-18 is yeast extract powder FM902, and its concentration is 100g / L. When yeast extract powder FM902 is used as a protective agent, the cell membrane integrity of Bifidobacterium breve BX-18 is tested, and the cell membrane integrity is shown in Table 2. Figure 2 .according to Figure 2 It was found that the survival rate of Bifidobacterium breve BX-18 was 14.21%.

[0101] Table 2 Flow cytometry results of different protein protective agents

[0102]

[0103] Note: Significant difference was P<0.05, different letters indicate significant difference between groups; survival rate = number of living cells / total number of cells*100%.

[0104] Example 4 Screening of buffer salt freeze-drying protective agents for Bifidobacterium breve BX-18

[0105] Buffer salt protective agents can maintain the acid-base balance of freeze-dried samples and reduce the osmotic stress effect during the freeze-drying process.

[0106] 1. Preparation of cell concentrate: The preparation method is the same as that in Example 2.

[0107] 2. Preparation of freeze-dried bacterial powder

[0108] Sodium chloride, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, calcium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate are 8 buffer salt protective agents, and each protective agent is prepared into a solution with a concentration of 30 g / L.

[0109] The cell concentrate obtained by fermentation was fully mixed with the prepared single protective agent solution, and then vacuum freeze-dried. The parameters of vacuum freeze-drying were the same as those in Example 2 to obtain freeze-dried bacterial powder.

[0110] 3. Detection Methods

[0111] (1) Dye. Same as Example 2.

[0112] (2) On-machine process. Same as Example 2. The results are shown in Table 3. It can be seen that when the concentration of the eight buffer salt protective agents is 30 g / L, the survival rate of Bifidobacterium breve BX-18 is from high to low: potassium dihydrogen phosphate > sodium carbonate > disodium hydrogen phosphate; In summary, the present application concludes that the best buffer salt protective effect for Bifidobacterium breve BX-18 is potassium dihydrogen phosphate, and its concentration is 30 g / L. The cell membrane integrity of Bifidobacterium breve BX-18 is shown in Table 3. Figure 3 .according to Figure 3 It was found that the survival rate of Bifidobacterium breve BX-18 was 57.72%.

[0113] Table 3 Flow cytometry results of different buffer salt protective agents

[0114]

[0115] Note: Significant difference was P<0.05, different letters indicate significant difference between groups; survival rate = number of living cells / total number of cells*100%.

[0116] Example 5 Screening of Antioxidant Freeze-Drying Protectants for Bifidobacterium breve BX-18

[0117] Antioxidant protective agents can be used to slow down the oxidation reaction of microbial cells and protect the cell membrane structure of bacteria.

[0118] 1. Preparation of cell concentrate: The preparation method is the same as that in Example 2.

[0119] 2. Preparation of freeze-dried bacterial powder

[0120] Ascorbic acid, glutathione, tea polyphenols, sodium ascorbate, β-carotene, and L-cysteine ​​hydrochloride are antioxidant protective agents; each protective agent is prepared into a solution with a concentration of 15 g / L.

[0121] The cell concentrate obtained by fermentation was fully mixed with the prepared single protective agent solution, and then vacuum freeze-dried. The vacuum freeze-drying parameters were the same as those in Example 2 to obtain freeze-dried bacterial powder.

[0122] 3. Detection Methods

[0123] (1) Dye. Same as Example 2.

[0124] (2) On-machine process. Same as Example 2. The results are shown in Table 4. It can be seen that among the six antioxidant protective agents, when the protective agent concentration is 15g / L, the survival rate of Bifidobacterium breve BX-18 is from high to low: glutathione> ascorbic acid> tea polyphenols. In summary, the present application concludes that glutathione has the best antioxidant protection effect on Bifidobacterium breve BX-18, and its concentration is 15g / L. The cell membrane integrity of Bifidobacterium breve BX-18 is shown in Table 4. Figure 4 .according to Figure 4 It was found that the survival rate of Bifidobacterium breve BX-18 was 16.74%.

[0125] Table 4 Protective agent flow cytometry results

[0126]

[0127] Note: Significant difference was P < 0.05, different letters abcdef indicate significant difference among groups; survival rate = number of living cells / total number of cells * 100%.

[0128] Example 6 Screening of amino acid freeze-drying protective agents for Bifidobacterium breve BX-18

[0129] 1. Preparation of cell concentrate: The preparation method is the same as that in Example 2.

[0130] 2. Preparation of freeze-dried bacterial powder

[0131] Cysteine, sodium glutamate, glutamic acid, proline, glycine, threonine, tryptophan, and glutamine are amino acid protective agents. Each protective agent is prepared at a concentration of 15 g / L. The cell concentrate obtained by fermentation is fully mixed with the prepared single protective agent, and then vacuum freeze-dried. The parameters of vacuum freeze-drying are the same as those in Example 2 to obtain freeze-dried bacterial powder.

[0132] 3. Detection Methods

[0133] (1) Dye. Same as Example 2.

[0134] (2) On-machine process. Same as Example 2. The results are shown in Table 5. It can be seen that when the protective agent concentration is 15 g / L, the survival rate of Bifidobacterium breve BX-18 is from high to low: sodium glutamate 14.24%> threonine 13.54%> glycine 11.99%; the cell membrane integrity of sodium glutamate is shown in Table 5. Figure 5 .according to Figure 5 It was found that the survival rate of Bifidobacterium breve BX-18 was 14.21%.

[0135] Table 5 Flow cytometry results of different amino acid protective agents

[0136]

[0137] Note: Significant difference was P<0.05, different letters indicate significant difference between groups; survival rate = number of living cells / total number of cells*100%.

[0138] Example 7 Verification of compounding of Bifidobacterium breve BX-18 protective agent

[0139] 100g / L trehalose, 100g / L yeast extract FM902, 30g / L potassium dihydrogen phosphate, 15g / L glutathione, and 15g / L sodium glutamate screened by single factors in Examples 2 to 6 were compounded, mixed evenly with the cell concentrate (i.e., bacterial mud) at a mass ratio of 1:1, and then freeze-dried. The preparation method of the cell concentrate was the same as in Example 2. The survival rate was determined by three repeated tests. The determination method was the same as the 3 detection methods in Example 2, and the results were shown in Table 6. The survival rate of Bifidobacterium breve BX-18 can be obtained. The average freeze-dried survival rate of Bifidobacterium breve BX-18 in the three repeated experiments reached 82.14%. It can be seen that flow cytometry screening of freeze-dried protective agents has certain application value.

[0140] Table 6 Verification test table

[0141]

[0142]

[0143] Positive verification experiment: The detection method refers to GB4789.35-2023 to detect the number of viable bacteria in the cell concentrate, and the results are shown in Table 7. It can be seen from Tables 6 and 7 that the freeze-dried survival rate evaluated by flow cytometry is 82.14%, the freeze-dried survival rate evaluated by the traditional method is 83.07%, and the accuracy of flow cytometry is 98.88%, which is high.

[0144] Table 7 Verification test table

[0145]

[0146] Note: Freeze-dried survival rate (%) = (number of live bacteria in freeze-dried powder × weight of freeze-dried powder) / (weight of concentrated bacterial sludge × number of live bacteria before freeze-drying) × 100%, where the weight of concentrated bacterial sludge is the weight before freeze-drying and the weight of freeze-dried powder is the weight after freeze-drying.

[0147] In summary, the cell activity-based lyophilization protective agent rapid screening technology provided by the present invention can accurately and efficiently complete the screening of probiotic lyophilization protective agents, and provides a new idea for the screening of probiotic lyophilization protective agents. The present invention can solve technical problems such as activity attenuation during the freeze-drying process of probiotic strains. The present invention also provides a freeze-dried powder composite protective agent for Bifidobacterium breve BX-18, which can make the survival rate of Bifidobacterium breve BX-18 in the freeze-dried Bifidobacterium breve BX-18 reach 82.14%, with high survival rate and high detection accuracy.

[0148] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of flow cytometry in screening of protective agents for freeze-dried microbial powders.

2. A method for screening a freeze-dried powder protective agent, characterized in that: include: The microorganisms are mixed with the protective agent to be screened and then dried to obtain a lyophilized powder; The total number of microbial cells before drying and the number of viable microbial cells after drying were determined by flow cytometry, and the microbial survival rate was calculated; the calculation formula of the microbial survival rate was: microbial survival rate = number of viable microbial cells after drying / total number of microbial cells before drying × 100%; Determining the freeze-dried powder protective agent according to the survival rate of the microorganisms; When the protective agent to be screened is a type of substance, the one with the highest microbial survival rate is the lyophilized powder protective agent obtained by screening; When the protective agents to be screened are more than two types of substances, the protective agent with the highest microbial survival rate in each type of substance is the screened protective agent in that type, and then the screened protective agents of different types are compounded to obtain the freeze-dried powder protective agent.

3. The screening method according to claim 2, characterized in that: The protective agents to be screened include one or more of carbohydrate lyophilization protective agents, protein lyophilization protective agents, buffer salt lyophilization protective agents, antioxidant lyophilization protective agents and amino acid lyophilization protective agents.

4. The screening method according to claim 3, characterized in that: The carbohydrate lyoprotectant comprises one or more of glucose, fructose, galactose, ribose, xylose, trehalose, sucrose, lactose, maltose, raffinose, stachyose, mannose, sorbitol, galacto-oligosaccharides, xylo-oligosaccharides, isomalto-oligosaccharides, maltodextrin and rhamnose; The protein freeze-drying protective agent includes one or more of yeast extract powder, yeast peptone, beef extract powder, beef liver extract powder and fish peptone; The buffer salt freeze-drying protective agent includes one or more of sodium chloride, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate and calcium chloride; The antioxidant freeze-drying protective agent includes one or more of glutathione, tea polyphenols, ascorbic acid, L-cysteine ​​hydrochloride, β-carotene and sodium ascorbate; The amino acid freeze-drying protective agent includes one or more of sodium glutamate, threonine, cysteine, proline, glutamic acid, tryptophan, glutamine and glycine.

5. The screening method according to claim 2, characterized in that: The microorganisms include probiotics; the probiotics include bifidobacteria; the bifidobacteria include one or more of Bifidobacterium breve, Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis.

6. The screening method according to claim 2, characterized in that: The drying method includes vacuum freeze drying.

7. The screening method according to claim 6, characterized in that: The vacuum freeze drying includes sequentially performing first stage drying, second stage drying, third stage drying, fourth stage drying, fifth stage drying, sixth stage drying and seventh stage drying; the temperature of the first stage drying can be -40 to -45°C, and the time can be 120 to 180 minutes; the temperature of the second stage drying can be -25°C to -30°C, and the time can be 180 to 240 minutes; the temperature of the third stage drying can be -20°C to -25°C, and the time can be 600 to 900 minutes; the temperature of the fourth stage drying can be -10 to 0°C, and the time can be 180 to 300 minutes; the temperature of the fifth stage drying can be 10 to 15°C, and the time can be 180 to 300 minutes; the temperature of the sixth stage drying can be 15 to 20°C, and the time can be 120 to 180 minutes; the temperature of the seventh stage drying can be 25 to 30°C, and the time can be 60 to 90 minutes.

8. The screening method according to claim 2, characterized in that: The application form of the microorganism includes microbial cell concentrate.

9. The screening method according to claim 2 or 8, characterized in that: The dyes used in flow cytometry to determine the number of viable microbial cells include PI and SYTO9.

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