Method for improving spray drying survival rate of probiotics
By optimizing the combination of carbon source culture medium and heat stress-remediation treatment, the problem of low survival rate of lactic acid bacteria in spray drying was solved, and the high survival rate and improved heat resistance of lactic acid bacteria in high temperature environment were achieved.
Patent Information
- Application Number
- CN202511863956.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-11
- Publication Date
- 2026-02-10
AI Technical Summary
Existing technologies have low survival rates during the spray drying process of lactic acid bacteria, and existing single methods have limited or insufficient effects in improving their effectiveness, making it difficult to maintain bacterial activity in high-temperature environments.
After culturing in an optimized carbon source medium, the strain underwent heat stress-repair treatment to activate its stress resistance mechanism. Through cross-stress and glutathione repair treatment, combined with a skim milk protectant, the survival rate of the strain during spray drying was improved.
It significantly improved the survival rate of lactic acid bacteria by spray drying, from 5%-10% to 60%-70%, reduced oxidative damage to cell membranes, enhanced the heat resistance of strains and the amount of biofilm formation, and protected cell integrity.
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Figure CN121495700A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of probiotic processing, and more particularly relates to a method for improving the survival rate of probiotics in spray drying. BACKGROUND
[0002] Lactic acid bacteria, as an important probiotic, are widely distributed in soil, plant surfaces, animal intestines and fermented foods. They can provide a variety of probiotic functions for the host, including regulating intestinal flora balance, inhibiting pathogenic bacteria colonization, reducing cholesterol, promoting nutrient metabolism and absorption, antioxidant and enhancing immune function, and have high application value in food, medicine and other fields.
[0003] The biological activity of lactic acid bacteria is the core of its probiotic function, but it faces severe challenges in industrial application, especially in the preparation of preparations for easy storage. Spray drying is a common method for preparing lactic acid bacteria preparations, but the high temperature, dehydration and oxidation stress conditions in the process can easily cause irreversible damage to the bacterial cells, such as destroying the peptidoglycan structure of the cell wall, inducing membrane lipid oxidation, causing DNA damage, etc., ultimately leading to a significant decrease in survival rate, which severely limits the effectiveness and application of the preparation.
[0004] To improve the heat resistance and spray drying survival rate of lactic acid bacteria, the existing technology mainly adopts four strategies: 1. Optimizing medium composition: such as adding Mg 2 ⁺ can significantly improve the heat resistance of Lactobacillus rhamnosus; 2. Mutagenesis breeding: such as obtaining a Lactobacillus acidophilus strain with an increased upper temperature limit through ultraviolet mutagenesis; 3. Microcapsule embedding: such as using emulsification to achieve a high embedding rate (more than 90%), which provides a physical barrier for the bacterial cells; 4. Stress pretreatment: including heat stress, cold stress, acid stress, oxygen stress, salt stress, alcohol stress, etc., aiming to activate the stress defense mechanism of the bacterial cells. Among them, optimizing the culture medium (especially the carbon source) and stress pretreatment are considered to have great development potential because they can effectively activate the defense mechanism of the bacterial cells, are relatively simple to operate and have lower cost.
[0005] However, the existing single means has limited improvement effect or deficiencies. For example, the traditional MRS culture medium is not the best for protecting the strain against heat damage in spray drying; microcapsule embedding can improve the embedding rate, but may increase the process complexity and cost; mutagenesis breeding has a long cycle and uncertain results; a single stress treatment may not be enough to cope with multiple damage mechanisms.
[0006] Therefore, developing a more efficient, low-cost and synergistic strategy to improve the survival rate and heat resistance of spray-dried lactic acid bacteria is still a key requirement to break through the current industrial application bottleneck. In particular, exploring a novel combination method that can simultaneously activate multiple intrinsic defense mechanisms of the bacterial body and effectively repair sub-injured structures produced during the drying process is of great significance to the development of the probiotic preparation industry. SUMMARY
[0007] In view of the deficiencies in the prior art, the purpose of the present application is to provide a new method for improving the survival rate of spray-dried probiotics, which is simple to operate and low in cost. The strain is first cultured in an optimized carbon source medium, and then subjected to heat stress-repair treatment to mobilize the strain's stress resistance mechanism. When the strain is subsequently exposed to high temperature, dehydration and other adverse environments during the spray drying process, the already formed defense mechanism can significantly reduce the damage to the bacterial body caused by the adverse environment, providing a new strategy and theoretical basis for improving the survival rate of lactic acid bacteria in high temperature environments.
[0008] The present application provides a method for improving the survival rate of spray-dried probiotics, which is achieved by the following specific technical means: A method for improving the survival rate of spray-dried probiotics, comprising the following steps: (1) Strain activation: freeze-stored Lactobacillus plantarum (Lactobacillus plantarum LIP-1, which was deposited in the Inner Mongolia Agricultural University Dairy Biotechnology and Engineering Ministry Key Laboratory on September 20, 2015, with the preservation number IMAU NO.12174) is inoculated into 10% skim milk medium (10g skim milk powder is added to 90ml distilled water), and is cultured in a 37℃ constant temperature incubator for activation. After coagulation treatment, it is transferred to a pH 6.8 basic MRS liquid medium at a 4% inoculation amount, and is cultured at 37℃ for 16-20h for activation and expansion. After two consecutive passages, the bacterial slurry is collected by centrifugation, washed twice with sterilized physiological saline, resuspended in 2ml sterile water to obtain a concentrated bacterial solution, shaken and mixed, and then poured into a basic MRS agar medium for culture. The number of viable bacteria is accurately calculated, and the bacterial body before the stable period is preserved at 4℃ for standby.
[0009] (2) Optimization of liquid culture: Additional 20% sucrose, L-alanyl-L-glutamine and galactose optimization liquid (ratio 2.5:1:0.5, hereinafter referred to as 20% optimization liquid) is added in the early logarithmic growth phase. Among them, MRS liquid medium is selected as the basic enrichment culture medium of Lactobacillus plantarum, and the formula is as follows (based on 1L medium): anhydrous glucose 20g, vegetable peptone 10g, yeast powder 5g, anhydrous sodium acetate 5g, anhydrous potassium phosphate dibasic 2g, sodium citrate 2g, anhydrous manganese sulfate 0.05g, magnesium sulfate heptahydrate 0.2g, distilled water 1L. After the second generation of bacteria is activated, the logarithmic phase cell suspension is vortexed, inoculated into the MRS liquid medium with pH=6.8 at 4% inoculation amount, grown for 10 hours, then 20mL of 20% optimization liquid is added, and the strain is cultured for 18 hours (stationary phase) to harvest the strain.
[0010] (3) Cross-stress treatment: centrifuge the bacterial suspension (4000xg, 5min, 4℃), wash with sterile physiological saline for three times, then resuspend in phosphate buffered saline solution (hereinafter referred to as: PBS) with pH value of 3-4, add 5mol / L KCl solution to the culture medium, adjust the final concentration to 0.7 mol / L, and additionally add 26 g / L trehalose + oligofructose compound solution (ratio 1:0.5), and stress culture at 37℃ for 1h.
[0011] (4) Comprehensive repair treatment: centrifuge the bacterial suspension after cross-stress treatment (4000xg, 5min), and repair in PBS+0.75mol / L glutathione solution at 37℃ constant temperature for 45min.
[0012] (5) The preparation of protective agent is as follows: add defatted milk powder slowly into preheated 45℃ distilled water at 20%, and mix well with a magnetic stirrer, then seal and place in a 85℃ water bath for pasteurization for 30min, then quickly cool with cold water and store in a 4℃ refrigerator.
[0013] (6) The preparation of bacterial cell and protective agent mixed solution is as follows: use a low-speed refrigerated centrifuge to centrifuge the treated sample at 4000xg for 15min, discard the supernatant, wash the cell pellet with sterile physiological saline (pH 6.5) for 2 times, then resuspend in an equal volume of 20% defatted milk protective agent, mix the protective agent and cells thoroughly, and store in a 4℃ refrigerator.
[0014] (7) Spray drying treatment: set the inlet temperature to 170±10℃, the outlet temperature to 65±5℃, the feeding amount to 8mL / min, and the atomization pressure to 0.3MPa. The prepared mixed sample is introduced into the spray drying tower for spray drying.
[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method to improve the survival rate of probiotics by spray drying. Using this invention, the survival rate of probiotics by spray drying can be increased from 5%-10% to 60-70%. Furthermore, the optimized carbon source combination with the heat stress-repair process reduces oxidative damage to the cell membrane, increases the amount of biofilm formed by the strain, which is beneficial to protecting the cell membrane integrity of the strain, reducing the degree of heat damage to the cell membrane, and improving the heat resistance of the strain. Attached Figure Description
[0016] Figure 1 To optimize the effect of carbon source combined with heat stress-repair treatment on the viable count and survival rate of probiotics after spray drying (Note: A represents the control group grown in MRS medium, B represents the optimized carbon source group with 2% sucrose added to the control group, C represents the control group subjected to heat stress-repair treatment, and D represents the control group subjected to heat stress-repair treatment after optimized carbon source MRS medium. Different uppercase and lowercase letters indicate significant differences in survival rate and viable count between groups (P < 0.05), and results are expressed as mean ± standard deviation).
[0017] Figure 2 To optimize the effect of carbon source combined with heat stress-repair treatment on the metabolic activity of probiotic cells (Note: Higher cell metabolic activity corresponds to a darker color, higher absorbance, and less cell damage). A represents the metabolic activity of each group before spray drying, from left to right: a1 optimized carbon source group underwent heat stress-repair treatment, a2 optimized carbon source group underwent heat stress treatment, a3 optimized carbon source group. B represents the cell metabolic activity of each group after spray drying, from left to right: b1 optimized carbon source combined with heat stress-repair treatment group, b2 optimized carbon source group, b3 heat stress-repair treatment, b4 control group. Groups in C are the same as in A and B. Figure 1 (To).
[0018] Figure 3 To observe the cellular microstructure of probiotics using transmission electron microscopy (Note: A is the control group, B is the optimized carbon source group, C is the heat stress treatment group, D is the heat stress-repair treatment group, and E is the optimized carbon source combined with heat stress-repair treatment group. Red arrows represent cell wall thickness (a), black arrows represent the degree of cell membrane integrity (b), and blue arrows represent the degree of homogeneity of intracellular substances (c)).
[0019] Figure 4 The image shows the surface morphology of spray-dried bacterial powder particles under a scanning electron microscope (Note: A represents the control group grown in MRS medium; B represents the optimized carbon source group with 2% sucrose added to the carbon source of the control group; C represents the control group that underwent heat stress-repair treatment; and D represents the combined treatment group that underwent heat stress-repair treatment after optimizing the carbon source of the control group. The spray drying protectant was skim milk). Detailed Implementation
[0020] The present invention will be further described below with reference to specific embodiments.
[0021] In this invention, unless otherwise specified, all equipment and raw materials are available from the market or commonly used in the industry. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.
[0022] Example The present invention provides a method for improving the survival rate of probiotics by spray drying, comprising the following steps: (1) Activation of strain: The frozen Lactobacillus plantarum LIP-1 (deposited on September 20, 2015 at the Key Laboratory of Dairy Biotechnology and Engineering of the Ministry of Education of Inner Mongolia Agricultural University, accession number IMAU NO.12174) was inoculated into 10% skim milk medium (10g skim milk powder was added to 90ml of distilled water) and activated in a constant temperature incubator at 37℃. After curdling treatment, it was transferred to basic MRS liquid medium at pH 6.8 at an inoculation rate of 4% and cultured at 37℃ for 16-20h for activation and amplification. After two consecutive subcultures, the bacterial sludge was collected by centrifugation, washed twice with sterile physiological saline, and resuspended in 2ml of sterile water to obtain concentrated bacterial solution. After shaking and mixing, it was poured into basic MRS agar medium for culture. The number of viable bacteria was accurately calculated and the early stage of the stationary phase was stored at 4℃ for later use.
[0023] (2) Optimized culture medium: During the early stage of logarithmic growth, an optimized culture medium containing 20% sucrose, L-alanyl-L-glutamine, and galactose (in a ratio of 2.5:1:0.5, referred to as the 20% optimized culture medium) was added. MRS liquid medium was selected as the basic enrichment medium for *Lactobacillus plantarum*, with the following specific formula (based on 1L of medium): 20g anhydrous glucose, 10g plant peptone, 5g yeast extract, 5g anhydrous sodium acetate, 2g anhydrous dipotassium hydrogen phosphate, 2g sodium citrate, 0.05g anhydrous manganese sulfate, 0.2g magnesium sulfate heptahydrate, and 1L distilled water. After activating the second-generation bacterial culture, the cell suspension in the late logarithmic phase was vortexed and transferred to MRS liquid medium at pH 6.8 at a 4% inoculum. After 10 hours of growth, 20mL of the 20% optimized culture medium was added, and the culture was continued for 18 hours (stationary phase) before harvesting the strain.
[0024] (3) Cross-stress treatment: Centrifuge the bacterial suspension (4000×g, 5min, 4℃), wash three times with sterile physiological saline, and resuspend in phosphate buffer solution (PBS) with pH 3-4. Add 5mol / L KCl solution to the culture medium to adjust the final concentration to 0.7 mol / L, and add an additional 26 g / L trehalose + fructooligosaccharide complex solution (ratio 1:0.5). Stress culture at 37℃ for 1 h.
[0025] (4) Comprehensive repair treatment: Centrifuge the bacterial suspension after cross-stress treatment (4000×g, 5min), resuspend it in an equal volume of PBS + 0.75mol / L glutathione solution, and repair it at 37℃ for 45min.
[0026] (5) The preparation of the protective agent is as follows: 20% of the skim milk powder is slowly added to preheated 45°C distilled water and mixed with a magnetic stirrer. After sealing, it is placed in an 85°C water bath for pasteurization for 30 min, then cooled with cold water and placed in a 4°C refrigerator for later use.
[0027] (6) The preparation of the bacterial cell and protectant mixture is as follows: The treated sample was centrifuged at 4000×g for 15 min using a low-speed refrigerated centrifuge. After discarding the supernatant of the culture medium, the cell pellet was obtained. The cell pellet was washed twice with sterile physiological saline (pH 6.5) and then resuspended in an equal volume of 20% skim milk protectant. After the protectant and cells were fully mixed, the mixture was temporarily stored in a 4℃ refrigerator.
[0028] (7) Spray drying: The inlet temperature was set to 170±10℃, the outlet temperature to 65±5℃, the feed rate to 8mL / min, and the atomization pressure to 0.3 MPa. The pre-prepared mixed sample was introduced into the spray drying tower for spray drying.
[0029] Test case I. Determination of survival rate during spray drying: Samples were taken from the culture medium before spray drying and from the bacterial powder sample after spray drying. The viable bacteria were counted in MRS solid medium at 37℃ for 36-48 h according to the viable bacteria counting method, and the survival rate was calculated. The formula is as follows.
[0030] In the formula: N1 is the number of viable bacteria collected in the bacterial powder after spray drying (CFU / g); W is the number of grams of bacterial powder obtained after spray drying (g); N2 is the number of viable bacteria before spray drying (CFU / mL); V is the volume of the sample before spray drying (mL).
[0031] The results show (see) Figure 1After optimizing the carbon source and heat stress-repair treatment, the survival rate of the strain during spray drying reached the highest level, which was 67.16±1.85%.
[0032] II. Determination of Cell Viability after Spray Drying: Cells were collected from the treated sample by centrifugation (4000×g, 4℃, 5 min). The cell pellet was washed with phosphate-buffered saline (PBG) containing glucose and resuspended in PBG at an equal volume. For the iodonitrobenzyl chloride (INT) assay, the cell suspension was mixed with INT stock solution at a 1:2 volume ratio, reacted at 37℃ for 10 min, and then the sample was simultaneously diluted. The absorbance of the reaction solution was read at 595 nm using a microplate reader. Higher absorbance values and a deeper red color indicate higher cell metabolic activity and less cell damage.
[0033] The results show (see) Figure 2 ): Optimized carbon source combined with heat stress-repair treatment can further reduce cell damage on the basis of single treatment, and provide superimposed protection for strains during spray drying.
[0034] III. Observation of cell morphology by transmission electron microscopy (see...) Figure 3 The pretreated cell culture medium was centrifuged, the supernatant was discarded, and the cells were collected. 2.5% glutaraldehyde was added, and the cells were fixed overnight at 4°C. The fixed cells were then collected by centrifugation and washed four times with 0.1 mol / L phosphate buffer for 15 min each time. Next, the cells were fixed for 2–4 h at room temperature (20°C) with 1% osmium tetroxide-0.1 mol / L phosphate buffer (pH 7.2). The samples were washed with PBS buffer until all acid was removed. The resulting cells were dehydrated in a gradient of different ethanol concentrations, followed by dehydration with 100% acetone. The samples were then soaked sequentially in a mixture of acetone and epoxy resin, and finally soaked in pure epoxy resin for 12 h to ensure complete resin penetration. The permeated samples were transferred to embedding plates, and epoxy resin was added as the embedding agent. After addition, the embedding plates were placed in a 60°C incubator for 48 h of polymerization. The sample was cut into slices approximately 70 nm thick and dried. The dried slices were placed on the transmission electron microscope (TEM) sample holder, the TEM parameters were adjusted, and images were captured from representative fields of view.
[0035] The results showed that the optimized carbon source combined with heat stress-repair treatment did indeed have a synergistic protective effect on *Lactobacillus plantarum*. The cell wall of the strain thickened while its integrity improved, and the cell membrane was more stable under adverse conditions after the damage was repaired. This avoided the decline in cell metabolic activity caused by the leakage of cell contents (such as key enzymes).
[0036] IV. Scanning Electron Microscopy Observation of the Spray-Dried Bacterial Powder Surface: The treated and untreated samples were centrifuged (4℃, 8000×g, 10 min), washed twice with PBS (pH 6.4), and the cell pellet was collected. Bacteria were fixed with 2.5% (v / v) glutaraldehyde (4℃, 4 h). The cells were then dehydrated sequentially in a series of ethanol gradients. Finally, the samples were sprayed onto a copper grid, dried at the critical point, and gold-plated. Images were then taken using a scanning electron microscope (SEM).
[0037] The results show (see) Figure 4 ): Optimized carbon source combined with heat stress-repair treatment improves the dispersibility of Bacillus plantarum powder particles, reduces the degree of powder aggregation, and improves the storage stability of the strain.
[0038] In conclusion, the above test results demonstrate that the optimized carbon source combined with heat stress-repair treatment provided by this invention can coordinate and stimulate multiple protective mechanisms of the strain, protect the cell membrane integrity of the strain, and improve the survival rate of the strain during spray drying. This provides a reliable pretreatment technology solution for the high-temperature application of probiotics, and has significant practical value and promising prospects for promotion.
[0039] The above description is merely an example to further illustrate the technical content of the present invention, so as to facilitate the reader's understanding. However, it does not mean that the implementation of the present invention is limited to this. Any technical extension or re-creation made in accordance with the present invention is protected by the present invention.
Claims
1. A method for improving the survival rate of Lactobacillus plantarum in spray drying, characterized in that, Includes the following steps: (1) Activation of strain: Lactobacillus plantarum LIP-1 was inoculated in 10% skim milk medium and activated at 37°C until curd. It was then transferred to basic MRS liquid medium at pH 6.8 at a 4% inoculation rate and cultured at 37°C for 16-20 h. After two consecutive subcultures, the bacterial sludge was collected by centrifugation, washed with sterile physiological saline and resuspended to obtain concentrated bacterial solution. The basic MRS liquid culture medium formula is as follows: glucose 20g / L, plant peptone 10g / L, yeast powder 5g / L, anhydrous sodium acetate 5g / L, anhydrous dipotassium hydrogen phosphate 2g / L, sodium citrate 2g / L, anhydrous manganese sulfate 0.05g / L, and magnesium sulfate heptahydrate 0.2g / L. (2) Optimized culture: The bacterial culture obtained in step (1) was inoculated into the basic MRS liquid medium at an inoculation rate of 4%. After culturing for 10 hours, a composite optimized culture containing sucrose, L-alanyl-L-glutamine and galactose was added with a mass ratio of 2.5:1:0.
5. The culture was continued until the stationary phase. (3) Cross-stress treatment: The bacterial cells were collected by centrifugation, washed with sterile physiological saline, resuspended in PBS buffer at pH 3-4, and KCl solution with a final concentration of 0.7 mol / L and trehalose-fructooligosaccharide complex were added. The cells were then subjected to stress at 37°C for 1 h. (4) Comprehensive repair treatment: After stress, the bacterial suspension was centrifuged and resuspended in PBS solution containing 0.75 mol / L glutathione, and repaired at 37°C for 45 min; (5) Mixing of protectants: Centrifuge to collect the repaired bacterial cells, wash with sterile physiological saline, and resuspend in 20% skim milk protectant; (6) Spray drying: Drying is carried out at an inlet temperature of 170±10℃, an outlet temperature of 65±5℃, a feed rate of 8mL / min, and an atomization pressure of 0.3MPa.
2. The method for improving the survival rate of probiotics by spray drying as described in claim 1, characterized in that: In step (1), the bacterial sludge is washed with physiological saline and then resuspended in sterile water. The number of viable bacteria is accurately calculated by pour culture, and the bacteria in the early stage of the stable period are stored at 4°C.
3. The method for improving the survival rate of probiotics by spray drying as described in claim 1, characterized in that: The amount of the composite optimization solution added in step (2) is 20 mL / L of basic MRS medium.
4. The method for improving the survival rate of probiotics by spray drying as described in claim 1, characterized in that: The preparation method of the 20% skim milk protectant in step (5) is as follows: dissolve skim milk powder in distilled water at 45°C to prepare a 20% solution, pasteurize at 85°C for 30 minutes, and then rapidly cool to 4°C for later use.
5. The method for improving the survival rate of probiotics by spray drying as described in claim 1, characterized in that: In step (5), the volume ratio of bacterial cells to skim milk protectant is 1:1, and the mixture is temporarily stored at 4°C.
6. The method for improving the survival rate of probiotics by spray drying as described in claim 1, characterized in that: The centrifugation conditions in steps (3) and (4) are 4000×g, 5min, and 4℃.
7. The method for improving the survival rate of probiotics by spray drying as described in claim 1, characterized in that: The sterile saline solution used in step (5) has a pH of 6.5.