Application of beta-carotene in vacuum freeze drying of lactobacillus paracasei

Abstract

The invention discloses an application of beta-carotene in vacuum freeze drying of lactobacillus paracasei. It is found for the first time that beta-carotene can significantly increase the viable count of lactobacillus paracasei in the vacuum freeze drying process. When the beta-carotene is independently used for freeze-drying the lactobacillus paracasei, the viable count of the lactobacillus paracasei can reach 2.38 * 10 < 12 > CFU / g. Compared with other types of protective agents or composite protective agents, the lactobacillus paracasei freeze-drying protective agent has the advantages that the viable count of lactobacillus paracasei in the freeze-drying process can be remarkably increased, the optimal protective effect can be achieved with the low protective agent concentration, and the lactobacillus paracasei freeze-drying protective agent has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to the application of β-carotene in the vacuum freeze-drying of Lacticaseibacillus paracasei. Background Art

[0002] The potential effects of probiotics on human health have been widely confirmed, and they are applied in many aspects such as food fermentation, yogurt production, milk powder formula, dietary supplements, etc. Lacticaseibacillus paracasei is one of the members of the probiotic family, a Gram-positive bacterium, facultative anaerobic, commonly found in the human intestine and oral cavity, and is a common probiotic candidate strain. Its physiological characteristics include acid resistance, bile salt resistance, the ability to adhere to intestinal epithelial cells for stable colonization, and the metabolism to produce beneficial substances such as lactic acid and γ-aminobutyric acid.

[0003] Most probiotic cultures need to be refrigerated during storage and transportation. During this process, it is difficult to maintain the activity of probiotics, the required cost is high and it is not convenient to use. Usually, probiotics are dried to prepare powder, and the powder has better stability, lower storage and transportation costs and is convenient to use.

[0004] In current preparation methods, the vacuum freeze-drying technology has become an ideal method for industrially preparing high-activity and high-stability powder due to its wide application range, high strain survival rate, long powder preservation time, and high strain stability. However, the freeze-drying process may damage the cell membrane structure and function of probiotics, and inactivate sensitive proteins and key enzyme activities. Therefore, usually a cryoprotectant is added before the strain is freeze-dried to reduce various damages suffered by the cells during the freeze-drying process and maximize the retention of various original physiological and biochemical characteristics and biological activities of the cells.

[0005] Currently, a large number of studies focus on the screening and optimization of cryoprotectants, and there are many studies on traditional cryoprotectants such as sugars, proteins, and polyols, and most of those with a high viable cell count are also compound cryoprotectant combinations. For example: Zhu Hong (CN106754525A) used a composite freeze-drying cryoprotectant: 20-30 parts of maltodextrin, 20-30 parts of trehalose, 19-29 parts of L-sodium glutamate, 15-25 parts of glycerol, 3-9 parts of galactomannan, and the viable cell count of the powder prepared under pre-freezing and low-temperature high-speed centrifugation (5°C, 8000r / min) was 6.0×1011 CFU / mL. Man Chaoxin (CN 111286469 B) optimized to obtain a compound freeze-drying cryoprotectant combination: 15% skim milk powder, 20% sucrose, 7% polyvinylpyrrolidone, 1.3% glutathione. The viable cell count of the Lacticaseibacillus paracasei powder prepared by this method was only 2.35×10 11 CFU / g. Although the above methods improved the viable cell count of Lacticaseibacillus paracasei, the viable cell count was relatively low and did not reach 1×10 12CFU / g, and the cryoprotectants used are mostly compound cryoprotectants with many components and complex operations. Therefore, there is an urgent need to find a new cryoprotectant with simple components that can significantly increase the viable count of freeze-dried Lactobacillus paracasei. SUMMARY OF THE INVENTION

[0006] An object of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide the use of β-carotene in the preparation of a freeze-drying protectant for Lactobacillus paracasei.

[0007] Another object of the present invention is to provide a preparation method of the above composite protectant.

[0008] Another object of the present invention is to provide the use of the above composite protectant.

[0009] The object of the present invention is achieved by the following technical solutions:

[0010] Use of β-carotene in the preparation of a freeze-drying protectant for Lactobacillus paracasei.

[0011] Use of β-carotene in the freeze-drying protection of Lactobacillus paracasei.

[0012] Use of β-carotene in increasing the viable count of freeze-dried Lactobacillus paracasei.

[0013] Use of β-carotene in reducing the cell membrane permeability and / or fluidity during the freeze-drying of Lactobacillus paracasei.

[0014] Use of β-carotene in maintaining the integrity of the cell membrane during the freeze-drying of Lactobacillus paracasei.

[0015] A preparation method of a Lactobacillus paracasei powder, comprising the following steps:

[0016] Prepare a bacterial sludge, and mix the bacterial sludge with β-carotene at a mass ratio of 1:3 to 7, preferably 1:5; quick-freeze at -90°C to -70°C for 1 to 3 h, preferably quick-freeze at -80°C for 2 h, and then perform freeze-drying. The freeze-drying parameters are: temperature -90°C to -70°C, vacuum degree 0.4 to 0.6 Pa, time 20 to 30 h; preferably: temperature -65°C, vacuum degree 0.5 Pa, time 24 h.

[0017] Further, the preparation method of the bacterial sludge is: inoculate the activated Lactobacillus paracasei liquid into MRS liquid medium at an inoculation amount of 2 to 6%, culture for 12 to 16 h, centrifuge at 4000 to 8000 r / min for 10 to 30 min to collect the bacterial sludge, and wash it 1 to 3 times in sterilized 0.85% physiological saline or phosphate buffer.

[0018] Further, the activation method is as follows: streak the bacterial strain on an MRS plate, pick a single colony and transfer it to an MRS liquid medium, culture at 37°C for 20 - 30 h, and subculture 2 - 3 times.

[0019] Further, the preferred culture condition is to culture at 37°C for 14 h.

[0020] Further, the preferred centrifugation condition is to centrifuge at 6000 r / min for 10 min.

[0021] A Lactobacillus paracasei powder is obtained by the above preparation method.

[0022] The application of the above Lactobacillus paracasei powder in the preparation of food additives.

[0023] The present invention has the following advantages and effects compared with the prior art:

[0024] The present invention provides the application of β-carotene in the preparation of a cryoprotectant for Lactobacillus paracasei. β-carotene can significantly increase the viable count of Lactobacillus paracasei during the freezing process. When using β-carotene alone for freeze-drying Lactobacillus paracasei, the viable count of Lactobacillus paracasei can reach 2.38×10 12 CFU / g. Compared with the composite protectant, it has a simple composition and a significant advantage in viable count.

[0025] Further experiments found that β-carotene has the effect of regulating the permeability, fluidity, and stability of the cell membrane. β-carotene can effectively increase the viable count of Lactobacillus paracasei powder by reducing the permeability of the cell membrane, increasing the fluidity of the cell membrane, and enhancing the activity of Na + -K + -ATPase in the cell. Description of the Drawings

[0026] Figure 1 It is a graph showing the determination results of the cell membrane permeability of Lactobacillus paracasei powder; Note: Different letters for the same group of data indicate significant differences (P < 0.05); the same letters indicate no significant differences (P > 0.05);

[0027] Figure 2 It is a graph showing the determination results of the cell membrane fluidity of Lactobacillus paracasei powder; Note: Different letters for the same group of data indicate significant differences (P < 0.05); the same letters indicate no significant differences (P > 0.05);

[0028] Figure 3 It is for Lactobacillus paracasei powder Na + -K +-ATPase activity assay results; Note: Different letters in the same group of data indicate significant differences (P<0.05); the same letters indicate no significant differences (P>0.05). Detailed implementation mode

[0029] The present invention will be further described in detail below in conjunction with embodiments, but the implementation modes of the present invention are not limited thereto.

[0030] The Lactobacillus paracasei used in the following examples and comparative examples is Lactobacillus paracasei CICC 6248, provided by the China Center for Industrial Culture Collection.

[0031] (1) Preparation of culture medium:

[0032] MRS culture medium (g / L): Beef extract 10g, peptone 10g, yeast extract powder 5g, glucose 20g, anhydrous sodium acetate 5g, diammonium hydrogen citrate 2g, magnesium sulfate heptahydrate 0.58g, dipotassium hydrogen phosphate 2g, manganese sulfate monohydrate 0.25g, Tween-80 1g, made up to 1L with sterile water, and the pH was adjusted to 6.5. Among them, 15-20g of agar powder needs to be added to the solid medium, while the liquid medium does not need to be added, and it is sterilized at 121°C for 15-20 minutes;

[0033] Peptone, yeast extract powder, glucose, anhydrous sodium acetate, diammonium hydrogen citrate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, manganese sulfate monohydrate, Tween: Analytical pure, Sinopharm Chemical Reagent Co., Ltd.;

[0034] (2) The β-carotene mentioned in the following examples and comparative examples was purchased from Shaanxi Shengqing Biotechnology Co., Ltd., with a content of ≥10%. The seabuckthorn peptide mentioned in the comparative example was purchased from Shaanxi Yunqi Biotechnology Co., Ltd., vitamin C and glutathione were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and L-sodium glutamate, L-cysteine, maltitol, and β-cyclodextrin were purchased from Shanghai Macklin Biochemical Co., Ltd.

[0035] (3) Detection method for the viable count of Lactobacillus paracasei: Adopt the national standard "GB4789.35-2016 National Food Safety Standard Microbiological Examination of Foods Detection of Lactic Acid Bacteria".

[0036] Example 1: Preparation of freeze-dried powder of Lactobacillus paracasei

[0037] (1) Take β-carotene and sterilize it at 85°C for 30 minutes.

[0038] (2) Cultivation of Lactobacillus paracasei

[0039] Strain activation: On the MRS solid medium plate, streak and isolate the preserved Lactobacillus paracasei bacterial liquid, culture it at 37°C for 36 h. After single colonies grow on the plate, inoculate the single colonies into the MRS liquid medium and culture statically at 37°C for 24 h; perform subculture with an inoculation amount of 2%, and repeat the subculture 3 times to obtain the activated bacterial liquid.

[0040] (3) Inoculate the activated Lactobacillus paracasei bacterial liquid into the MRS liquid medium with an inoculation amount of 2%, culture it statically at 37°C for 12 h until the late logarithmic phase to the early stationary phase, centrifuge at 6000 r / min for 10 min to collect the bacterial sludge, and wash it 2 times with 0.85% physiological saline. Add the sterilized β-carotene according to the mass ratio of bacterial sludge:β-carotene of 1:5, and then vortex thoroughly to obtain a bacterial suspension. The bacterial suspension is quickly frozen at -80°C for 2 h, and then placed in a vacuum freeze dryer for freeze-drying. The freeze-drying parameters are freeze-drying at -65°C and a vacuum degree of 0.65 Pa for 24 h. After the freeze-drying is completed, take out the bacterial powder and rehydrate it with the same volume of physiological saline as before freeze-drying. After vortexing and mixing evenly, perform gradient dilution, take 100 μL and spread it on the plate to count the viable bacteria. Set three parallels, calculate the viable bacteria count of the freeze-dried bacterial powder after culturing at 37°C for 48 h.

[0041] Example 2

[0042] Compared with Example 1, the difference is that the sterilized β-carotene is added according to the mass ratio of bacterial sludge:β-carotene of 1:5, and the final concentration of β-carotene is 2 wt%, and the other steps are the same as those in Example 1.

[0043] Example 3

[0044] Compared with Example 1, the sterilized β-carotene is added according to the mass ratio of bacterial sludge:β-carotene of 1:5, and the final concentration of β-carotene is 3 wt%, and the other steps are the same as those in Example 1.

[0045] Example 4

[0046] Compared with Example 1, the difference is that the sterilized β-carotene is added according to the mass ratio of bacterial sludge:β-carotene of 1:5, and the final concentration of β-carotene is 4 wt%, and the other steps are the same as those in Example 1.

[0047] Example 5

[0048] Compared with Example 1, the difference is that the sterilized β-carotene is added according to the mass ratio of bacterial sludge:β-carotene of 1:5, and the final concentration of β-carotene is 5 wt%, and the other steps are the same as those in Example 1.

[0049] Comparative Example 1

[0050] Compared with Example 1, the difference is that the lyophilization protectant consists of 0.85% normal saline. Sterilized normal saline is added according to the mass ratio of bacterial sludge to normal saline of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0051] Comparative Example 2

[0052] Compared with Example 1, the difference is that the lyophilization protectant consists of 2% glutathione. Sterilized glutathione is added according to the mass ratio of bacterial sludge to glutathione of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0053] Comparative Example 3

[0054] Compared with Example 1, the difference is that the lyophilization protectant consists of 2% vitamin C. Vitamin C is added according to the mass ratio of bacterial sludge to vitamin C of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0055] Comparative Example 4

[0056] Compared with Example 1, the difference is that the lyophilization protectant consists of 2% sodium L-glutamate. Sodium L-glutamate is added according to the mass ratio of bacterial sludge to sodium L-glutamate of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0057] Comparative Example 5

[0058] Compared with Example 1, the difference is that the lyophilization protectant consists of 2% L-cysteine. L-cysteine is added according to the mass ratio of bacterial sludge to L-cysteine of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0059] Comparative Example 6

[0060] Compared with Example 1, the difference is that the lyophilization protectant consists of 1% β-cyclodextrin. β-cyclodextrin is added according to the mass ratio of bacterial sludge to β-cyclodextrin of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0061] Comparative Example 7

[0062] Compared with Example 1, the difference is that the lyophilization protectant consists of 3% β-cyclodextrin. β-cyclodextrin is added according to the mass ratio of bacterial sludge to β-cyclodextrin of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0063] Comparative Example 8

[0064] Compared with Example 1, the difference lies in that the freeze-drying protectant component is 5% β-cyclodextrin. β-cyclodextrin is added according to the mass ratio of bacterial sludge to β-cyclodextrin of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0065] Comparative Example 9

[0066] Compared with Example 1, the difference lies in that the freeze-drying protectant component is 7% β-cyclodextrin. β-cyclodextrin is added according to the mass ratio of bacterial sludge to β-cyclodextrin of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0067] Comparative Example 10

[0068] Compared with Example 1, the difference lies in that the freeze-drying protectant component is 9% β-cyclodextrin. β-cyclodextrin is added according to the mass ratio of bacterial sludge to β-cyclodextrin of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0069] Comparative Example 11

[0070] Compared with Example 1, the difference lies in that the freeze-drying protectant component is 1% seabuckthorn peptide. Seabuckthorn peptide is added according to the mass ratio of bacterial sludge to seabuckthorn peptide of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0071] Comparative Example 12

[0072] Compared with Example 1, the difference lies in that the freeze-drying protectant component is 3% seabuckthorn peptide. Seabuckthorn peptide is added according to the mass ratio of bacterial sludge to seabuckthorn peptide of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0073] Comparative Example 13

[0074] Compared with Example 1, the difference lies in that the freeze-drying protectant component is 5% seabuckthorn peptide. Seabuckthorn peptide is added according to the mass ratio of bacterial sludge to seabuckthorn peptide of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0075] Comparative Example 14

[0076] Compared with Example 1, the difference lies in that the freeze-drying protectant component is 7% seabuckthorn peptide. Seabuckthorn peptide is added according to the mass ratio of bacterial sludge to seabuckthorn peptide of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0077] Comparative Example 15

[0078] Compared with Example 1, the difference lies in that the freeze-drying protectant component is 9% seabuckthorn peptide. Seabuckthorn peptide is added according to the mass ratio of bacterial sludge to seabuckthorn peptide of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0079] Comparative Example 16

[0080] Compared with Example 1, the difference lies in that the lyophilization protectant consists of 1% maltitol, and maltitol is added at a mass ratio of bacterial sludge:maltitol of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0081] Comparative Example 17

[0082] Compared with Example 1, the difference lies in that the lyophilization protectant consists of 3% maltitol, and maltitol is added at a mass ratio of bacterial sludge:maltitol of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0083] Comparative Example 18

[0084] Compared with Example 1, the difference lies in that the lyophilization protectant consists of 5% maltitol, and maltitol is added at a mass ratio of bacterial sludge:maltitol of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0085] Comparative Example 19

[0086] Compared with Example 1, the difference lies in that the lyophilization protectant consists of 7% maltitol, and maltitol is added at a mass ratio of bacterial sludge:maltitol of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0087] Comparative Example 20

[0088] Compared with Example 1, the difference lies in that the lyophilization protectant consists of 9% maltitol, and maltitol is added at a mass ratio of bacterial sludge:maltitol of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0089] Comparative Example 21

[0090] Compared with Example 1, the difference lies in that the lyophilization protectant is a composite protectant composed of 3% β-cyclodextrin, 1% seabuckthorn peptide, 4% β-carotene, and 7% maltitol. The composite protectant is added at a mass ratio of bacterial sludge:composite protectant of 1:5, and then vortexed thoroughly to obtain a bacterial suspension. The remaining steps are the same as those in Example 1.

[0091] Among them, regarding the composite protectant, an orthogonal experiment with four factors and three levels was carried out with β-cyclodextrin, seabuckthorn peptide, β-carotene, and maltitol, and it was optimized with the viable bacteria count as the index. The influence factor levels of the orthogonal experiment are shown in Table 2. The results of the orthogonal experiment are shown in Table 3.

[0092] The above-mentioned different lyophilization protectants and the number of lyophilized viable bacteria are shown in Table 1 and Table 2.

[0093] Table 1. Different kinds of lyoprotectants and viable cell counts

[0094]

[0095]

[0096] Table 2. Orthogonal level factors

[0097]

[0098]

[0099] Table 3. Results of orthogonal experiments

[0100] Number β-Cyclodextrin Seabuckthorn peptide β-Carotene Maltitol <![CDATA[Viable count (10 11 CFU / g)]]> 1 1 1 1 1 8.27±1.5 2 1 2 3 2 7.20±2.62 3 1 3 2 3 6.33±1.10 4 2 1 3 3 9.47±1.45 5 2 2 2 1 2.47±0.31 6 2 3 1 2 8.00±0.60 7 3 1 2 2 7.33±0.81 8 3 2 1 3 7.20±0.60 9 3 3 3 1 2.80±0.20 <![CDATA[k1]]> 3.63 4.18 3.91 2.26 <![CDATA[k2]]> 3.32 2.81 2.69 3.76 <![CDATA[k3]]> 2.89 2.86 3.24 3.83 Range R 0.74 1.37 1.22 1.58 Optimal level <![CDATA[A1B1C1D3]]>

[0101] Effect implementation example

[0102] (1) Cell membrane permeability:

[0103] By adding NaCl to the culture medium to prepare culture media with different osmotic pressures, the sensitivity of normal Lactobacillus paracasei to the NaCl concentration was observed. The change in the sensitivity of the bacterial cells to NaCl can reflect whether the cell membrane is damaged, which is related to the impairment of its osmotic regulation and transport mechanism. Through preliminary determination in the laboratory, the NaCl limiting medium concentration of Lactobacillus paracasei was obtained as 0.75%. The cell membrane permeability of Lactobacillus paracasei powder prepared with different protectants β-carotene was measured using the NaCl limiting medium.

[0104] The results are as Figure 1 , and it can be seen that in the blank group of bacterial powder, the calculated cell membrane permeability was close to 100%. Therefore, it can be inferred that during the freeze-drying process, the cell membrane of Lactobacillus paracasei was severely damaged, resulting in an increase in cell membrane permeability and the inability to maintain the normal physiological functions of the bacterial cells, which is one of the main reasons for freeze-drying loss. As can be seen from Figure 1 , the addition of the protectant significantly reduced the cell membrane permeability of Lactobacillus paracasei R33 (P < 0.05). Among them, seabuckthorn peptide, β-carotene, and the compound protectant all reduced the cell membrane permeability of the bacterial cells by about 80%, indicating that reducing the cell membrane permeability through the protectant, especially β-carotene, can effectively increase the viable cell count of Lactobacillus paracasei powder.

[0105] (2) Cell membrane fluidity

[0106] The fluidity of the cell membrane refers to the viscosity of its phospholipid bilayer, which is a necessary condition for the cell to carry out normal life activities. The cell membrane fluidity of normal probiotics is within a certain range. When the fluidity increases or decreases, it will cause damage to the cell membrane physiology. To measure the cell membrane fluidity, the steady-state fluorescence polarization method is used. Laurdan is a dye that shows changes in the maximum value of its fluorescence emission spectrum according to the fluidity of the surrounding membrane, and the generalized polarization parameter GP value is used to evaluate the cell membrane fluidity of bacterial cells. As the cell membrane fluidity increases, the GP value will decrease.

[0107] The results are as Figure 2 , and the cell membrane fluidity of different protectants after freeze-drying was measured respectively. Compared with the GP value of the blank group of 0.254 ± 0.008, there was no significant difference in β-cyclodextrin, seabuckthorn peptide, and maltitol (P > 0.05), indicating that these three types of protectants do not reduce cell damage by improving the cell membrane fluidity of Lactobacillus paracasei R33. The lowest was the composite protectant group, with a GP value of 0.093 ± 0.016, indicating that the addition of the composite protectant increased the content of unsaturated fatty acids and decreased the content of saturated fatty acids in the cell membrane during freeze-drying. The change in membrane composition increased its fluidity, enabling it to better resist adverse environments. The GP value of the β-carotene group also decreased significantly (P < 0.05). It can be speculated that the carotene in the compound protectant played a role in regulating the cell membrane fluidity, which was also consistent with the highest viable count of Lactobacillus paracasei powder in the β-carotene group. It is speculated that β-carotene exerts its freeze-drying protection effect by changing the fatty acid composition of the cell membrane of Lactobacillus paracasei R33, thereby affecting the cell membrane fluidity. The difference in fluidity between the two groups may be due to the concentration. The concentration of carotene in the single protectant was 3%, and that in the compound protectant was 4%. A higher concentration may have a better protective effect on cell membrane fluidity.

[0108] (3)Na + -K + -ATPase activity

[0109] ATPase is widely distributed in the cell membrane system and is crucial for maintaining normal cell physiological functions. Among them, Na + -K + -ATPase is a carrier protein embedded in the phospholipid bilayer of the cytoplasmic membrane, which can maintain the potential balance on both sides of the cell membrane, regulate cell osmotic pressure, and provide energy for cell nutrient absorption by catalyzing the hydrolysis of ATP. By comparing the activity of Na + -K + -ATPase, the degree of damage to the cell membrane of bacterial cells during freeze-drying was evaluated, and it can also be used as an index to evaluate the integrity of the cell membrane. Explore the effects of different protectants after freeze-drying on the activity of Na + -K + -ATPase.

[0110] The results are shown in Figure 3 , there were significant differences in the Na + -K + -ATPase activity of Lactobacillus paracasei after freeze-drying with different protectants and the blank group (P < 0.05), indicating that the activity of Na + -K + -ATPase was severely damaged during the freeze-drying process, and the addition of protectants could effectively improve this situation. Among them, when β-carotene was used as a protectant, the Na + -K + -ATPase activity in the cells was the highest, being (73.52 ± 1.70) U / g, and its protective effect was the best, which was also consistent with the results of the optimization of protectants. When carotene was used as a protectant, the viable count of the Lactobacillus paracasei powder was the highest.

[0111] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of β-carotene in preparing a cryoprotectant for Lactobacillus paracasei freeze-drying.

2. Use of β-carotene in Lactobacillus paracasei freeze-drying protection.

3. Use of β-carotene in increasing the viable count of Lactobacillus paracasei during freeze-drying.

4. Use of β-carotene in reducing the cell membrane permeability and / or fluidity of Lactobacillus paracasei during freeze-drying.

5. Use of β-carotene in maintaining the cell membrane integrity of Lactobacillus paracasei during freeze-drying.

6. A preparation method of Lactobacillus paracasei powder, characterized in that: It includes the following steps: Prepare bacterial sludge, and fully mix the bacterial sludge with β-carotene at a mass ratio of 1:3 to 7; after quick-freezing at -90°C to -70°C for 1 to 3 h, perform freeze-drying, and the freeze-drying parameters are: temperature -90°C to -70°C, vacuum degree 0.4 to 0.6 Pa, time 20 to 30 h.

7. The method for preparing Lactobacillus paracasei powder according to claim 6, characterized in that: Prepare bacterial sludge, and fully mix the bacterial sludge with β-carotene at a mass ratio of 1:5; after quick-freezing at -80°C for 2 h, perform freeze-drying, and the freeze-drying parameters are: temperature -65°C, vacuum degree 0.5 Pa, time 24 h.

8. The method for preparing Lactobacillus paracasei powder according to claim 6 or 7, characterized in that: The method for preparing the bacterial sludge is as follows: The activated Lactobacillus paracasei bacterial liquid is inoculated into MRS liquid medium at an inoculation amount of 2% to 6% and cultured for 12 to 16 h, centrifuged at 4000 to 8000 r / min for 10 to 30 min to collect the bacterial sludge, and washed 1 to 3 times in sterilized 0.85% physiological saline or phosphate buffer; The activation method is: streak the strain on an MRS plate, pick a single colony and transfer it to MRS liquid medium, culture at 37°C for 20 to 30 h, and passage 2 to 3 times.

9. A Lactobacillus paracasei powder, characterized in that: Obtained by the preparation method described in any one of claims 6-8.

10. Use of the Lactobacillus paracasei powder described in claim 9 in preparing a food additive.

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