Probiotic fermented litchi juice as well as preparation method and application thereof

Abstract

The invention discloses probiotic fermented litchi juice as well as a preparation method and application thereof, and relates to the technical field of probiotic fermentation. The probiotic fermented litchi juice disclosed by the invention is prepared by taking fresh litchis as a raw material and fermenting the fresh litchis by using lactobacillus mucilaginosus NCU326. The invention also provides application of the probiotic fermented litchi juice in preparation of drugs for preventing and / or improving nerve injury. The probiotic fermented litchi juice disclosed by the invention is excellent in flavor, coordinated in nutrition, unique in fermentation flavor, soft in sourness and pleasant in fragrance (a lot of olefinic terpenoids and aldehyde compounds are degraded, and the contents of alcohol, ketone, ether and acid compounds are obviously increased), and the litchi beverage fermented by NCU326 is rich in nutrients, rich in nutrients, rich in nutrients, rich in nutrients, rich in nutrients, rich in nutrients, and rich in nutrition. The survival rate of human neuroblastoma SH-SY5Y cells damaged by 1-methyl-4-phenylpyridine ions (MPP +) can be remarkably increased, and the compound has certain potential in the aspect of improving nerve injury, so that the compound can be used for preparing the medicine for preventing and / or improving nerve injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotic fermentation, and particularly relates to a probiotic fermented litchi juice and a preparation method and application thereof. BACKGROUND

[0002] Lactic acid bacteria are a kind of bacteria that colonize in the intestines of humans and animals and have a probiotic effect, and have the effects of improving human immunity, balancing intestinal flora, reducing cholesterol, and delaying aging. Therefore, lactic acid bacteria are widely used in the food industry, and the fermented food is favored by the public due to its good health care effect. Since China has a high annual output of fruits and vegetables, there is still a large research prospect and space in solving the high loss and corruption rate of fruits and vegetables during processing, transportation and storage. Lactic acid bacteria have been widely recognized as a kind of probiotic for a long time.

[0003] Litchi fruits are rich in nutrients such as sugars, organic acids, vitamins, proteins, amino acids and minerals, and are rich in bioactive substances such as polyphenols and anthocyanins. The effects of litchi on antioxidant, anti-aging, blood sugar reduction, blood lipid reduction, immune enhancement and inhibition of hepatitis B virus surface antigen have been confirmed. The litchi mature season is concentrated in the summer with high temperature and high humidity. In addition, litchi has special structure and physiological characteristics. If the picked litchi is not treated for preservation in time, it is easy to cause browning. At the same time, the reproduction of self-originating microorganisms in litchi and external contaminated microorganisms will cause litchi to rot and deteriorate, resulting in serious economic losses and great waste. At present, the main processed products are litchi dried, litchi canned, litchi juice, and quick-frozen litchi, and there are few deep-processed products. SUMMARY

[0004] The present application aims to at least solve one of the problems in the prior art, and provides a probiotic fermented litchi juice and a preparation method and application thereof.

[0005] On the one hand, the probiotic fermented litchi juice prepared by the present application can convert part of the sugar substances in litchi into probiotic bodies and other beneficial metabolites. Not only does it solve the quality problems of litchi juice, but also it can increase the content of flavor substances, which has a positive significance for opening up litchi processing channels and increasing beverage varieties. Compared with fresh fruit and vegetable juice beverages on the market, the probiotic fermented litchi juice of the present application has a long shelf life. Compared with the same product of fruit and vegetable juice beverage, the probiotic fermented litchi juice of the present application does not add any essence, pigment or preservative, and is more in line with the requirements of consumers for healthy drinks.

[0006] On the other hand, the probiotic fermented litchi juice of the present application can also significantly increase the survival rate of human neuroblastoma SH-SY5Y cells damaged by 1-methyl-4-phenylpyridinium ions (MPP+), has certain potential in improving nerve damage, and therefore can be used for preparing a drug for preventing and / or improving nerve damage.

[0007] The technical solution of the present application is as follows: The first aspect of the present application provides a probiotic fermented litchi juice, which is obtained by fermenting litchi pulp with probiotics; wherein the litchi pulp is obtained by beating or juicing litchi pulp, and the probiotics are Lactobacillus muci- Limosilactobacillus fermentum ) NCU326.

[0008] The fermented Lactobacillus muci- NCU326 of the present application is a strain in the prior art, and the screening method, identification method, 16S sequence and biological preservation information of the strain are specifically referred to Chinese patents with publication numbers CN 116925951 A or CN 116687986 A.

[0009] The fermented Lactobacillus muci- NCU326 of the present application is a probiotic strain with excellent fruit and vegetable fermentation performance and tolerance to digestive tract environment pressure, and the strain has the following physiological characteristics: 1. The survival rate is 99.85% after 3 h of treatment in PBS with pH 2.0; 2. The survival rate is 62.28% after 3 h of treatment in a 0.2% bile salt environment; 3. The strain has good surface properties and the ability to adhere to intestinal epithelial cells: the self-aggregation rate is 32.11% in 12 h, the surface hydrophobicity rate is 5.63%, and the adhesion rate to human colon cancer cells Caco-2 is 9.82%; 4. The 24 h fermentation supernatant of the strain has excellent antibacterial activity against common foodborne pathogens, especially the inhibitory activity against Listeria monocytogenes and Bacillus cereus, with antibacterial ring diameters of 15.33 mm and 13.50 mm, respectively; in addition, the hemolytic activity detection of the strain shows that it has no hemolytic activity; the antibiotic sensitivity detection shows that the strain is sensitive to ampicillin, ceftriaxone, tetracycline, erythromycin and penicillin, and is resistant to kanamycin and gentamicin; 5. Colony morphology: light yellow, round, flat, smooth surface, regular edge, strong acid production, 1-3 mm; gram-positive, bacterial body is short rod-shaped; 6、The lichi juice fermented by the probiotic bacteria has excellent fermentation performance, and the acid production speed is fast in the lichi raw material, and the fermentation time is obviously shortened compared with other fermented Lactobacillus mucus; the pH of the lichi juice inoculated with NCU326 decreases fast, the acid production capacity is strong, and the pH is reduced to 3.61 and the acidity is 13.61 g / L after 24 hours of fermentation.

[0010] Preferably, the lichi juice fermented by the probiotic bacteria is obtained from the mixture of the lichi pulp fermented by the probiotic bacteria and vitamin C.

[0011] Preferably, in the mixture, the mass ratio of the lichi pulp to vitamin C is 100:0.01-0.5.

[0012] Preferably, compared with the lichi pulp, the content of alcohol, ketone, ether and acid compounds in the lichi juice fermented by the probiotic bacteria is increased, and the type and content of terpenes are reduced.

[0013] Preferably, the lichi juice fermented by the probiotic bacteria contains 2-heptanone, 2-nonanone, linalyl acetate, alpha-terpineol, ethyl acetate, 2-nonanol and (-)-rose oxide.

[0014] The second aspect of the present application provides a preparation method of the lichi juice fermented by the probiotic bacteria, comprising the following steps: After the lichi pulp is pulped or juiced, the lichi pulp is obtained; vitamin C is added to the lichi pulp, and the mixture is stirred to obtain lichi juice, and then the lichi juice is sterilized; The lichi juice is inoculated with the probiotic bacteria, and then fermented to obtain the lichi juice fermented by the probiotic bacteria.

[0015] Preferably, the sterilization method is 400 MPa for 3 minutes.

[0016] Preferably, the fermentation method comprises: fermentation at 37℃ for 12-24 hours, and the pH value of 3.0-5.0 is the end point of fermentation; the inoculation amount of the probiotic bacteria is 10 3 ~10 9 cfu / mL.

[0017] Preferably, the preparation method further comprises: sterilizing the lichi juice fermented by the probiotic bacteria, and the sterilization method comprises 85℃-132℃, 2 seconds-10 minutes of ultra-high temperature instantaneous sterilization and then aseptic packaging; or after the packaging is sealed, sterilization is performed at 75℃-132℃ for 20-40 minutes.

[0018] The third aspect of the present application provides the use of the lichi juice fermented by the probiotic bacteria in the preparation of a medicine for preventing and / or improving nerve injury.

[0019] The probiotic fermented litchi juice of the present application can significantly increase the survival rate of human neuroblastoma SH-SY5Y cells damaged by 1-methyl-4-phenylpyridinium ions (MPP+), has certain potential in improving nerve damage, and can be used to prepare medicines for preventing and / or improving nerve damage.

[0020] The present application has at least one of the following beneficial effects: The present application uses fresh litchi as raw material, and the fermented litchi juice fermented by Lactobacillus muciadis NCU326 has excellent flavor, balanced nutrition, and does not add any essence, pigment or preservative. The fermented litchi juice has the following advantages: (1) After fermentation by the probiotic NCU326, the types and contents of flavor substances in litchi are increased, so that the product has a unique fermented flavor, soft sour taste and pleasant aroma (a large amount of olefin terpenes and aldehyde compounds are degraded, and the contents of alcohol, ketone, ether and acid compounds are significantly increased). The probiotic fermented litchi juice of the present application can be used to prepare beverages, which not only improves the flavor of the beverages, but also fully retains the original aroma components and nutrients of litchi by using a non-concentration processing technology. In addition, the probiotic fermented litchi beverage prepared by the method can effectively provide a softer sour taste, and has certain function for human intestinal health. The product does not add any essence, pigment or preservative, and is healthier and safer.

[0021] (2) The litchi beverage juice fermented by NCU326 can significantly increase the survival rate of human neuroblastoma SH-SY5Y cells damaged by 1-methyl-4-phenylpyridinium ions (MPP+), and has certain potential in improving nerve damage. Therefore, the probiotic fermented litchi juice of the present application can also be used to prepare medicines for preventing and / or improving nerve damage. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0023] The fermented Lactobacillus muciadis (Lactobacillus fermentum) NCU326 of the present application is a strain in the prior art. The screening method, identification method, 16S sequence and biological preservation information of the strain are specifically referred to Chinese patents with publication numbers CN 116925951 A or CN 116687986 A. Moreover, when the strain was preserved in 2019, the strain name was Lactobacillus fermentum (Lactobacillus fermentum). Limosilactobacillus fermentum Limosilactobacillus fermentum ​)NCU326, but according to the "List of strains permitted for use in food" and "List of strains permitted for use in infant food" issued in 2021, "Lactobacillus fermentum" is renamed as "Lactobacillus mucosus", so the strain name in the present application is changed to "Lactobacillus mucosus (L. Limosilactobacillus fermentum )NCU326".

[0024] The "Lactobacillus mucosus CECT5716", "Lactobacillus mucosus ATCC9338", "Lactobacillus mucosus ATCC14931", "Lactobacillus mucosus CICC 22555" and "Lactobacillus mucosus CICC24575" used in the following examples are all strains in the prior art, which are purchased from China Industrial Microbial Culture Collection Center.

[0025] The present application will be further described in detail with specific examples below, but the present application is not limited to the following specific examples.

[0026] Example 1 The physiological and biochemical characteristics of Lactobacillus mucosus (L. Limosilactobacillus fermentum )NCU326 (hereinafter referred to as "NCU326") were determined, and the determination method was as follows: 1. Acid resistance test - the survival rate was 99.85% after 3 h treatment in PBS with pH 2.0; The stored NCU326 was activated twice with MRS liquid medium, then cultured at 37 ℃ for 24 h at an inoculation amount of 2% (v / v), and the bacterial cells were collected by centrifugation at 10000 g for 5 min. The bacterial cells were resuspended with sterile PBS with pH 2.0 and adjusted to a viable bacterial concentration of 10 8 CFU / mL, incubated at 37 ℃ for 3 h, and the viable bacterial count before and after incubation was determined by dilution plating method to determine the survival rate. The survival rate was calculated as follows: Where N1 is the number of surviving bacteria after incubation, and N0 is the initial number of bacteria.

[0027] 2. Bile salt tolerance test - the survival rate was 62.28% after 3 h treatment in 0.2% bile salt environment; The NCU326 cultured for 24 h was centrifuged at 4 ℃, 10000 g for 5 min to collect the bacterial cells, and the obtained bacterial cells were resuspended with sterile PBS containing 0.2% bile salt and adjusted to a viable bacterial concentration of 10 8 CFU / mL, incubated at 37 ℃ for 3 h, and the viable bacterial count was determined by dilution plating method to evaluate the bile salt tolerance of NCU326. The survival rate of NCU326 was calculated according to the following formula: Wherein N1 is the viable cell count, N0 is the initial cell count.

[0028] 3. Auto-aggregation test The overnight cultured NCU326 was centrifuged at 10000 g for 5 min to collect the bacterial cells, and washed twice with sterile PBS buffer. Then, the obtained bacterial cells were resuspended with sterile PBS and adjusted to A 600 = 0.7 ± 0.05 (A0), vortexed for 10 s, and then incubated at 37 °C for 24 h. The upper bacterial suspension was taken at 2, 6 and 12 h, and the absorbance (A t ) was measured at 600 nm. Each determination was performed in triplicate, and repeated three times. The auto-aggregation ability of the bacteria was calculated according to the following formula.

[0029] Wherein A0 represents the initial absorbance of the bacterial cells, and A t represents the absorbance of the upper bacterial suspension at t time.

[0030] The results showed that the auto-aggregation rate of the strain was 32.11% at 12 h.

[0031] 4. Surface hydrophobicity test The surface hydrophobicity of NCU326 was determined by the Bacteria Adhesion To Hydrocarbons (BATH) method. First, the overnight cultured NCU326 was centrifuged at 10000 g for 5 min to collect the bacterial cells, and washed twice with sterile PBS buffer; then, the obtained bacterial cells were resuspended with 0.1 mol / L KNO3 and adjusted to A 600 = 0.6 ± 0.05 (A0). Then, 1 mL of xylene was added to 3 mL of the bacterial cell suspension with adjusted concentration, mixed, and pre-incubated at room temperature for 10 min, vortexed for 2 min, and then incubated at room temperature for 30 min to separate the layers, and the water phase was carefully aspirated. The OD 600 value (A) was determined with sterile PBS as a blank. The hydrophobicity of the bacteria was calculated according to the following formula. Wherein A0 represents the initial absorbance of the bacterial cells, and A represents the absorbance of the lower water phase after xylene treatment.

[0032] The results showed that the surface hydrophobicity rate of the strain was 5.63%.

[0033] 5. Adhesion test The cultured Caco-2 cells were digested with trypsin-EDTA digestion solution, and then the cell concentration was adjusted to 1.0 × 10 5mL, 2 mL per well, in a CO2 incubator (5% CO2, 95% air) at 37°C until the cells grow to a differentiated monolayer, and the medium is changed every two days. Discard the DMEM medium in each well of the tissue culture plate, wash the plate twice with sterile PBS buffer, and add 1 mL of NCU326 bacterial suspension (10 8 CFU / mL; OD = 1) resuspended in DMEM incomplete medium (Caco-2 cells: bacteria number ≧ 1:100), and incubate at 37°C for 2 h. After the incubation, discard the mixture in each well of the tissue culture plate, wash 5 times with sterile PBS buffer to remove the unadhered bacterial cells. Add 0.5 mL of 0.25% trypsin and incubate at 37°C for 5 min to digest the cells, and then perform gradient dilution of the cell digestion solution and spread on MRS solid plates to calculate the number of adhered bacteria. The adhesion ability is calculated by the following formula where N t represents the number of bacteria adhered to Caco-2 cells, and N0represents the total number of NCU326 bacteria added.

[0034] The results show that the adhesion rate of the strain to human colon cancer cells Caco-2 is 9.82%.

[0035] 6. Bacteriostatic activity test NCU326 is cultured in MRS medium at 37°C for 24 h, and centrifuged at 10,000 g at 4°C for 10 min. The fermentation supernatant is filtered with a 0.22 μm filter to remove bacteria to obtain cell-free supernatant (CFS) and stored at -80°C for standby. The bacteriostatic activity of NCU326 CFS on pathogenic bacteria is determined by the punch method, that is, 200 μL of NCU326 CFS is added to the LB plate well coated with Escherichia coli, Salmonella, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus, and incubated at 37°C for 12 h to measure the diameter of the bacteriostatic circle.

[0036] 7. Hemolytic activity test The test strain is streaked on a Columbia blood agar plate and incubated at 37°C for 48 h to determine the hemolytic activity. Fermented L. mucosus CECT5716 is used as a positive control, and Staphylococcus aureus is used as a negative control, and the experiment is repeated three times.

[0037] 8. Antibiotic sensitivity test The K-B (agar diffusion method with drug-sensitive paper) is used for drug sensitivity test to evaluate the antibiotic resistance of the strain. The overnight cultured Lactobacillus fermentation broth is adjusted to a bacterial concentration of 10 8CFU / mL, 100 μL was taken and spread on MRS plate. Ampicillin (10 mg / mL), erythromycin (15 mg / mL), tetracycline (30 mg / mL), gentamicin (10 mg / mL), kanamycin (30 mg / mL), penicillin (10 mg / mL), ceftriaxone (15 mg / mL) were placed on the MRS plate with the lactobacillus liquid, 37℃ incubation for 24 h, the diameter of the inhibition zone was measured by vernier caliper, and the sensitivity of the tested lactobacillus to the above antibiotics was determined.

[0038] The results showed that the 24 h fermentation supernatant of the strain had excellent antibacterial activity against common foodborne pathogens, especially the inhibition activity against Listeria monocytogenes and Bacillus cereus was the best, and the diameters of the inhibition zone were 15.33 mm and 13.50 mm, respectively; in addition, the hemolytic activity detection of the strain showed that it had no hemolytic activity; the antibiotic sensitivity detection showed that the strain was sensitive to ampicillin, ceftriaxone, tetracycline, erythromycin and penicillin, and resistant to kanamycin and gentamicin.

[0039] Example 2 The present embodiment provides a preparation method of probiotic fermented litchi juice, comprising the following steps: (1) fresh, non-rotten litchi was selected as raw material, washed, peeled and pitted, and then pulped to obtain litchi slurry; 0.5 parts of vitamin C was added into 100 parts of the litchi slurry to obtain litchi juice, and the ultra-high pressure sterilization processing parameters were 400 MPa and 3 minutes.

[0040] (2) After sterilization, the freeze-dried bacterial powder of Lactobacillus mucosae NCU326 was inoculated into the sterilized litchi juice at a ratio of 10 6 cfu / mL, and then fermented at 37℃, and the fermentation was terminated when the pH value reached 3.7. The probiotic fermented litchi juice was obtained, and then stored in a refrigerator at 0℃-4℃.

[0041] (3) High-temperature sterilization: the probiotic fermented litchi juice was sterilized at 132℃ for 2 seconds, and then aseptically canned.

[0042] Comparative Example 1 The difference from Example 2 is that "fermented Lactobacillus mucosae NCU326" is replaced by "fermented Lactobacillus mucosae ATCC9338", and the others are the same as Example 2.

[0043] Comparative Example 2 The difference from Example 2 is that "fermented Lactobacillus mucosae NCU326" is replaced by "fermented Lactobacillus mucosae ATCC14931", and the others are the same as Example 2.

[0044] Comparative Example 3 The difference from Example 2 is that "Lactobacillus fermentum NCU326" is replaced by "Lactobacillus fermentum CICC22555", and the others are the same as Example 2.

[0045] Comparative Example 4 The difference from Example 2 is that "Lactobacillus fermentum NCU326" is replaced by "Lactobacillus fermentum CICC24575", and the others are the same as Example 2.

[0046] Test: 1. The flavor substances in the probiotic fermented litchi juice (before high-temperature sterilization) prepared by Example 2 and Comparative Example 1 were determined, and the determination method was as follows: (1) The detection method of flavor substances is as follows: Static headspace-gas chromatography-mass spectrometry was used to detect flavor substances using an Agilent triple quadrupole mass spectrometer. The gas chromatography and mass spectrometry conditions are as follows: Chromatographic column: HP-5 quartz capillary column (30 m x 0.25 mm, 1 μm); Temperature program: initial temperature 50℃, hold for 3 min, then increase to 120℃ at 2℃ / min, finally increase to 250℃ at 20℃ / min, hold for 5 min; carrier gas (He), flow rate 1.0 mL / min, pressure 7.6522 psi; injection volume 1 μL; no split.

[0047] Electron ionization ion source; electron energy 70 eV; mass scan range m / z 35-450, 20 scans / s; ion source temperature 230℃.

[0048] (2) The amino acid detection method is according to GB / T 5009.124-2003 "Determination of Amino Acids in Food", and the amino acid analyzer is used to determine the types and contents of amino acids in the sample.

[0049] The determination results are shown in Tables 1 and 2.

[0050] Table 1 Analysis of main flavor substances before and after litchi juice fermentation Note: "-" indicates that the sample is not detected.

[0051] As can be seen from Table 1, after fermentation of litchi by Lactobacillus mucosus NCU326, the volatile aroma components of litchi have significant differences in flavor constituent components, quantity and proportion. After fermentation, the content of alcohol, ketone, ether and acid compounds increases obviously, the type and content of terpene compounds decrease obviously, and the distribution characteristics are changed significantly. Among them, terpene compounds account for a large proportion before fermentation, which is the main characteristic aroma component of litchi juice. After fermentation, the content of terpene compounds decreases, which reduces the characteristic aroma of litchi to a certain extent, but new flavor substances such as 2-heptanone, 2-nonanone and linalyl acetate, which have fruity aroma; alpha-terpineol, which has a minty aroma; ethyl acetate, which has a pineapple aroma; 2-nonyl alcohol, which has a cucumber aroma; the main ether compound in litchi is (-)-rose oxide, which gives green, rose and floral aroma, which further enriches the flavor of fermented litchi juice. After fermentation by the comparative strain Lactobacillus mucosus ATCC9338, the change of volatile flavor substances in litchi juice is relatively not obvious.

[0052] Table 2 Change of amino acids in litchi juice before and after fermentation Note: "-" indicates that the sample is not detected.

[0053] As can be seen from Table 2, after fermentation by NCU326, the content of bitter amino acids in litchi juice decreases significantly. After fermentation by the comparative strain Lactobacillus mucosus ATCC9338, the change of taste amino acids in litchi juice is not significant.

[0054] Therefore, the strain NCU326 used in the present application can increase the type and content of flavor substances in litchi, so that the product has a unique fermented flavor, soft sour taste and pleasant aroma.

[0055] 2. The pH and acidity of the fermentation process of Example 2 and Comparative Example 1 were determined The results show that the pH of strain NCU326 decreases faster than that of ATCC9338, and the acid production ability is strong. The pH of litchi juice fermented by strain NCU326 for 24 h is 3.61, and the acidity is 13.61 g / L. The pH of litchi juice fermented by comparative strain ATCC9338 for 8 h is 4.35, and the pH after 24 h is 3.69, and the acidity is only 3.05 g / L.

[0056] Therefore, the strain NCU326 used in the present application has excellent fermentation performance, and the acid production speed in fruit and vegetable raw materials is fast, and the fermentation time is significantly shortened compared with other fermented Lactobacillus mucosus.

[0057] 3. The SH-SY5Y cells damaged by MPP+ were determined by the probiotic fermented litchi juice (before high-temperature sterilization) prepared in Example 2 and Comparative Examples 1-4, and the determination method is as follows: 1) Construction of SH-SY5Y cell injury model MPP+ was used to construct the cell injury model, and CCK-8 method was used to screen the MPP+ injury concentration. Cell Counting Kit-8 (CCK-8) cell counting kit uses WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt) to realize rapid detection. When the cell adhesion reaches 80%-90%, the cells are collected after digestion and centrifugation, and the cell density is adjusted to 1-2×10 5 After 24h incubation in 37℃, 5% CO2 incubator, the original culture medium was discarded, and 100μL of culture medium (containing different concentrations of MPP+, the concentration is 0.1mmol / L, 0.5mmol / L, 1mmol / L, 2mmol / L, 4mmol / L) was added according to the experimental plan, 6 replicates were set for each concentration, and the cell viability was measured at 24h, 48h and 72h. At the end of drug action, 10ul CCK-8 solution was added to each well, and bubbles were avoided. After incubation in the cell incubator for 1-4h, the OD value was measured at 450nm using a microplate reader. The zero setting hole (containing only culture medium, not containing cells) and the blank control hole (normal cultured cells, without intervention) were set. The experiment was repeated at least 3 times.

[0058] 2) Detection of the effect of fermented litchi juice on the survival rate of MPP+ injured cells When the cell adhesion reaches 80%-90%, the cells are collected after digestion and centrifugation, and the cell density is adjusted to (1-2)×10 5 After 24h incubation in 37℃, 5% CO2 incubator, the original culture medium was discarded, and 100μL of culture medium (containing different concentrations of MPP+, the concentration is 0.1mmol / L, 0.5mmol / L, 1mmol / L, 2mmol / L, 4mmol / L) was added according to the experimental plan, 6 replicates were set for each concentration, and the cell viability was measured at 24h, 48h and 72h. At the end of drug action, 10ul CCK-8 solution was added to each well, and bubbles were avoided. After incubation in the cell incubator for 1-4h, the OD value was measured at 450nm using a microplate reader. The zero setting hole (containing only culture medium, not containing cells) and the blank control hole (normal cultured cells, without intervention) were set. The experiment was repeated at least 3 times. + The experiment was divided into zero setting hole, blank control group (healthy cells), injury group (1mmol / L MPP +The treatment was 24 hours. The rest of the group was placed in the cell culture box, the conditions were unchanged, and the culture was continued for 24 h. At the end of the drug, 10ul of CCK-8 solution was added to each well, and it was incubated in the cell culture box for 1-4 h. The OD value was measured at 450 nm using a microplate reader, and the cell survival rate was calculated. The experiment was repeated at least 3 times. For easy comparison, the survival rate of damaged cells was used as a reference to calculate the fold increase in cell survival rate of litchi juice fermented by different strains. The formula for calculating cell survival rate is: Cell survival rate (%) = (OD experiment-OD zero / OD control-OD zero) x 100% The results are shown in Table 3.

[0059] Table 3 Effect of litchi juice fermented by different strains on MPP + Effect of litchi juice fermented by different strains on MPP As can be seen from Table 3, litchi juice fermented by NCU326 can significantly increase the survival rate of human neuroblastoma SH-SY5Y cells damaged by 1-methyl-4-phenylpyridinium ion (MPP+). Unfermented litchi juice, and litchi juice fermented by ATCC9338, CICC 22555, and CICC24575 had no significant effect on the survival rate of MPP+-damaged SH-SY5Y cells.

[0060] 4, Shelf life evaluation of probiotic fermented litchi juice prepared in Example 2 Shelf life evaluation criteria: The shelf life of the present application is determined by the following indicators. The unsterilized probiotic fermented litchi juice prepared in step (2) of Example 2 is stored at 0-4℃, and the probiotic fermented litchi juice after high temperature sterilization in step (3) of Example 2 is stored at room temperature. The sensory evaluation and microbial quantity detection of the above stored probiotic fermented litchi juice are carried out.

[0061] The indicators and test results are shown in Tables 4-5: Table 4 Sensory and microbial quantity changes of unsterilized fermented litchi juice during 0-4℃ storage Table 5 Sensory and microbial quantity changes of sterilized fermented beverage during room temperature storage As can be seen from Tables 4-5, the shelf life of unsterilized fermented litchi juice during 0-4℃ storage can reach 21 days, and the shelf life of fermented litchi juice after high temperature sterilization at room temperature can reach 18 months, indicating that the prepared fermented litchi juice has a long shelf life.

[0062] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A probiotic fermented litchi juice, characterized in that, The probiotic fermented litchi juice is obtained by fermenting litchi pulp with probiotics; wherein the litchi pulp is obtained by beating or juicing litchi pulp, and the probiotics are Lactobacillus muci Limosilactobacillus fermentum ) NCU326.

2. The probiotic fermented litchi juice as claimed in claim 1, wherein, The probiotic fermented lychee juice is obtained from a mixture of probiotic fermented lychee pulp and vitamin C.

3. The probiotic fermented litchi juice as claimed in claim 2, wherein, The mass ratio of the lychee pulp to vitamin C in the mixture is 100:0.01-0.

5.

4. The probiotic fermented litchi juice according to any one of claims 1 to 3, characterized in that, Compared with the lychee pulp, the content of alcohol, ketone, ether and acid compounds in the probiotic fermented lychee juice is increased, and the types and content of terpenes are reduced.

5. The probiotic fermented litchi juice according to any one of claims 1 to 3, characterized in that, The probiotic fermented lychee juice contains 2-heptanone, 2-nonanone, linalyl acetate, alpha-terpineol, ethyl acetate, 2-nonanol and (-)-rose oxide.

6. A method of preparing the probiotic fermented litchi juice according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: After the lychee pulp is pulped or juiced, a lychee pulp is obtained; vitamin C is added to the lychee pulp, and the mixture is stirred to obtain lychee juice, which is sterilized; The probiotic is inoculated into the sterilized and cooled lychee juice, and fermented to obtain probiotic fermented lychee juice.

7. The production method according to claim 6, characterized by, The sterilization method is 400MPa for 3 minutes.

8. The preparation method according to claim 6, characterized in that, The fermentation method comprises: fermentation at 37℃ for 12-24 hours, with pH value of 3.0-5.0 as the end point of fermentation; and the inoculation amount of the probiotic bacteria is 10 3 ~10 9 cfu / mL.

9. The preparation method according to claim 6, characterized in that, The preparation method further comprises sterilizing the probiotic fermented lychee juice, and the sterilization method comprises aseptic packaging after 85-132℃, 2-10 minutes of ultra-high temperature instantaneous sterilization; or sterilization at 75-132℃ for 20-40 minutes after the packaging is sealed.

10. Use of the probiotic fermented lychee juice according to any one of claims 1-5 in the preparation of a medicine for preventing and / or improving nerve injury.

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