CN110786435B - Lactobacillus plantarum for producing high-activity plasminogen activator and fruit and vegetable juice beverage fermented by lactobacillus plantarum - Google Patents
Lactobacillus plantarum for producing high-activity plasminogen activator and fruit and vegetable juice beverage fermented by lactobacillus plantarum Download PDFInfo
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Abstract
The invention provides a Lactobacillus plantarum for producing high-activity plasmin, which is classified and named as Lactobacillus plantarum and is preserved in China general microbiological culture collection management center of institute of microbiology, China academy of sciences, with the preservation number of CGMCC No.17372 and the preservation time of 2019, 3 months and 20 days. The invention also provides a fruit and vegetable juice beverage prepared by fermenting the lactobacillus plantarum. The enzymatic activity of the lactobacillus plantarum fermented supernatant plasmin capable of producing the high-activity plasmin is 497.72U/mL; the strain fermentation supernatant has strong bacteriostatic ability; and the strain has no multiple drug resistance to various antibiotics, has higher tolerance to gastrointestinal fluids, and also has potential intestinal attachment capability. The beverage of the invention reaches the national relevant viable count standard and has good commercial value.
Description
Technical Field
The invention relates to the technical field of biotechnology and food, in particular to lactobacillus plantarum for producing a high-activity plasminogen activator and a fruit and vegetable juice beverage fermented by the lactobacillus plantarum
Background
Lactobacillus plantarum (lactobacillus plantarum) is one of the lactobacilli that are commonly found in fermented vegetables and fruit juices. The lactobacillus plantarum is used as probiotics of human gastrointestinal tracts and has multiple functions of maintaining the balance of flora in intestinal tracts, improving the immunity of organisms, promoting the absorption of nutrient substances and the like. In the food fermentation process, lactobacillus plantarum can convert sugars in food raw materials into lactic acid, and simultaneously produce antibacterial peptides, exopolysaccharides and other metabolites. The application of the lactobacillus plantarum in food is mainly in dairy products, meat products, fruit and vegetable products, bean products, grains, potato products and the like, and has a wide market. According to the relevant regulations of ' list of strains available for food ' (ministry of health and supervision issue [ 2010 ] 65 '), lactobacillus plantarum can be directly used for food production. However, the plasmin production by lactobacillus plantarum and related research reports are few at present.
Thrombotic diseases are frequently generated in old people before, but the threat of thrombotic diseases to people's health is no longer limited to the old people only because the unhealthy living habits and dietary patterns of contemporary people. According to data in reports of nutrition and chronic disease status of Chinese residents (2015), the death rate of adults aged 2012 and older than eighteen years old in the country is 271.8/10 ten thousand due to cardiovascular and cerebrovascular diseases belonging to thrombotic diseases, and the death rate is a main cause of death. Thrombolysis by means of thrombolytic agents is one of the main therapeutic methods for thrombotic diseases, and prevention of thrombotic chronic diseases by means of thrombolytic agents is also attracting much attention. The thrombolytic agent mainly used for treatment at present has the defects of large side effect or high price and the like, and is not suitable for preventing thrombotic diseases. The microorganisms are various in variety, the metabolites are rich and various, and the microorganisms are easy to culture and fast to reproduce, so that the microorganisms become an important resource for exploring novel thrombolytic agents. Microbial plasmin such as streptokinase, glucokinase, nattokinase and the like all show good thrombolysis characteristics, and particularly, nattokinase secreted by bacillus subtilis is already prepared into health-care food for commercialization. The screened lactobacillus which can be directly used for food fermentation production and can produce high-activity plasminogen activator can be used for researching and developing foods with plasmin activity and can be used as effective measures for preventing thrombotic diseases and reducing the morbidity of the diseases.
Disclosure of Invention
In view of the disadvantages and needs of the prior art, it is an object of the present invention to obtain a lactobacillus plantarum producing a highly active plasminogen activator, having good bacteriostatic properties and no multiple resistance to multiple antibiotics.
In order to achieve the aim, the invention provides a Lactobacillus plantarum for producing a high-activity plasminogen activator, which is classified and named as Lactobacillus plantarum, and is preserved in the China general microbiological culture collection management center of the institute of microbiology of China academy of sciences (zip code 100101 of the institute of microbiology of China academy of sciences, No. 3 of the institute of sciences, North West No.1 of the south of the morning, Beijing city), with the preservation number of CGMCC No.17372 and the preservation time of 2019, 3 and 20 days.
Unless otherwise stated, in the specification of the present invention, the lactobacillus plantarum producing a high-activity plasminogen activator according to the present invention is simply referred to as lactobacillus plantarum HQ-3.
The invention also aims to provide a fruit and vegetable juice beverage, which is prepared by using fruits and vegetables as raw materials and fermenting the lactobacillus plantarum producing the high-activity plasminogen activator.
As an optional embodiment of the present invention, the fruit and vegetable is kiwi.
The invention also provides a method for preparing the fruit and vegetable juice beverage, which comprises the following steps:
juicing kiwi fruits, adding water for blending, and fermenting by using the lactobacillus plantarum producing the high-activity plasminogen activator.
As an alternative embodiment of the invention, the method comprises the steps of:
(1) selecting and treating kiwi fruits: selecting 8-9-minute-maturity red-yang kiwi fruits, and removing pests and rotten fruits for later use; juicing the selected red kiwi fruits by using a spiral juicer, separating peels and fruits, and reserving fruit pulp;
(2) blending: uniformly mixing the red kiwi fruit pulp and water according to the volume ratio of 1:1, adding 80mg/100g of sodium carboxymethylcellulose and 12g/100g of white granulated sugar, and adjusting the pH value to 5.5 by using sodium citrate;
(3) and (3) sterilization: heating the prepared red-yang kiwi fruit pulp at 83 ℃ for 10min to kill harmful microorganisms and pathogenic bacteria;
(4) fermentation: cooling the sterilized red-yang kiwi fruit pulp to room temperature, adding 1% of activated lactobacillus plantarum producing high-activity plasminogen activator for fermentation, and standing at 37 ℃ for 28h for fermentation to obtain the high-activity plasminogen activator.
The invention has the beneficial effects that:
the enzymatic activity of plasmin fermentation supernatant of the lactobacillus plantarum producing the high-activity plasminogen activator is 497.72U/mL; the fermented supernatant has stronger bacteriostatic ability; meanwhile, the lactobacillus plantarum producing the high-activity plasminogen activator has no multiple drug resistance to various antibiotics; has high tolerance to gastrointestinal fluid and potential intestinal adhesion.
The beverage obtained by the invention reaches the national standard of viable count of the lactobacillus fermented viable bacteria beverage, and meanwhile, the beverage contains high-activity plasmin, so that the beverage has good commercial value.
Drawings
FIG. 1 is a graph showing preliminary screening results of screening of plasmin-producing lactic acid bacteria;
FIG. 2 is a diagram showing the re-screening results of the screening of plasmin-producing lactic acid bacteria;
FIG. 3 is a urokinase standard curve for determining plasmin activity of Lactobacillus plantarum HQ-3 according to the present invention;
FIGS. 4 and 5 are graphs showing the morphological identification results of Lactobacillus plantarum HQ-3 according to the present invention;
FIG. 6 is a 16S rDNA amplification electrophoretogram of Lactobacillus plantarum HQ-31 of the present invention;
FIG. 7 is a phylogenetic tree of Lactobacillus plantarum HQ-3 according to the invention;
FIG. 8 is a graph showing the growth of Lactobacillus plantarum HQ-3 according to the present invention;
FIG. 9 is a PCR electrophoretogram of specific adhesin gene of Lactobacillus plantarum HQ-3 according to the present invention;
FIGS. 10 and 11 are graphs showing the results of experiments for determining the way of hydrolyzing fibrin in fermented supernatant of Lactobacillus plantarum HQ-3 according to the present invention.
Detailed Description
The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
Example 1
Test method 1:
1.1 materials and reagents
1.1.1 Experimental samples
59 parts of fermented soybeans collected from various places of Sichuan, 59 parts of traditional fermented foods such as northeast fermented soybean paste and fermented shrimp paste and 8 parts of food raw materials such as fresh vegetable leaves purchased in Yaan farm trade market.
1.1.2 culture Medium
MRS culture medium, E-linker culture medium, sucrose thiamine culture medium, LB culture medium, peptone water culture medium, nitrate culture medium, MH culture medium, blood plate, CM culture medium, and skim milk agar plate.
1.1.3 Primary reagents
Bovine fibrinogen, thrombin (biochemical, 40U/mg), urokinase standard (biochemical, 50U/mg) were purchased from Shanghai-derived leaf Biotech, Inc.; pepsin (biochemical, 30000U/mg), trypsin (biochemical, 250U/mg) were purchased from Yongsheng Biotech, Inc., Shanghai Rui; SDS-PAGE kit, purchased from Dr. Wuhan bioengineering, Inc.
1.1.4 strains
Escherichia coli (Escherichia coli) ATCC 25922, Staphylococcus aureus (Staphylococcus aureus) ATCC 25923, Salmonella (Salmonella) was provided by Sichuan university of agriculture food microbiology laboratory.
1.2 methods
1.2.1 sample treatment
And respectively carrying out enrichment culture on the sample by using an MRS liquid culture medium, an E-liker culture medium and a sucrosethiamine culture medium to obtain different enrichment culture solutions.
1.2.2 isolation of plasmin-producing lactic acid bacteria
(1) Primary screening: inoculating the screened lactobacillus on a skim milk-containing plate, and culturing at 37 ℃ for 48 h. And (4) selecting a single colony with an obvious lysis ring on a skim milk plate, and purifying.
(2) Re-screening: the strains obtained by screening are inoculated in a CM liquid medium and cultured for 24h at 37 ℃. Collecting 1mL of bacterial liquid, centrifuging at 4 ℃ and 10000r/min for 10min, and taking 20 mu L of supernatant to sample in a small hole of a fibrin plate. Incubation was carried out at 37 ℃ for 18h and strains producing a lytic loop were selected.
1.2.3 determination of fibrinolytic Activity
(1) Drawing a standard curve: 20 μ L of urokinase 31.25, 62.5, 125, 250, 500U/mL were spotted in the well of the prepared fibrin plate. The logarithmic value of the standard urokinase concentration was plotted on the abscissa and the logarithmic value of the area of the lysis cuvette was plotted on the ordinate.
(2) And (3) enzyme activity determination: activating the strains obtained by re-screening, inoculating the activated strains into a liquid culture medium according to the inoculum size of 2 percent, and culturing for 12 hours at the temperature of 37 ℃ and at the speed of 180 r/min. Centrifuging 1mL of the bacterial liquid at 4 ℃ at 10000r/min for 10min, and spotting 20 mu L of supernate in small holes of a fibrin plate. The fibrin plate after sample application is stably placed for 10min at room temperature, then is placed at 37 ℃ for constant-temperature culture for 18h, the vertical diameter of the dissolved transparent lysis ring of each sample is measured, the experiment is repeated for 3 times, the average value is taken, the product is calculated as the area of the dissolved transparent lysis ring, and the fibrinolytic activity of each test sample is calculated according to a standard curve.
1.2.4 identification of plasmin-producing lactic acid bacteria
(1) And (5) morphological identification.
(2) Physiological and biochemical identification: according to Bergey's Manual of bacteria identification (8 th edition) and methods for the differential identification and experiments of lactic acid bacteria.
(3) And (3) molecular identification: 16Sr DNA amplification is carried out on the target strain by using the bacterial 16S rDNA universal primer. The products after reaction were detected by 1.0% agarose gel electrophoresis, PCR products with clear bands at around 1500bp were selected for sequencing, and sequencing was performed by Biotechnology engineering (Shanghai) Co., Ltd. The sequencing results were compared and identified with the corresponding sequences of known strains in the GenBank database using the BLAST analysis tool of NCBI and phylogenetic trees were constructed using MEGA 5.05 software.
1.2.5 biological Properties of Lactobacillus plantarum HQ-3
(1) Drug susceptibility test
The (K-B) paper diffusion method is adopted to carry out drug sensitivity test, and the tested bacteria are prepared into the sterile normal saline with the viable count of about 1.5-3.0 multiplied by 10 8 And (3) uniformly coating 100 mu L of CFU/mL bacterial suspension on an MRS solid culture medium, and uniformly placing the drug sensitive tablets on the MRS solid culture medium according to the instruction. After culturing for 18h at 37 ℃, recording the diameter of the inhibition zone of each drug sensitive paper sheet. The test was repeated 3 times to average. The drug sensitivity of the strain is judged according to the minimum zone diameter of instruction sensitivity and drug sensitivity by adopting the principle of one bacterium, one drug and one judgment value. The drug sensitive paper sheets were as follows: ampicillin, amoxicillin, ceftazidime, imipenem, vancomycinTetracycline, chloramphenicol, gentamicin, rifampin, cefoperazone, amikacin, streptomycin, penicillin, and erythromycin. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 are used as quality control standard bacteria.
(2) Experiment for inhibiting bacteria
Performing a pathogenic bacteria inhibition test on the screened strains by adopting an Oxford cup method, and correcting the bacterial liquid concentrations of indicator bacteria escherichia coli, salmonella and staphylococcus aureus to 1.5-3.0 multiplied by 10 by using normal saline 8 CFU/mL, centrifuging the bacterial liquid of the tested strain (4 ℃, 8000r/min) for 10min to obtain a supernatant for later use, and detecting the inhibition effect of the added culture supernatant on the indicator bacterium by taking a blank LB culture medium as a negative control.
(3) Determination of growth curves
Activating the separated strain, inoculating 2% of the activated strain into a liquid culture medium, and culturing at 37 ℃ for 24 h. The absorbance at 600nm was measured every 2h and a growth curve was plotted.
(4) Simulated gastrointestinal test
The lactobacillus plantarum HQ-3MRS culture medium is statically cultured for 20h at 37 ℃, centrifuged for 10 minutes at 6000r/min15 ℃, and washed twice with 10mM phosphate buffer (pH 7) for later use. The bacterial pellet was resuspended to the initial volume with a saliva solution prepared with 10mM phosphate buffer (pH 7). Adjusting the pH gradient of the bacterial suspension with HCl, wherein the pH gradient is 4.0-2.3, and incubating for about 1.5h at 37 ℃. After acid treatment, the bacterial suspension is centrifuged at 6000r/min at 15 ℃ for 10 minutes, suspended in phosphate buffer (0.1M pH 8) to prepare 1% (w/v) of pig bile to the initial volume, and incubated at 37 ℃ for 10 minutes; the bacterial suspension was centrifuged, suspended in bile-pancreatin solutions (0.3% and 0.1% w/v, respectively) prepared in phosphate buffer (0.1M pH 8), and incubated at 37 ℃ for 1 hour. Samples were taken at different times (table 1) and viable count was performed.
TABLE 1 simulation of Lactobacillus plantarum HQ-3 gastrointestinal tract test method conditions
Note: samples taken before and after each step were counted for colonies (48 h at 37 ℃).
A saliva solution (0.125M NaCl, 0.007M KCl, 0.045M NaHCO3) of bovine pepsin (3g/L) was added.
b preparation of bile solutions in phosphate buffer (0.1M pH 8).
c preparation of a bile-pancreatin solution in phosphate buffer (0.1M pH 8).
(5) Determination of Lactobacillus plantarum HQ-3 specific adhesin gene
Studies have shown that the production capacity of lactobacillus plantarum-specific adhesins has a significant influence on the ability of lactobacillus plantarum to adhere to the intestinal mucosal surface in the intestinal system. According to the reported literature, specific primers for PCR amplification of the specific adhesin gene slp2588A are designed, and whether the specific adhesin gene slp2588A exists in the genome of the Lactobacillus plantarum HQ-3 is determined through PCR.
1.2.6 measurement of the Effect of dissolving fibrin
Preparing two agarose-fibrin plates, heating one plate in an electric heating constant temperature incubator at 85 deg.C for 30min to obtain a heating plate, and the other plate is standard for fibrin. Punching a hole on the plate by using a puncher with the diameter of 3mm, adding 20 mu L of crude enzyme solution into two plates respectively, culturing at 37 ℃ for 18h by using urokinase with the same volume as the control, measuring the diameter of a dissolving ring by using a vernier caliper, and judging the action mode of dissolving fibrin according to the area of the dissolving ring.
Preparation method of 1.2.7 lactobacillus plantarum HQ-3 fruit and vegetable fermented beverage
(1) Selecting the red kiwi fruits: selecting the red-yang kiwi fruits with the maturity of 8-9 minutes, and removing pests and rotten fruits for later use. Juicing the selected red kiwi fruits by using a spiral juicer, separating peels, and performing lactic acid fermentation on the remained pulp.
(2) Blending: after the red kiwi fruit pulp and water are uniformly mixed according to the volume ratio of 1:1, 80mg/100g of sodium carboxymethylcellulose and 12g/100g of white granulated sugar are added, and the pH value is adjusted to 5.5 by using sodium citrate.
(3) And (3) sterilization: the prepared red-yang kiwi fruit pulp is heated at 83 ℃ for 10min to kill harmful microorganisms, pathogenic bacteria and the like.
(5) Fermentation: cooling the sterilized red kiwi fruit pulp to room temperature, adding 1% of activated lactobacillus plantarum HQ-3, and standing and fermenting at 37 ℃ for 28 h. The red-yang kiwi fruit viable bacteria beverage fermented by lactobacillus plantarum HQ-3 is obtained.
1.2.8 viable count of lactic acid bacteria in fermented beverage
According to the food microbiological inspection of GB 4789.35-2016 food safety national standard, the total number of lactobacillus in the red kiwi fruit beverage produced by fermenting lactobacillus plantarum HQ-3 is detected.
2 results and analysis
2.1 screening of plasmin-producing lactic acid bacteria
63 strains capable of forming larger proteolytic cycles were isolated from 89 samples by enrichment culture and primary screening of acid-producing strains with skim milk, as shown in FIG. 1. The plasmin-active strains were 7 identified by agarose-fibrin plate, and the strain with large hydrolytic cycle was selected and identified as HQ-3, as shown in FIG. 2.
2.2 determination of fibrinolytic Activity
Urokinase standard curves were prepared by preparing urokinase standards at different concentrations, measuring the area of the transparent circles on a fibrin plate, and plotting a urokinase standard curve with the logarithm of the fibrinolytic activity of urokinase (lgB) as the abscissa and the logarithm of the area of the transparent circles (lgA) as the ordinate, as shown in FIG. 3. And calculating the enzyme activity of the strain HQ-3 to be 497.72U/mL according to the drawn urokinase standard curve.
2.3 identification of HQ-3 Strain
2.3.1 morphological characterisation
The bacterial colonies are round, the diameters are 3.0mm +/-1 mm, and the single bacterial colonies are milky white, convex, whitish, wet, opaque and smooth in edges, and are shown in figure 4. Under the microscope, the cocci are rod-shaped, arranged in single or pair or chain, as shown in figure 5.
2.3.2 physiological and Biochemical identification
The attribution of the bacteria can not be judged according to the physiological and biochemical characteristics, more identification indexes are needed to be integrated to analyze the classification status, and the specific physiological and biochemical index results are shown in table 2.
TABLE 2 physiological and biochemical characteristics of bacteria
Note: in the table, "-" indicates that the reaction was negative; "+" indicates a positive reaction; "growth" means that growth is possible.
2.3.3 molecular characterization
The PCR amplification of the 16S rDNA sequence obtains a target band of about 1500bp, as shown in figure 6, the PCR product is sent to Shanghai engineering for sequencing, the sequencing result of the PCR product is compared in an NCBI database, a phylogenetic tree is established by utilizing MEGA 5.0, as shown in figure 7, the homology of the strain and the 16S rRNA of Lactobacillus plantarum lp-15(FJ763580.1) in Genbank reaches 99 percent, and HQ-3 is preliminarily determined to be lactobacillus plantarum by combining the physiological and biochemical identification results.
2.4 biological Properties of the Strain HQ-3
2.4.1 drug susceptibility test
HQ-3 drug sensitivity test is carried out by using clinically common antibiotic pair, the drug sensitivity test result is shown in table 3, and the test result proves that HQ-3 has no multiple drug resistance to the following 14 antibiotics.
TABLE 3 sensitivity of the Strain HQ-3 to antibiotics
Note: s: is sensitive; i: intermediate sensitivity; r: drug resistance; data in the table are mean. + -. standard deviation
2.4.2 bacteriostatic test
Common food-borne bacteria are used as indicator bacteria, the HQ-3 fermentation supernatant is used as a sample to carry out an antibacterial test, the antibacterial test result is shown in table 4, the diameters of inhibition zones of the lactobacillus plantarum on escherichia coli ATCC 25922, staphylococcus aureus ATCC 25923 and salmonella ATCC 13311 are all larger than 16mm, and the lactobacillus plantarum has strong antibacterial effects on the three pathogenic bacteria.
TABLE 4 bacteriostatic effect of HQ-3 strain on 3 pathogenic bacteria
2.4.3 measurement of growth Curve of Strain HQ-3
The growth curve of the strain to be measured in 36 hours is shown in FIG. 8, and a simulated digestive tract environment tolerance test was carried out on a bacterial liquid cultured for about 16 hours.
2.4.4 simulated gastrointestinal tract test
MRS solid plate culture was performed at 37 ℃ for 48h at different times and the colony counts were determined, the results are shown in Table 5. As can be seen from Table 5, after a series of treatments of the gastrointestinal tract, the survival rate of the strain was high, and the colony count was finally UFC/ml. High resistance after passage through the gastrointestinal tract is essential for any potential probiotic microorganism, as it must reach the intestine and still have a high viable count (about 10) 5 To 10 6 UFC/g or ml) will have a beneficial effect on the health of the consumer. Therefore, the strain has higher value in the aspect of food research and preparation.
TABLE 5 colony count results of simulated gastrointestinal tract tests
2.4.5 Lactobacillus plantarum HQ-3 specific adhesin Gene determination
The specific adhesin gene was amplified using lactobacillus plantarum HQ-3 bacterial solution as template and lactobacillus plantarum specific adhesin gene slp2588A primer, and the results of gel electrophoresis of PCR products are shown in fig. 9. The PCR amplification result shows that the genome of the Lactobacillus plantarum HQ-3 contains a specific adhesion gene slp2588A, which indicates that the Lactobacillus plantarum HQ-3 may produce Lactobacillus plantarum specific adhesion and has potential intestinal adhesion capability.
2.5 determination of the mode of action of fibrinolysis by plasmin generated by Lactobacillus plantarum HQ-3
The results of the determination test of the mode of dissolving fibrin by plasmin generated by lactobacillus plantarum HQ-3 are shown in FIGS. 10 and 11, the positive control nattokinase has obvious dissolution rings in both heated and unheated fibrin plates, while the fermentation supernatant of the strain HQ-3 and the fermentation supernatant after enzyme treatment have no obvious dissolution rings in the fibrin plates after heating as the negative control urokinase, and the plasmin is inferred to indirectly dissolve fibrin by activating plasminogen.
2.5 Lactobacillus plantarum HQ-3 fermented Kiwi fruit beverage functionality
The number of viable lactic acid bacteria and plasmin activity of the viable red kiwi fruit functional beverage prepared by fermenting the red kiwi fruit pulp with lactobacillus plantarum HQ-3 are shown in Table 6.
Table 6 beverage experimental data
Test data show that the content of lactic acid bacteria in the live red kiwi fruit beverage produced by fermenting lactobacillus plantarum HQ-3 reaches the national standard on the number of live lactic acid bacteria in live lactic acid bacteria fermented live kiwi fruit beverage, and the live red kiwi fruit beverage has potential commercial value.
Claims (5)
1. The Lactobacillus plantarum for producing the high-activity plasminogen activator is classified and named as Lactobacillus plantarum and is preserved in the China general microbiological culture collection management center of the institute of microbiology of China academy of sciences, the preservation number is CGMCC No.17372, and the preservation time is 3 months and 20 days in 2019.
2. A fruit and vegetable juice beverage, which is prepared by using fruits and vegetables as raw materials and fermenting the lactobacillus plantarum producing a high-activity plasminogen activator according to claim 1.
3. The fruit and vegetable juice beverage according to claim 2, wherein the fruit and vegetable is kiwi.
4. A method of preparing the juice beverage of claim 3, the steps of the method comprising:
juicing kiwi fruits, adding water for blending, and fermenting by using the lactobacillus plantarum producing the high-activity plasminogen activator to obtain the high-activity plasminogen activator.
5. Method according to claim 4, characterized in that it comprises the following steps:
(1) selecting and treating kiwi fruits: selecting red-yang kiwi fruits with the maturity of 8-9 minutes, and removing pests and rotten fruits for later use; juicing the selected red kiwi fruits by using a spiral juicer, separating peels and peels, and reserving fruit pulp;
(2) blending: uniformly mixing the red kiwi fruit pulp and water according to the volume ratio of 1:1, adding 80mg/100g of sodium carboxymethylcellulose and 12g/100g of white granulated sugar, and adjusting the pH value to 5.5 by using sodium citrate;
(3) and (3) sterilization: heating the prepared red-yang kiwi fruit pulp at 83 ℃ for 10min to kill harmful microorganisms and pathogenic bacteria;
(4) fermentation: cooling the sterilized red-yang kiwi fruit pulp to room temperature, adding 1% of activated lactobacillus plantarum producing high-activity plasminogen activator for fermentation, and standing at 37 ℃ for 28h for fermentation to obtain the high-activity plasminogen activator.
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| JP2011132188A (en) * | 2009-12-25 | 2011-07-07 | Kirin Holdings Co Ltd | Lactobacillus having effect of enhancing activation of plasminogen and composition thereof |
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| CN104789511A (en) * | 2015-05-08 | 2015-07-22 | 内蒙古农业大学 | Lactobacillus plantarum AB-2 with broad-spectrum antifungal properties and application thereof |
| CN107929329A (en) * | 2017-07-07 | 2018-04-20 | 西北民族大学 | A kind of prebiotic compound bacteria Chinese medicinal granule of embolism extinguishing lipid-loweringing and preparation method thereof |
| CN109706092A (en) * | 2018-10-08 | 2019-05-03 | 四川农业大学 | The preparation method of one plant of bacillus coagulans for producing fibrinolysin and fibrinolysin and viable bacteria tablet |
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