CN112358988B - Lactobacillus plantarum LDVS008 strain and application thereof - Google Patents
Lactobacillus plantarum LDVS008 strain and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum LDVS008 strain and application thereof, the LDVS008 strain is separated from cowpea in Nanning market, has higher salt tolerance, can survive under 10% of sodium chloride concentration, also has higher nitrite degradation capability under 8% of sodium chloride concentration, can improve the nitrite degradation capability of high-salinity food, has the characteristics of high salt tolerance and high nitrite degradation capability, can greatly reduce the nitrite content of pickled food, and uses a protective agent: the bacterial sludge is prepared into freeze-dried bacterial powder by skim milk of 25-30g/100mL, cane sugar of 15-19g/100mL, zinc sulfate of 0.01-0.02g/100mL and stevioside of 10-14g/100mL, and the freeze-dried bacterial powder is used for food processing and can be better stored, transported and applied.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum strain LDVS008 and application thereof.
[ background of the invention ]
Lactobacillus plantarum (Lactobacillus plantarum), belonging to gram-positive strains, is a facultative Lactobacillus with bent rod shape, round end short rod shape, chain shape and other shapes and no spore production, which takes fructose, glucose, lactose, xylose and the like as raw materials to carry out metabolic growth to produce acid, is one of indispensable strains in the production of fermentation products and is commonly used for the production of pickled vegetables such as cowpeas, cabbages, onions and the like.
The lactobacillus plantarum is a relatively common leavening agent at present, mainly aims at improving the production efficiency of vegetables, shortening the production period and improving the product quality and safety, applicants are dedicated to screening lactobacillus plantarum, and researches find that the lactobacillus plantarum has a good effect of reducing nitrous acid; however, in the process of researching on pickled foods, the applicant finds that some lactobacillus plantarum can have a good effect of reducing nitrite, but is greatly influenced by salt (sodium chloride), and when the salt concentration is increased, the effect of reducing nitrite is obviously weakened, but in the process of pickling foods, sometimes in order to pursue taste and pickling effect, the foods need to be pickled under a high salt concentration, most lactobacillus plantarum cannot survive at the moment, the good effect of reducing nitrite cannot be achieved, and the safety of the foods is influenced, so that a strain with high salt tolerance and strong capability of degrading nitrite is selected; in the application process of the strain, the freeze-dried powder strain is easier to store and transport than a liquid strain, so that research on freeze-dried powder preparation aiming at the strain is necessary to obtain the strain freeze-dried powder with high activity.
[ summary of the invention ]
In view of the above, there is a need to provide a bacterial strain with salt tolerance and nitrite degradation, and to improve the activity of lyophilized bacterial strain powder by improving the lyophilization process.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
lactobacillus plantarum (Lactobacillus plantarum) strain LDVS008 with the preservation number of CGMCC NO: 20029.
further, the bacterial strain is separated from the sour cowpea.
The invention also comprises freeze-dried powder containing the Lactobacillus plantarum (Lactobacillus plantarum) strain LDVS 012.
Further, the protective agent in the freeze-dried powder comprises the following components: 25-30g/100mL skim milk, 15-19g/100mL sucrose and 0.01-0.02g/100mL zinc sulfate.
Further, the protective agent in the freeze-dried powder also comprises the following components: 10-14g/100mL stevioside.
The invention also comprises application of the Lactobacillus plantarum (Lactobacillus plantarum) strain LDVS012 and/or the freeze-dried powder in nitrite reduction.
The invention also comprises application of the Lactobacillus plantarum (Lactobacillus plantarum) strain LDVS012 and/or the freeze-dried powder in food processing.
The invention also comprises a method for preparing the freeze-dried powder containing the Lactobacillus plantarum (Lactobacillus plantarum) strain LDVS012, which comprises the following steps:
(1) strain activation and culture: inoculating the preserved lactobacillus plantarum to an MRS culture medium, standing overnight for seed culture, and inoculating the lactobacillus plantarum to the MRS culture medium for amplification culture after one-time passage activation;
(2) centrifugally collecting thalli and subpackaging: subpackaging the fermentation liquor, centrifuging, removing supernatant fermentation liquor, adding physiological saline for resuspending, and repeatedly centrifuging to obtain bacterial sludge; mixing and oscillating the bacterial sludge and 1/5 original fermentation liquor volume protective agent solution to make them uniform and make them into bacterial suspension;
(3) pre-freezing: the bacterial suspension was poured into a sterile petri dish with a thickness of about 0.5cm and pre-frozen at-80 ℃ for 12 h.
(4) Vacuum freeze drying: pre-freezing the bacterial sludge, and freeze-drying for 24h under the condition of vacuum degree of 20-30Pa to ensure that the water content of the freeze-dried bacterial powder is about 3 percent.
The invention has the following beneficial effects:
1. the LDVS008 strain disclosed by the invention is separated from sour cowpea in a Nanning market, has higher salt tolerance, can survive under the concentration of 10% of sodium chloride, has higher nitrite degradation capability under the concentration of 8% of sodium chloride, can improve the nitrite degradation capability of high-salinity food, has the characteristics of high salt tolerance and nitrite degradation capability, can greatly reduce the nitrite content of pickled food, and uses a protective agent: after the bacterial sludge is prepared into freeze-dried bacterial powder by 25-30g/100mL of skim milk, 15-19g/100mL of cane sugar, 0.01-0.02g/100mL of zinc sulfate and 10-14g/100mL of stevioside, the freeze-dried bacterial powder can be better stored, transported and applied in food processing, and the survival rate of bacterial strains in the bacterial powder can reach 85 percent or even more than 90 percent, so that the bacterial strains still maintain higher salt resistance and decomposition capacity on nitrite.
[ description of the drawings ]
FIG. 1 is a morphology of LDVS008 strain on petri dish according to the example of the present application;
FIG. 2 is a diagram of an embodiment of LDVS008 microscopy;
FIG. 3 is a graph of the growth of Lactobacillus plantarum strain of example 2;
FIG. 4 is a graph showing the change in pH of Lactobacillus plantarum in example 2.
[ detailed description ] embodiments
All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.
Any feature disclosed in this specification (including any accompanying claims, abstract) is merely an example of a generic series of equivalent or similar features, unless explicitly described as such.
Example 1:
this example is a screening method of Lactobacillus plantarum (Lactobacillus plantarum) strain LDVS 008:
the bacterial strain of the embodiment is separated from acid cowpea, the acid cowpea is obtained from Guangxi Nanning market and separated by adopting MRS medium plate coating method and inclined surface scribing method, and finally, the bacterial strains LDVS012, LDVS007, LDVS008 and LDVS005 are screened out. The applicant found that the strain LDVS008 has the highest tolerance to sodium chloride through measurement.
The strain of the embodiment uses NCBI Blast program to compare the spliced sequence file with data in NCBI 16S database, and the comparison result constructs phylogenetic tree through MEGA 7.0. The results showed that the homology of the strain LDVS008 with Lactobacillus plantarum was 99.18%, respectively. Physiological and biochemical experiments show that: the suitable growth temperature of the strain LDVS008 is 28-32 ℃, and the strain LDVS008 belongs to the genus of Lactobacillus and the species of Lactobacillus plantarum after being identified. The lactobacillus plantarum is preserved in the China general microbiological culture Collection center, and the address is as follows: the microbial research institute of China academy of sciences No. 3 of Xilu No. 1 of Beijing, Chaoyang district, with the collection number of CGMCC NO: 20029 and the preservation date is 2020 and 6 months and 08 days.
The morphological characteristics of the Lactobacillus plantarum (Lactobacillus plantarum) LDVS008 are shown in figure 1, the edges of bacterial colonies of the Lactobacillus plantarum are smooth and clear, the bacterial colonies are white and circular, and the middle of the bacterial colonies is convex; as shown in the microscopic image of FIG. 2, the strains observed under the microscope are short rods, mostly pair, split at the beginning and the end, no movement and no spore.
Example 2:
the growth and acid production capability of the strains LDVS005, LDVS007, LDVS008 and LDVS012 of the present example were identified:
inoculating seed fermentation liquor into a triangular flask filled with 100mL of liquid MRS according to the inoculation amount of 2% (v/v), culturing for 36h at 30 ℃, measuring OD values at the wavelength of 600nm respectively for 0, 1, 3, 6, 9, 12, 24 and 36h, and drawing a growth curve; the results are shown in FIG. 3: at 0-3h 4 lactic acid bacteria were in the lag phase in the growth phase, beginning to enter the logarithmic growth phase after 3h and beginning to enter the stationary phase after 12 h. The result shows that the lactobacillus grows and breeds more vigorously before 12 hours, and the characteristics are just consistent with the characteristics of the change curve of the pH value of the lactobacillus shown in the figure 4. At 12h, the absorbance of LDVS012 at 600nm was significantly higher than that of the other three strains (P <0.05), there was no significant difference between LDVS005 and LDVS007 (P >0.05), and LDVS008 was significantly lower than that of the other three strains (P < 0.05). At 24h and 36h, the light absorption values of LDVS012 and LDVS005 at 600nm are obviously higher than those of other two strains (P <0.05), and no obvious difference (P >0.05) exists between LDVS007 and LDVS008, and the growth curve of 4 strains with high acid production shows that LDVS005 and LDVS012 grow vigorously compared with other two strains of lactic acid bacteria, so that the strain has obvious advantages in the later period of fermentation and has the best growth capacity of LDVS 012.
Inoculating the separated and purified strains into 5mL MRS liquid culture medium, standing and culturing for 12h at 30 ℃, inoculating seed fermentation liquor into 100mL liquid MRS culture medium according to the inoculation amount of 2% (v/v), culturing and fermenting for 36h at 30 ℃, and respectively measuring the pH values of 0, 0.5, 3, 6, 9, 12, 24 and 36h, wherein the results are shown in figure 4: in the first 12h, the lactobacillus grows and breeds more vigorously and has a high acid production speed, wherein the acid production capacity of LDVS012 at the stage is superior to that of LDVS005, LDVS007 and LDVS 008. After 12h, the pH value slowly decreases, and the relative stationary phase is entered, the pH values of 4 lactobacillus strains are not obviously different at each time point (P >0.05), the pH values of MRS culture media of LDVS005, LDVS007, LDVS008 and LDVS012 at 36h are respectively 3.80, 3.85, 3.83 and 3.70, and the pH values of LDVS012 and LDVS005 are superior to those of other strains in the post-fermentation phase.
Meanwhile, the lactic acid bacteria generate acid to enable the culture medium to form a transparent ring, the size of the transparent ring reflects the acid generating capacity of the lactic acid bacteria, and the results of the transparent ring and the acidity are shown in table 1:
TABLE 1 acid production Capacity of different lactic acid bacteria
| Strain numbering | Diameter/cm of calcium dissolving ring | Acidity/%) |
| LDVS005 | 1.33±0.072 a | 2.001±0.006 a |
| LDVS007 | 1.24±0.042 b | 1.958±0.054 b |
| LDVS008 | 1.23±0.038 b | 1.950±0.030 b |
| LDVS012 | 1.34±0.076 a | 2.018±0.054 a |
As can be seen from table 1, the transparent circles of LDVS005, LDVS007, LDVS008, LDVS012 were significantly larger in diameter than the other lactic acid bacteria (P <0.05), with the transparent circle of LDVS012 having the largest diameter (1.34 ± 0.076 cm). Consistent with the results obtained for medium acidity, where LDVS005 and LDVS012 were significantly higher than LDVS007 and LDVS008 strains (P < 0.05).
Example 3:
testing of the strains' tolerance to sodium chloride:
in the culture medium flat plates containing sodium chloride with different concentrations, strains with good sodium chloride resistance can be screened out by comparing the growth conditions of the lactic acid bacteria, and the experimental results show that 4 strains of lactic acid bacteria grow well on the culture medium flat plates containing 0%, 2% and 5% of sodium chloride, and the results are both excessive and countable. In the growth conditions in MRS medium containing 8% sodium chloride and 10% sodium chloride, as shown in Table 2, the salt tolerance of different strains begins to show obvious difference, which is identical with the discovery that 8% NaCl can reduce the total biological activity of lactobacillus.
TABLE 2 growth of lactic acid bacteria in media containing sodium chloride
As can be seen from table 2, strain LDVS008 had growth on medium plates containing 10% sodium chloride, while the other strains did not. The colony growth of lactic acid bacteria on the medium plate containing 8% sodium chloride is significantly higher than that of other lactic acid bacteria (p <0.05) from the total number of LDVS008 colonies in the table, and LDVS008 colonies grow best on the medium containing 8% sodium chloride. The salt tolerance of the lactic acid bacteria is LDVS008 more than LDVS005 more than LDVS007 more than LDVS 012; therefore, the LDVS008 strain has stronger salt tolerance.
Example 4:
adding 100 mu g/mL nitrite into MRS culture solution with mass concentration of 8% sodium chloride and 10% sodium chloride; then inoculating the 4 lactobacillus strains with the inoculation amount of 5% (v/v) to obtain lactobacillus strains with the activity of about 10 6 cfu/mL; then culturing for 24h at constant temperature of 30 ℃, determining the content of nitrite by using a nitrite kit, and calculating the degradation rate of the nitrite at 6, 12 and 24 h:
in the formula W 0 Initial nitrite content
W t -nitrite content of fermentation broth at cultivation time t
The test results are shown in table 3:
TABLE 3 degradation rate of Lactobacillus nitrite under high sodium chloride concentration conditions
As can be seen from Table 3, the LDVS008 strain also has a strong capability of degrading nitrite in a sodium chloride culture medium with a mass concentration of 8%; the degradation capability of the strains LDVS005, LDVS007 and LDVS012 is far lower than that of LDVS 008; in a sodium chloride culture medium with the mass concentration of 10%, the bacterial strains LDVS005, LDVS007 and LDVS012 can not grow, and the nitrite can not be degraded without bacterial strain metabolism, which is consistent with the growth condition of the bacterial strains recorded in the table 2, thereby showing that the salt-tolerant bacterial strain LDVS008 can still keep higher degradation capability on the nitrite under the concentration of 8%.
Example 5:
the capability of directly adding the strain into food to prepare the nitrite of the pickled food is detected as follows:
the strains of the present application were used to prepare tamarind beans, namely: the above strain was prepared to about 10 6 cfu/mL bacterial liquid; washing cowpea, draining, adding 8% of salt by mass percent, softening, and inoculating the LDVS012, the LDVS005, the LDVS008, the LDVS007 and the commercial microbial inoculum according to 3%; putting the cowpea into a cowpea, pressing the cowpea for 3 days at normal temperature, taking liquid generated by pickling, detecting the content of nitrite in the cowpea, and testing results are shown in a table 4:
TABLE 4 nitrite content after pickling cowpea with different strains
| Group of | LDVS012 | LDVS005 | LDVS007 | LDVS008 | Commercial microbial inoculum |
| Nitrite (ug/ml) | 2.13 | 2.09 | 2.20 | 1.15 | 2.21 |
As can be seen from Table 4, in 8% by mass of the strain LDVS008, the nitrite concentration is significantly lower than that of LDVS012, LDVS005, LDVS007 and commercial microbial inoculum; therefore, the strain LDVS008 also has stronger nitrite decomposition capability under high-concentration salt; the LDVS012, LDVS005 and LDVS007 strains have reduced growth activity, so that the nitrite decomposition capability is greatly reduced.
Example 6:
selecting a strain LDVS008 with the strongest salt tolerance to prepare freeze-dried powder; the preparation method comprises the following steps:
(1) strain activation and culture: inoculating the preserved lactobacillus plantarum to an MRS culture medium, standing overnight for seed culture, inoculating the lactobacillus plantarum to the MRS culture medium according to the inoculation amount of 2% (v/v) after one-time passage activation, carrying out amplification culture, and carrying out shake culture at 30 ℃ for 24 hours at 40 r/min.
(2) Centrifugally collecting thalli and subpackaging: subpackaging the fermentation liquor, centrifuging at 7500rpm for 10min at 4 deg.C, removing supernatant fermentation liquor, adding physiological saline for resuspending, and repeatedly centrifuging to obtain bacterial sludge; mixing the bacterial sludge and 1/5 lyophilized protectant solution in original fermentation volume, and shaking to make it uniform to obtain bacterial suspension.
(3) Pre-freezing: the bacterial suspension was poured into a sterile petri dish with a thickness of about 0.5cm and pre-frozen at-80 ℃ for 12 h.
(4) Vacuum freeze drying: after the bacterial sludge is pre-frozen, the bacterial sludge is frozen and dried for 24 hours under the condition of vacuum degree of 30Pa (only one test condition is disclosed in the embodiment, and the freezing and drying effect can be actually achieved under 20-30 Pa), so that the moisture content of the freeze-dried bacterial powder is about 3%.
The freeze-drying protective agent is considered to have the greatest influence on the protective agent through orthogonal optimization selection of the applicant, and the components comprise 30-33g/100mL of skim milk, 22-25g/100mL of lactose and 0.01-0.02g/100mL of zinc sulfate; in addition, the applicant also unexpectedly finds that stevioside has an obvious protective effect on lactobacillus, and the effect on the strain LDVS008 is particularly obvious when the concentration is 10-14g/100 mL; the applicant selects several groups of obviously representative formulas for comparison in the experimental process, and the specific formulas are shown in table 5:
TABLE 5 selection of formulations for lyophilized powder protectants
According to the table 5, the freeze-dried powder prepared by the freeze-dried powder protective agent is subjected to survival rate determination, nitrous acid degradation capability and salt tolerance determination, and the determination method comprises the following steps:
(1) and (3) survival rate determination: and at 24h, respectively measuring the viable count of the lactobacillus plantarum before freeze-drying and the viable count of the bacterial sludge with the same volume after freeze-drying.
Freeze-drying survival rate/% (viable count of bacterial sludge 1mL after freeze-drying)/(viable count of bacterial sludge 1mL before freeze-drying)
(2) Inoculating the freeze-dried powder into MRS culture solution containing 150 mu g/mL of nitrite, carrying out shake culture at constant temperature of 30 ℃ for 24h, then measuring the nitrite content, calculating the degradation rate of the nitrite, and taking a sterile water inoculation group as a control. The nitrite content is determined by a nitrite kit.
Nitrite degradation rate/% (nitrite content before fermentation-nitrite content in fermentation broth)/nitrite content before fermentation x 100%.
(3) The lyophilized powder was inoculated into MRS medium containing 8% sodium chloride and 10% sodium chloride at high concentration, and the growth was observed. The number of colonies in the medium was tested.
The results obtained are shown in Table 6:
TABLE 6 Strain survival, nitrite degradation and salt tolerance of different lyoprotectants
As can be seen from Table 6, the survival rates, nitrite degradation rates and salt tolerance of groups 5-7 were the highest, and the survival rates, nitrite degradation rates and salt tolerance of groups 1-3 were higher than those of groups 4 and 8; this shows that nitrite degradation capacity and salt tolerance are in positive correlation with survival rate of bacterial strains, and when the freeze-dried powder is prepared, the freeze-drying protective agent: skim milk, cane sugar and zinc sulfate can basically meet the requirements of the strains, the survival rate of the strains can reach more than 85 percent, the addition of 10-14g/100mL of stevioside can promote the protection of LDVS008 strains, and the basic protective agent comprises the following components: 30g/100mL skim milk, 19g/100mL sucrose and 0.02g/100mL zinc sulfate, the survival rate of the strain LDVS008 will be affected if the stevioside addition amount is not within the range of 10-14g/100mL, and the degradation rate of nitrite and the tolerance to sodium chloride will be affected, so that stevioside in the components may mainly play a role in balancing cell concentration, and cell damage of bacteria can be caused if the stevioside is too low or too high.
Example 7:
the strain freeze-dried powder is used for detecting the capability of nitrite in the high-concentration salt pickled food:
the frozen powder prepared by the groups 1-8 and the leavening agent (purchased from Shaanxi Saen Biotechnology Co., Ltd.) purchased on the market are selected to produce the pickled food, and the food processing method comprises the following steps:
cleaning vegetables (radish), draining, adding 8% of salt, pepper and other seasonings, inoculating the freeze-dried strains of the groups 1-8 according to the inoculation amount of 5% of the total mass, pickling, and absorbing pickling liquid to detect the nitrite content in the pickled vegetables after pickling for 3 days. The nitrite content obtained is tested as in table 6:
TABLE 6 nitrite content in radish pickle
| Group of | Group 1 | |
|
|
|
Group 6 | Group 7 | Group 8 | Commercial species |
| Nitrite (ug/ml) | 0.91 | 0.96 | 0.92 | 1.02 | 0.72 | 0.87 | 0.76 | 1.14 | 1.91 |
As can be seen from Table 6, the nitrite content was lowest for groups 5-7, and higher for groups 1-3 than for groups 5-7; the nitrite content of the group 4 and the group 8 is lower than that of the group 1-the group 3 and the group 5-the group 7, and the nitrite content of the commercial species is the highest, which shows that the freeze-dried powder prepared from the bacterial strain LDVS008 can still maintain higher activity and greatly reduce the nitrite content in the pickled food.
In conclusion, the lactobacillus plantarum LDVS008 disclosed by the application can grow under a higher sodium chloride concentration, keeps a higher capacity of degrading nitrite and can still keep a higher activity after being prepared into freeze-dried powder.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Claims (5)
1. Comprises Lactobacillus plantarum (A)Lactobacillus plantarum) Strain LDVS008 freeze-dried powder, which is characterized in that the lactobacillus plantarum (C)Lactobacillus plantarum) The preservation number of the strain LDVS008 is CGMCC NO: 20029; the protective agent in the freeze-dried powder comprises the following components: 25-30g/100mL skim milk, 15-19g/100mL sucrose and 0.01-0.02g/100mL zinc sulfate.
2. The lyophilized powder of claim 1, wherein the protectant components of the lyophilized powder further comprise: 10-14g/100mL stevioside.
3. Use of a lyophilized powder according to claim 1 or 2 for improving salt tolerance.
4. Use of a lyophilized powder according to claim 1 or 2 for food processing.
5. A process for producing a composition comprising Lactobacillus plantarum (L.) (I) according to claim 1Lactobacillus plantarum) The method for preparing the LDVS008 strain freeze-dried powder is characterized by comprising the following steps:
(1) strain activation and culture: inoculating the preserved lactobacillus plantarum to an MRS culture medium, standing overnight for seed culture, and inoculating the lactobacillus plantarum to the MRS culture medium for amplification culture after one-time passage activation;
(2) centrifugally collecting thalli and subpackaging: subpackaging the fermentation liquor, centrifuging, removing supernatant fermentation liquor, adding physiological saline for resuspending, and repeatedly centrifuging to obtain bacterial sludge; mixing and oscillating the bacterial sludge and 1/5 original fermentation liquor volume protective agent solution to make them uniform and make them into bacterial suspension;
(3) pre-freezing: pouring the bacterial suspension into a sterile culture dish, wherein the thickness is 0.5cm, and pre-freezing for 12h at-80 ℃;
(4) vacuum freeze drying: pre-freezing the bacterial sludge, and freeze-drying under the vacuum degree of 20-30Pa for 24h to make the water content of the freeze-dried bacterial powder be 3%.
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