Background
At present, the microorganism strains are generally subjected to a regular transplantation preservation method, a liquid paraffin preservation method, freeze drying and a liquid nitrogen ultralow temperature preservation method. The preservation under the low temperature condition can slow down the metabolic activity of the microbial strains, inhibit the propagation speed of the microbial strains, and achieve the purposes of reducing the mutation of the strains and prolonging the preservation time of the strains.
A regular transplant preservation method: is a classic simple preservation method, also called as a passage preservation method. The method comprises slant culture, liquid culture, puncture culture and the like. The method has the advantages of simple operation and the disadvantages of large contact surface between the slant culture medium and air, rapid dehydration and dry shrinkage within half a year; the cotton plug is easy to absorb water and damp, so that strains are easy to contaminate; the preservation temperature is higher, the strains still have certain metabolic activity and can be propagated, in order to prevent the strains from dying, the strains are required to be passaged and transferred in 3-4 months, the workload is increased, and the strains have high strain variation frequency and weak activity due to frequent strain passage.
Preservation method of liquid paraffin: the basic method is the same as the regular transplant preservation method, and the slant culture is covered with liquid paraffin in order to prevent evaporation of water from the culture medium and to prevent oxygen from entering. The method has the advantages that the strain preservation time is long, generally 2-10 years are different, and the method has the defect that the method is only suitable for preservation of microorganism strains which can not decompose liquid paraffin; when the strains are preserved, the test tube rack is required to be vertically placed, the space occupied by preservation is large, and the whole operation is complicated.
The freeze drying process is to freeze the suspension of the strain to be preserved in low temperature and sublimate it in vacuum condition for drying, and the low temperature, drying and air isolating are three essential factors for strain preservation.
The liquid nitrogen ultra-low temperature preservation is a long-term preservation method for preserving strains in liquid nitrogen at-196 ℃ or nitrogen at-150 ℃, the principle of the method is to effectively preserve microorganisms by utilizing the tendency of the microorganisms to stop metabolism below-130 ℃, and the method also has high equipment requirement and complex operation. In addition, the resuscitation is troublesome.
The lactobacillus is a general name of a kind of bacteria which can utilize fermentable carbohydrate to generate a large amount of lactic acid, has various physiological functions, is widely applied to the industries of food, feed, medicine and the like, and the extension of the living bacteria preservation period of the lactobacillus is a technical key problem in the development and development of the existing lactobacillus preparation.
The lactic acid bacteria using the above four preservation methods have disadvantages. The liquid paraffin preservation method is only suitable for slant culture and puncture culture, but the slant culture method is not suitable for culture of lactobacillus because the contact surface of a slant culture medium and air is large, and the puncture culture is inconvenient to operate when strains are activated. The freeze-drying method and the liquid nitrogen ultra-low temperature preservation method have the advantages of complex operation, high technical content, high equipment investment and high operation cost, are only suitable for professional preservation organizations, and are not suitable for production enterprises. The liquid culture in the periodical transplanting preservation method belongs to a common preservation method of lactic acid bacteria, but because the liquid culture needs to be vertically placed by using a test tube rack, the space occupied by preservation is larger, most importantly, the liquid culture needs to be passaged once every two months or so, otherwise, the strain dies, the workload is increased, and the strain has high strain variation frequency and weakened vitality because the strain is passaged frequently. For the reasons, a lactobacillus preservation method which is simple and convenient and does not need total passage and is suitable for enterprises needs to be researched urgently.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for preserving lactic acid bacteria.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for preserving lactic acid bacteria comprises the steps of adopting vegetables as strain preservation carriers, inoculating the lactic acid bacteria into a mixed solution of sterilized vegetable particles and an improved MRS culture medium for culture, centrifuging after culture, removing supernatant, collecting the vegetable particles, and preserving in a sealed manner at the preservation temperature of-20-4 ℃.
The mixed liquid of the vegetable particles and the improved MRS culture medium is as follows: 20-30 g of vegetable particles, 6-10 g of peptone, 6-10 g of beef extract, 12-20 g of glucose, 2g of triammonium citrate, 5g of yeast extract, 2g of sodium acetate and K 2 HPO 4 2g、MgSO 4 ·7H 2 O 0.58g、MnSO 4 ·4H 2 0.25g of O and 1L of distilled water.
The method specifically comprises the following steps:
a) Sterilizing the vegetable particles at 121 ℃ for 10min according to the dosage, and cooling for later use;
b) Sterilizing the modified MRS culture medium at 121 ℃ for 15min according to the dosage, and cooling for later use;
c) Mixing the sterilized vegetable particles of the step a) with the sterilized modified MRS medium of the step b);
d) Inoculating lactobacillus into the above mixture at an inoculation amount of 1-3% (v/v) under aseptic condition, and culturing at 38-42 deg.C for 1d;
e) Centrifuging the cultured strain at 5000r/min for 10min, discarding supernatant, collecting vegetable particles, drying at 42 deg.C for 2 hr under aseptic condition, and sealing for storage.
The lactobacillus is Lactobacillus plantarum, lactobacillus brevis or Lactobacillus paracasei.
The vegetables are Chinese cabbage highs, radishes, cowpeas, gingers, cabbages or leaf mustard;
the granules are in the shape of blocks with the length, width and height respectively not more than 0.5 cm.
The preferred storage temperature is 1-4 ℃ or-16 ℃ to-20 ℃.
The preferable mixed liquid of the vegetable particles and the improved MRS culture medium is as follows: 25g of vegetable particles, 9g of peptone, 8g of beef extract, 14g of glucose, 2g of triammonium citrate, 5g of yeast extract, 2g of sodium acetate and K 2 HPO 4 2g、MgSO 4 ·7H 2 O 0.58g、MnSO 4 ·4H 2 0.25g of O and 1L of distilled water.
The strain recovery method comprises transferring the preserved vegetable granules into liquid MRS culture medium under aseptic condition, and culturing at proper temperature.
Compared with the prior art, the beneficial effect of this application is:
the preservation method overcomes the defects in the prior art, and provides a microorganism strain preservation method which is suitable for production enterprises, has long preservation time, simple and practical operation, stable and reliable strain quality and low preservation cost, and specifically comprises the following steps:
1. the vegetable, especially the cabbage side used in the invention is a natural edible material, because the interior of the vegetable contains a large number of micro-tube bundles, the internal surface area is large, the microbial cells are favorably adsorbed, and the cabbage side has rich sources and low price, so the cabbage side is an ideal strain preservation embedding medium.
2. The invention uses vegetable particles to preserve strains, can solve the problems of high difficulty in culture and proliferation of free strains, poor external resistance and difficult long-term preservation, improves the convenience of strain application, prolongs the preservation period of the strains, and can preserve the strains by refrigeration at 1-4 ℃ for 3-4 years and refrigeration at-16 ℃ to-20 ℃ for 4-5 years.
3. The invention selects proper culture medium and culture condition to make microorganism grow in culture, then centrifugal drying, the water content of dried vegetable is 60-70%, because the water content in strain cell is very low, it can be refrigerated or frozen. The method has the advantages of low equipment investment, capability of realizing long-term preservation of strains in a common refrigerator, strong practicability, simple operation, stability, reliability, low cost and long preservation period.
4. Compared with the common MRS culture medium preservation method, the vegetable particle preservation method has the advantages that the preservation space is reduced, a passage rotating pipe is not needed, and the workload is reduced.
5. Compared with the freeze-drying preservation method, the method does not need rapid freezing at extremely low temperature (-80 ℃) and long-time freeze-drying treatment, and can be realized only by the refrigeration or freezing condition of a common refrigerator.
Detailed Description
The following examples are further illustrative of the present invention, but the present invention is not limited to the preparation and use in this embodiment.
And screening the optimal quantity range of the improved MRS culture medium components according to a single-factor experiment, determining the final optimal culture medium ratio by using a response surface experiment, and taking the number of lactobacillus paracasei obtained by fermentation as an index.
The lactobacillus plantarum, lactobacillus brevis, or lactobacillus paracasei used in the following examples were all commercially available from beijing baiopaowabri: lactobacillus plantarum bio-60181, lactobacillus brevis bio-67750, lactobacillus paracasei bio-112991.
One single factor test:
on the basis of MRS culture medium, different quantities of peptone, beef extract, glucose and Chinese cabbage upper particles are selected, and the contents of other components are unchanged. The optimal amount ranges of the four factors of peptone (4,6,8,10,12,14), beef extract (4,6,8,10,12,14), glucose (8,12,16, 20,24,28) and Chinese cabbage side granules (10,15,20,25,30,35) are determined by single factors.
1. The effect of peptone content on the number of bacteria is shown in FIG. 1, and it can be seen from FIG. 1 that the number of bacteria is up to 117.35 hundred million, the optimal range is 6-10 when the peptone content is 8 g.
2. The effect of the content of the beef extract on the number of bacteria is shown in FIG. 2, and it can be seen from FIG. 2 that the number of bacteria is up to 115.26 hundred million, and the optimal range is 6-10 when the content of the beef extract is 8 g.
3. The effect of glucose content on the number of bacteria is shown in FIG. 3. From FIG. 3, it can be seen that the number of bacteria is up to 110.45 billion, with an optimal range of 8-16, when the glucose content is 12 g.
4. The influence of the content of Chinese cabbage leaf granules on the number of bacteria is shown in FIG. 4, and it can be seen from FIG. 4 that the number of bacteria can reach as high as 115.13 hundred million, and the optimal range is 20-30, when the content of Chinese cabbage leaf granules is 25 g.
Two response surface test design
According to the result of the single-factor test, selecting factors of peptone, glucose, beef extract and Chinese cabbage side particles which influence the bacterial count of the strain, and designing a 4-factor 3 horizontal Box-Behnken response surface test by using Design-Expert 8.0.6 software with the bacterial count as a response value. The specific scheme is shown in table 1. And (3) performing quadratic multiple regression equation fitting on the bacterial count to obtain a regression equation of the functional relationship between each factor and the response value, and determining the optimal culture medium formula according to the generated response surface diagram.
TABLE 1 response surface factor horizon
29 groups of tests are designed by using Box-Benhnken with the number of bacteria as response values, and the design and the results of the Box-Benhnken tests are shown in Table 2.
TABLE 2 Box-Benhnken test design and results
|
Peptone
|
Glucose
|
Beef extract
|
Chinese cabbage granule
|
Number of bacteria
|
1
|
0
|
0
|
-1
|
-1
|
105.44
|
2
|
0
|
0
|
1
|
-1
|
109.71
|
3
|
1
|
0
|
1
|
0
|
113.67
|
4
|
0
|
0
|
0
|
0
|
119.03
|
5
|
-1
|
-1
|
0
|
0
|
107.24
|
6
|
1
|
1
|
0
|
0
|
115.76
|
7
|
-1
|
0
|
0
|
1
|
106.29
|
8
|
1
|
0
|
0
|
1
|
115.66
|
9
|
0
|
-1
|
0
|
1
|
109.42
|
10
|
0
|
1
|
0
|
-1
|
112.00
|
11
|
0
|
0
|
-1
|
1
|
111.09
|
12
|
0
|
0
|
1
|
1
|
112.70
|
13
|
-1
|
1
|
0
|
0
|
108.33
|
14
|
0
|
1
|
0
|
1
|
114.15
|
15
|
0
|
-1
|
-1
|
0
|
106.89
|
16
|
0
|
1
|
1
|
0
|
114.88
|
17
|
0
|
0
|
0
|
0
|
117.14
|
18
|
-1
|
0
|
-1
|
0
|
103.95
|
19
|
1
|
-1
|
0
|
0
|
106.27
|
20
|
0
|
0
|
0
|
0
|
118.82
|
21
|
0
|
0
|
0
|
0
|
116.14
|
22
|
-1
|
0
|
0
|
-1
|
104.62
|
23
|
-1
|
0
|
1
|
0
|
108.16
|
24
|
0
|
-1
|
0
|
-1
|
109.23
|
25
|
1
|
0
|
-1
|
0
|
110.26
|
26
|
0
|
-1
|
1
|
0
|
109.01
|
27
|
0
|
1
|
-1
|
0
|
111.35
|
28
|
1
|
0
|
0
|
-1
|
109.32
|
29
|
0
|
0
|
0
|
0
|
117.11 |
The result of the table 2 is subjected to multiple regression fitting by Design-Expert 8.0.6 software to obtain a regression equation
Y=+117.65+2.70*A+2.37*B+1.60*C+1.58*D+2.10*A*B-0.20*A*C+1.17*A*D+0.35*B*C+0.49*B*D-0.66*C*D-4.94*A 2 -3.07*B 2 -3.99*C 2 -3.68*D 2 。
The optimal formula of the response surface test result is as follows: 8.85g of peptone, 14.27g of glucose, 8.38g of beef extract, 26.52g of Chinese cabbage side granules and 119.283 hundred million bacteria. In order to facilitate the actual operation, the formula is adjusted as follows: the method comprises the following steps of performing tests according to the conditions of 9g of peptone, 14g of glucose, 8g of beef extract and 25g of Chinese cabbage side particles, repeating the tests for 3 times to obtain an average value, wherein the measured bacteria number is 119.87 hundred million, the relative error with a predicted value is 0.49%, the equation fitting is better, the formula parameters of the culture medium obtained by optimizing a response surface are accurate, and the method has practical value.
TABLE 3 analysis of variance
Source
|
Sum of squares
|
Degree of freedom
|
Mean square
|
F value
|
P value
|
Significance of
|
Model (model)
|
510.56
|
14
|
36.47
|
22.20
|
<0.0001
|
**
|
Peptone
|
87.21
|
1
|
87.21
|
53.08
|
<0.0001
|
**
|
Glucose
|
67.26
|
1
|
67.26
|
40.94
|
<0.0001
|
**
|
Beef extract
|
30.56
|
1
|
30.56
|
18.60
|
0.0007
|
**
|
Chinese cabbage granule
|
30.05
|
1
|
30.05
|
1829
|
0.0008
|
**
|
AB
|
17.64
|
1
|
17.64
|
10.74
|
0.0055
|
**
|
AC
|
0.16
|
1
|
0.16
|
0.097
|
0.7596
|
-
|
AD
|
5.45
|
1
|
5.45
|
332
|
0.0899
|
-
|
BC
|
0.5
|
1
|
0.5
|
0.30
|
0.5910
|
-
|
BD
|
0.96
|
1
|
0.96
|
0.58
|
0.4572
|
-
|
CD
|
1.77
|
1
|
1.77
|
1.08
|
0.3171
|
-
|
A 2 |
158.36
|
1
|
158.36
|
96.38
|
<0.0001
|
**
|
B 2 |
60.98
|
1
|
60.98
|
37.11
|
<0.0001
|
**
|
C 2 |
103.45
|
1
|
103.45
|
62.96
|
<0.0001
|
**
|
D 2 |
87.77
|
1
|
87.77
|
53.42
|
<0.0001
|
**
|
Residual error
|
23.00
|
14
|
1.64
|
|
|
|
Missimilitude term
|
16.90
|
10
|
1.69
|
1.11
|
0.5022
|
-
|
Pure error
|
6.11
|
4
|
1.53
|
R 2 =0.9569
|
A djR 2 =0.9138
|
|
Sum of
|
533.57
|
28
|
|
|
|
|
Note: * Significand (P < 0.05), indicates extremely significant (P < 0.01); -means not significant.
As can be seen from Table 3, the coefficient of determination R of the model 2 =0.9569, correction determination coefficient a djR 2 =0.9138, which indicates that the actual and predicted values of the test fit well. From the influence on the bacterial count, the influence of the peptone, the glucose, the beef extract and the Chinese cabbage side particles on the bacterial count reaches an extremely obvious level. The regression model P is less than 0.0001, which shows that the secondary model adopted in the test is very obvious, the disimilation term P =0.5022 is more than 0.05, and the model disimilation term is not obvious, which shows that the model is statistically significant, and the improved MRS culture medium is suitable for lactic acid bacteria, in particular for lactobacillus plantarum, lactobacillus brevis or lactobacillus paracasei.
Example 1
a) Filling 25g Chinese cabbage leaf granules into a triangular flask, sterilizing at 121 ℃ for 10min, and cooling for later use;
b) Filling the improved MRS culture medium into a 500mL triangular flask, sterilizing at 121 ℃ for 15min, and cooling for later use;
c) Mixing the sterilized Chinese cabbage leaf particles in the step a) with the sterilized improved MRS culture medium in the step b);
d) Inoculating Lactobacillus plantarum into the mixed solution according to the inoculation amount of 1% (v/v) under aseptic conditions, and culturing at 38 deg.C for 1d;
e) Transferring the cultured strain into an aseptic centrifuge tube under aseptic condition, centrifuging for 10min at 5000r/min, discarding supernatant, collecting Chinese cabbage leaf granules, replacing the centrifuge tube with sterilized gauze plug, drying in a drying oven at 42 deg.C for 2 hr, transferring into an aseptic EP tube, packaging, and storing at 4 deg.C and-18 deg.C in a refrigerator.
The ratio of the improved MRS culture medium is as follows: 9g of peptone, 8g of beef extract, 14g of glucose, 2g of triammonium citrate, 5g of yeast extract, 2g of sodium acetate and K 2 HPO 4 2g、MgSO 4 ·7H 2 O 0.0.58g、MnSO 4 ·4H 2 0.0.25g of O and 1L of distilled water.
Example 2
a) Filling 25g Chinese cabbage leaf granules into a triangular flask, sterilizing at 121 ℃ for 10min, and cooling for later use;
b) The improved MRS culture medium is filled in a 500mL triangular flask, sterilized at 121 ℃ for 15min and cooled for standby;
c) Mixing the sterilized Chinese cabbage leaf particles in the step a) with the sterilized improved MRS culture medium in the step b);
d) Inoculating Lactobacillus brevis into the mixed solution according to the inoculation amount of 2% (v/v) under aseptic condition, and culturing at 40 deg.C for 1d;
e) Transferring the cultured strain into an aseptic centrifuge tube under aseptic condition, centrifuging for 10min at 5000r/min, discarding supernatant, collecting Chinese cabbage leaf granules, replacing the centrifuge tube with sterilized gauze plug, drying in a drying oven at 42 deg.C for 2 hr, transferring into an aseptic EP tube, packaging, and storing at 4 deg.C and-18 deg.C in a refrigerator.
The ratio of the improved MRS culture medium is as follows: peptone 9g, beef extract 8g, grape14g of sugar, 2g of ammonium citrate tribasic, 5g of yeast extract, 2g of sodium acetate and K 2 HPO 4 2g、MgSO 4 ·7H 2 O 0.58g、MnSO 4 ·4H 2 0.25g of O and 1L of distilled water.
Example 3
a) Placing 25g Chinese cabbage leaf granules in a triangular flask, sterilizing at 121 deg.C for 10min, and cooling;
b) Filling the improved MRS culture medium into a 500mL triangular flask, sterilizing at 121 ℃ for 15min, and cooling for later use;
c) Mixing the sterilized Chinese cabbage leaf particles in the step a) with the sterilized improved MRS culture medium in the step b);
d) Inoculating lactobacillus paracasei into the mixed solution according to the inoculation amount of 3% (v/v) under the aseptic condition, and culturing for 1d at 42 ℃;
e) Transferring the cultured strain into an aseptic centrifuge tube under aseptic condition, centrifuging for 10min at 5000r/min, discarding supernatant, collecting Chinese cabbage leaf granules, replacing the centrifuge tube with sterilized gauze plug, drying in a drying oven at 42 deg.C for 2 hr, transferring into an aseptic EP tube, packaging, and storing at 4 deg.C and-18 deg.C in a refrigerator.
The ratio of the improved MRS culture medium is as follows: 9g of peptone, 8g of beef extract, 14g of glucose, 2g of triammonium citrate, 5g of yeast extract, 2g of sodium acetate and K 2 HPO 4 2g、MgSO 4 ·7H 2 O 0.58g、MnSO 4 ·4H 2 0.25g of O and 1L of distilled water.
Table 4 the deposited species of examples 1-3:
example numbering
|
Name of scholars
|
Name of Chinese
|
1
|
Lactobacillusplanetarium
|
Lactobacillus plantarum
|
2
|
Lactobacillusbrevis
|
Lactobacillus brevis
|
3
|
Lactobacillus paracasei
|
Lactobacillus paracasei |
TABLE 5 results of initial cell number measurement of cultures deposited in examples 1-3
Table 6 strain preservation survival (%) units at different ages at 4 ℃: year of year
Example numbering
|
Name of Chinese
|
1 year
|
2 years old
|
3 years old
|
4 years old
|
1
|
Lactobacillus plantarum
|
94
|
85
|
45
|
14
|
2
|
Lactobacillus brevis
|
92
|
83
|
38
|
10
|
3
|
Lactobacillus paracasei
|
87
|
80
|
41
|
18 |
Table 7-18 ℃ survival (%) of strain preservation at different age: year-round
Example numbering
|
Name of Chinese
|
1 year
|
2 years old
|
For 3 years
|
4 years old
|
5 years old
|
1
|
Lactobacillus plantarum
|
96
|
90
|
81
|
66
|
58
|
2
|
Lactobacillus brevis
|
95
|
80
|
72
|
65
|
52
|
3
|
Lactobacillus paracasei
|
92
|
87
|
76
|
63
|
55 |
As can be seen from the above examples, the preservation period of the strain is prolonged by adopting the preservation method of the embodiment, compared with the preservation of the conventional liquid MRS culture medium at 1-4 ℃ for only 2-3 months, and the preservation can not be carried out by freezing, the method of the embodiment can be preserved for 3-4 years by refrigeration at 1-4 ℃ and can be preserved for 4-5 years by freezing at-16 ℃ to-20 ℃, the operation is simple and practical, the quality of the strain is stable and reliable, and the preservation cost is low.