CN113278538A - Lactic acid bacteria with high folic acid yield, preparation method and application thereof - Google Patents
Lactic acid bacteria with high folic acid yield, preparation method and application thereof Download PDFInfo
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- 239000011724 folic acid Substances 0.000 title claims abstract description 57
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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Abstract
The invention belongs to the technical field of lactic acid bacteria, and particularly relates to a lactic acid bacteria with high folic acid yield, a preparation method and application thereof; the invention provides a lactic acid bacterium with high folic acid yield, a preparation method and application thereof; the lactobacillus is bifidobacterium longum BL21 with the preservation number of CGMCC No. 10452; the folic acid obtained by the lactobacillus for high folic acid yield through the microbial product has no side effect on human body, so that the screened probiotic strains which can produce folic acid with high folic acid yield and can be well planted in the human body have high social value, and the yield of the obtained folic acid is higher than that of the existing strains; the lactic acid bacteria with high folic acid yield disclosed by the invention also have the acid resistance and bile salt resistance.
Description
Technical Field
The invention relates to the technical field of lactic acid bacteria, in particular to a lactic acid bacteria with high folic acid yield, a preparation method and an application thereof.
Background
Folic acid is one of B vitamins, can improve memory, delay brain cognitive ability degeneration, and has the function of preventing senile dementia; can soften blood vessel, prevent hypertension and arteriosclerosis; preventing fetal neural tube defect, heart defect, and upper lip crack defect. The human body cannot synthesize folic acid independently, natural folic acid is obtained mainly through food, and anemia and other symptoms are caused by insufficient folic acid intake or malabsorption. The commercially available folic acid tablets are mainly chemically synthesized, are low in safety and free of reducibility, and can cause some adverse reactions after long-term administration. The natural vitamin B group, such as folic acid, biotin, thiamine, nicotinic acid, vitamin B6, riboflavin, vitamin B12, etc., has no side effect on human body. Some microorganisms can synthesize high-activity tetrahydrofolic acid to be directly utilized by human bodies, and the method has good application prospect in food industry. Therefore, the screened probiotic strains capable of producing folic acid with high yield have high social value.
Since folic acid is mainly ingested by food, folic acid deficiency occurs when folic acid is not ingested or the utilization rate of folic acid is reduced. The natural folic acid is mainly derived from folic acid of green foods, animal livers, soybeans and wheat, but the extraction of folic acid from these natural raw materials is costly and not conducive to industrial production. Some microorganisms have the capability of synthesizing B vitamins, including folic acid, biotin, thiamine, nicotinic acid, vitamin B6, riboflavin, vitamin B12 and the like, and the folic acid obtained by the microorganisms has no side effect on human bodies, so that the screened probiotic strains which can produce the folic acid with high yield and can be planted in the bodies well have high social value.
Disclosure of Invention
In view of the above, the present invention provides a lactic acid bacterium with high folate yield, a preparation method thereof and applications thereof.
The technical scheme of the invention is realized as follows:
a lactobacillus for high yield of folic acid is Bifidobacterium longum BL21 with a preservation number of CGMCC No. 10452.
In order to prepare the lactic acid bacteria with high folic acid yield, the invention also provides a preparation method, which comprises the following steps:
s1, screening: obtaining a sample to be screened from saliva and excrement of nature and infants, putting the sample to be screened into a bottle containing normal saline, vibrating and uniformly mixing, and marking an obtained product as bacterial liquid, wherein the mass of the normal saline is 15-20 times of that of the sample to be screened;
s2, dilution: pouring the bacterial liquid obtained in the step 1 into a test tube filled with normal saline to carry out gradient dilution step by 10 times, and marking the obtained product as diluted bacterial liquid;
s3, coating: coating 100 mu L of the diluted bacterial liquid obtained in the step 2 on an agar solid plate by using a coating rod, inverting and putting into an incubator for anaerobic culture for 36-60 h;
s4, purification: after the culture is finished, selecting the agar solid plate with the number of the single colonies larger than 50 on the agar solid plate, and carrying out streak purification on the single colonies on the agar solid plate for multiple times until the microscopic examination forms of the colonies on the whole plate are consistent.
Further, the agar solid plate described in step 3 was prepared by the following steps: 20g of glucose, 0g of chen egg white, 0g g of beef extract, 5g of yeast extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 801 g of tween, 0.59g of magnesium sulfate, 0.19g of manganese sulfate monohydrate, 1g of L-cysteine, 1000mL of distilled water and 15g of agar are mixed uniformly, the pH value is adjusted to 6.8 by using sodium hydroxide, and the mixture is sterilized at 121 ℃ for 15 min.
Further, the agar liquid medium described in step 3 is prepared by the following steps: 20g of glucose, 0g of chen egg white, 0g g of beef extract, 5g of yeast extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 801 g of tween, 0.59g of magnesium sulfate, 0.19g of manganese sulfate monohydrate, 1g of L-cysteine, 1000mL of distilled water and 15g of agar are mixed uniformly, the pH value of the distilled water is adjusted to 6.8, and the mixture is sterilized at 121 ℃ for 15 min.
Further, after the purification in step 4 is completed, picking the single colony obtained in step 4 to an agar liquid culture medium for propagation, preserving the strain in the agar liquid culture medium containing 30% of glycerol, and freezing and preserving the strain in a refrigerator at-80 ℃.
The invention also discloses application of the high-yield lactic acid bacteria in folic acid production, and folic acid is synthesized by adopting the lactic acid bacteria.
Has the advantages that:
1. the folic acid obtained by the lactic acid bacteria with high folic acid yield through microbial products has no side effect on human bodies, so that the screened probiotic strains which can produce folic acid with high folic acid yield and can be well planted in the bodies have high social value, and the folic acid yield is higher than that of the existing strains.
2. The lactic acid bacteria with high folic acid yield disclosed by the invention also have the acid resistance and bile salt resistance.
Drawings
FIG. 1 is a microscopic picture of a strain of lactic acid bacteria of the present invention with high folate productivity after 16h of plating;
FIG. 2 shows the base sequence of a highly folate-producing lactic acid bacterial strain according to the present invention;
FIG. 3 acid resistance experiment of high folate yielding lactic acid bacterial strains;
FIG. 4 shows the bile salt tolerance test of the high folate yielding lactic acid bacterial strain of the present invention;
FIG. 5 shows the growth curve of the high folate producing lactic acid bacterial strain of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The preparation method of the MRS liquid culture medium comprises the following steps: 20g of glucose, 0g of chen egg white, 0g g of beef extract, 5g of yeast extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 801 g of tween, 0.59g of magnesium sulfate, 0.19g of manganese sulfate monohydrate, 1g of L-cysteine, 1000mL of distilled water, pH adjusted by sodium hydroxide to be 6.8, and sterilization at 121 ℃ for 15 min.
The preparation method of the MRS solid culture medium comprises the following steps: 20g of glucose, 0g of chen egg white, 0g g of beef extract, 5g of yeast extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 801 g of tween, 0.59g of magnesium sulfate, 0.19g of manganese sulfate monohydrate, 1g of L-cysteine, 1000mL of distilled water, 15g of agar and sodium hydroxide for adjusting the pH value to 6.8, and sterilizing at 121 ℃ for 15 min.
The preparation method of the culture medium of the fermentation infusion bottle comprises the following steps: 25g of glucose, 0g of chen egg white, 0g g of beef extract, 15g of yeast extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 801 g of tween, 0.59g of magnesium sulfate, 0.19g of manganese sulfate monohydrate, 1g of L-cysteine, 1000mL of distilled water, pH adjusted by sodium hydroxide to be 6.8, and sterilization at 121 ℃ for 15 min.
EXAMPLE 1 isolation, purification and preservation of the Strain
Screening lactobacillus from saliva and feces of nature and infant. Putting 2g of a sample to be separated into a blue-cap bottle filled with 38mL of physiological saline, shaking and uniformly mixing, gradually diluting with a test tube filled with the physiological saline in a gradient of 10 times, sucking 100 mu L of diluted bacterial liquid by using a pipette, coating the diluted bacterial liquid on an MRS solid plate by using a coating rod, and putting the MRS solid plate into an incubator to perform anaerobic culture for 48 hours in an inverted manner. After the culture is finished, selecting the plate with the number of the single colonies larger than 50 on the plate, selecting the single colonies on the agar solid plate by using the inoculating loop, and streaking and purifying for many times until the colony microscopic examination forms on the whole plate are consistent. Picking single colony to MRS liquid culture medium for propagation, preserving strain in MRS liquid culture medium containing 30% glycerol, and freezing and storing in-80 deg.C refrigerator. A strain of Bifidobacterium longum (number BL 21) is screened out by the method. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 10452.
Experimental example 1 preliminary screening of Folic acid-producing Strain
Bromcresol purple was added as an indicator to the Medium FACM (Folic Acid Casei Medium, available from Difco), the pH was adjusted to 6.8, the activated strain was inoculated by sucking 1-2 drops of the liquid with a pipette and incubated at 37 ℃ for 2 days. The medium solution turned yellow, indicating a positive indication that the strain was able to synthesize folate.
Experimental example 2 identification of strains
3.1 colony morphology
Referring to fig. 1, after the strain is cultured in MRS solid medium for 16h, the colony on the plate is smooth, convex, complete in edge, milky white and soft in texture. The single colony is selected for microscopic examination to observe the strain morphology, and the microscopic examination is shown in figure 1.
3.216 identification of S rRNA
Referring to fig. 2, genomic DNA of the target strain was extracted using an Ezup column type bacterial genomic DNA extraction kit, amplified using the extracted lactic acid bacteria strain genomic DNA as a template, and then subjected to PCR experiments using primers for PCR, which were bacterial universal primers 27F and 1492R. And after the PCR amplification reaction is finished, taking the product to dilute to a proper gradient and then carrying out agar gel photographing detection. The amplified product was sent to Biotechnology engineering (Shanghai) Co., Ltd for detection. The nucleotide sequence is shown in FIG. 2. The detection result is compared with a BLAST sequence on an NCBI website, and the result shows that the homology with the 16S rDNA sequence of the bifidobacterium longum is over 99 percent. According to the combination of the morphological observation and the sequence comparison of the strains, the screened lactobacillus strains are determined to be bifidobacterium longum which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 10452.
EXAMPLE 3 determination of Folic acid content
1) Preparing bacterial liquid: after the strain on the plate is selected and activated, the strain is inoculated into an MRS liquid culture medium for 24 hours, and after the strain is cultured, the strain is inoculated into a culture medium of a fermentation infusion bottle in an inoculum size of 2 percent, the culture temperature is 32 ℃, and the culture time is 16 hours.
2) Preparation of a sample to be tested: centrifuging the cultured strain fermentation liquid with high speed centrifuge (10000 rpm, 10min), collecting the centrifuged supernatant, filtering the supernatant with 0.22 μm filter membrane, detecting folic acid content in the supernatant by high performance liquid chromatography, and subtracting folic acid content value in MRS culture medium. The results showed that the folic acid content was 4.1. mu.g/mL.
EXAMPLE 4 growth Curve determination
Referring to fig. 5, after inoculating 2% lactobacillus seed solution in MRS liquid medium, the fermentation broth was taken every 2 hours and the OD600 value of the fermentation broth was measured with a spectrophotometer. The growth curve of the strain was plotted according to the measured data: the strain enters a logarithmic growth phase within about 2 hours, the logarithmic growth phase is finished within about 15 hours, and the strain is shifted into a stationary phase.
EXAMPLE 5 acid resistance test of Strain
Referring to fig. 3, the activated BL21 strain and the control group strain were inoculated into MRS liquid media with pH values of 2.0, 4.0, 5.0, 5.5, 6.0, 6.5, and 7.0, respectively, at a constant temperature of 37 ℃, after 2 hours of culture, viable counts in the media were determined by a spread plate counting method, and the test was repeated 2 times with the MRS media with pH value of 7.0 as a blank, to calculate the effective activities, respectively. BL21 acted for 2h under pH3.0 condition, the survival rate reached 72%.
EXAMPLE 6 bile salt test of Strain
Referring to FIG. 4, the activated BL21 strain and the control group strain were inoculated into MRS media containing 3mg/mL and 6mg/mL cholate according to 3% (v/v) inoculum sizes, respectively, and cultured at a constant temperature of 37 ℃, after 2h of culture, viable count in the media was determined by spread plate counting, and the test was repeated 2 times with the MRS media without cholate as a blank, to calculate the effective activities, respectively. BL21 acted for 2h under the condition of 0.3% of bile salt concentration, and the survival rate was 78%.
The address of the preservation unit of the invention is Beijing in China, and the preservation date is 2015, 01 and 27 days.
Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (6)
1. The lactic acid bacteria with high folic acid yield are characterized in that the lactic acid bacteria are bifidobacterium longum BL21 with the preservation number of CGMCC No. 10452.
2. The method for preparing lactic acid bacteria with high folate yield of claim 1, comprising the following steps:
s1, screening: obtaining a sample to be screened from saliva and excrement of nature and infants, putting the sample to be screened into a bottle containing normal saline, vibrating and uniformly mixing, and marking an obtained product as bacterial liquid, wherein the mass of the normal saline is 15-20 times of that of the sample to be screened;
s2, dilution: pouring the bacterial liquid obtained in the step 1 into a test tube filled with normal saline to carry out gradient dilution step by 10 times, and marking the obtained product as diluted bacterial liquid;
s3, coating: coating 100 mu L of the diluted bacterial liquid obtained in the step 2 on an agar solid plate by using a coating rod, inverting and putting into an incubator for anaerobic culture for 36-60 h;
s4, purification: after the culture is finished, selecting the agar solid plate with the number of the single colonies larger than 50 on the agar solid plate, and carrying out streak purification on the single colonies on the agar solid plate for multiple times until the microscopic examination forms of the colonies on the whole plate are consistent.
3. The method for preparing lactic acid bacteria with high folate according to claim 2, wherein the agar solid plate of step 3 is prepared by the following steps: 20g of glucose, 0g of dried egg white, 10g of beef extract, 5g of yeast extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 801 g of tween, 0.59g of magnesium sulfate, 0.19g of manganese sulfate monohydrate, 1g of L-cysteine, 1000mL of distilled water and 15g of agar are mixed uniformly, the pH value is adjusted to 6.8 by using sodium hydroxide, and the mixture is sterilized at 121 ℃ for 15 min.
4. The method for preparing lactic acid bacteria with high folate according to claim 2, wherein the agar liquid medium of step 3 is prepared by the following steps: 20g of glucose, 0g of dried egg white, 10g of beef extract, 5g of yeast extract, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 801 g of tween, 0.59g of magnesium sulfate, 0.19g of manganese sulfate monohydrate, 1g of L-cysteine, 1000mL of distilled water and 15g of agar are mixed uniformly, the pH value of the distilled water is adjusted to 6.8, and the mixture is sterilized at 121 ℃ for 15 min.
5. The method for preparing lactic acid bacteria with high folate according to claim 2, wherein after the purification in step 4 is completed, the single colony obtained in step 4 is picked up and spread in agar liquid medium, and the strain is preserved in agar liquid medium containing 30% glycerol and stored in a refrigerator at-80 ℃.
6. The use of a lactic acid bacterium for producing folic acid according to claim 1, characterized in that folic acid is synthesized using said lactic acid bacterium.
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