CN110878273B

CN110878273B - Bifidobacterium breve and application thereof in preparation of conjugated fatty acid

Info

Publication number: CN110878273B
Application number: CN201911398561.0A
Authority: CN
Inventors: 陈卫; 杨波; 陆文伟; 王顺余; 何建新; 郑宋友; 李国平; 付成丽; 朱玲丽; 赵建新; 张灏
Original assignee: Zhejiang Liziyuan Food Co ltd; Jiangnan University
Current assignee: Zhejiang Liziyuan Food Co ltd; Jiangnan University
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2021-08-13
Anticipated expiration: 2039-12-30
Also published as: CN110878273A

Abstract

The invention relates to bifidobacterium breve and application thereof in preparation of conjugated fatty acid, belonging to the technical field of microorganisms. The invention provides a Bifidobacterium breve FBJCP2M1 capable of producing conjugated linoleic acid, conjugated linolenic acid and conjugated stearidonic acid, the conversion rate of converting linoleic acid into conjugated linoleic acid to produce conjugated linoleic acid can reach 87.98%, wherein c9, t11-CLA isomer accounts for 98.38%; the conversion rate of converting linolenic acid to produce conjugated linolenic acid can reach 95.6 percent, wherein c9, t11, c15-CLNA accounts for 95.01 percent; the conversion rate of converting the stearidonic acid to produce the conjugated stearidonic acid can reach 55.86%, wherein c6, c9, t11 and c15-CSA account for 90.45%.

Description

Bifidobacterium breve and application thereof in preparation of conjugated fatty acid

Technical Field

The invention relates to bifidobacterium breve and application thereof in preparation of conjugated fatty acid, belonging to the technical field of microorganisms.

Background

Conjugated Linoleic Acid (CLA) is a generic term for octadecadienoic acid containing Conjugated double bonds, and is a positional isomer and a geometric isomer of Linoleic acid (Linoleic acid, 18: 2). The most common isomer is cis 9, trans 11-CLA (c9, t11-CLA), also known as Rumenic acid (Rumenic acid). In addition, trans-10, cis-12-CLA (t10, c12-CLA) is also an isomer having a relatively high content in nature. The conjugated linoleic acid is concerned due to biological functions, different conjugated linoleic acid isomers have different physiological functions, wherein c9, t11-CLA, t10 and c12-CLA are recognized as the most physiologically active conjugated linoleic acid isomers, the most main functions of c9 and t11-CLA are in the aspects of cancer resistance, anti-inflammation, immunoregulation and the like, and t10 and c12-CLA have obvious effects on weight loss and lipid metabolism. Furthermore, t9, t11-CLA have been reported to have anti-inflammatory activity.

Conjugated Linolenic acid (CLNA) is a general term for various positional isomers and geometric isomers of octadecatrienoic acid with Conjugated double bonds derived from Linolenic acid (LNA), has various nutritional and health-care functions, such as cancer resistance, diabetes resistance, atherosclerosis resistance, body fat content reduction, insulin resistance, body immunity regulation and the like, and has become a research hotspot in the fields of medicine, chemistry, nutrition and the like. Among the various stereoisomers of conjugated linolenic acid, c9, t11, c15-CLNA (CLNA1), t9, t11, c15-CLNA (CLNA2), t10, c12, c15-CLNA and c6, c9, t11-CLNA, etc. are the isomers considered to be the most biologically active.

Conjugated Stearidonic Acid (CSA) is a generic name of various positional isomers and geometric isomers of Stearidonic acid with Conjugated double bonds derived from Stearidonic Acid (SA), and has various nutritional and health-care functions, such as anticancer and other physiological functions. Among the various stereoisomers of conjugated linolenic acid, c6, c9, t11, c15-CSA (CSA1) and c6, t9, t11, c15-CLNA (CSA2) are the most biologically active.

The natural conjugated linoleic acid is mainly present in the milk fat and meat products of rumen animals such as cattle and sheep, the CLA content in each gram of milk fat is 2mg-25mg, and the CLA content increases with the age of the dairy cow. CLA derived from non-natural sources is mainly obtained by artificial synthesis. The artificially synthesized CLA has different contents of different isomers in the obtained product due to different raw materials and synthesis methods.

Natural conjugated linolenic acid is present in some plant seeds in nature, such as pomegranate seeds, tung seeds, bitter melon seeds, calendula seeds, trichosanthes kirilowii maxim, jacaranda javanica seeds, and the like. However, only trichosanthes seeds in many plant seeds containing conjugated linolenic acid can be directly eaten, and the oil and fat components in the plant seeds are very complex, so that the separation and purification of the conjugated linolenic acid are very difficult to realize by taking the plant seed oil and fat as raw materials. In the prior art, the conjugate linolenic acid can be produced by an alkali treatment linolenic acid isomerization method, but the yield is low, and a reagent residue exists, so that the industrial production of the conjugate linolenic acid is not realized.

It has been found that CLA, CLNA, CSA are transformed by microorganisms, and particularly some lactic acid bacteria have the ability to transform conjugated linoleic acid, conjugated linolenic acid and conjugated stearidonic acid, for example, Gorisen et al have studied the bioconversion CLA and CLNA of 30 strains of Bifidobacterium, and found that CLA or CLNA can be produced by 6 strains of Bifidobacterium out of 36 strains, and that CLA or CLNA can be produced by 53% and 78% respectively, which are the highest conversion rates among 6 strains of Bifidobacterium (Gorisen L, et al. production of conjugated linoleic acid and conjugated linoleic acid), respectively (Gorisen L, et al. production of conjugated linoleic acid and conjugated linoleic acid bacteria by Bifidobacterial biotechnology, 2010,87(6): 2257-. However, the isomers of the fatty acids obtained are not predominantly c9, t11-CLA, t10, c12-CLA or c9, t11, c15-CLNA, t9, t11, c 15-CLNA.

Bifidobacterium breve is a species of the genus Bifidobacterium. The Bifidobacterium genus contains 35 species including Bifidobacterium adolescentis, Bifidobacterium animalis subsp lactis (i.e. Bifidobacterium lactis), Bifidobacterium bifidum, Bifidobacterium ursinum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dolphin, Bifidobacterium corynebacterium, Bifidobacterium rabbit, Bifidobacterium odonta, Bifidobacterium gallnut, Bifidobacterium gallinarum, Bifidobacterium longum subsp, Bifidobacterium longum subsp.infantis, Bifidobacterium major, Bifidobacterium minium, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum subsp.pseudolongum, Bifidobacterium pseudolongum subsp.coccidium subsp.pseudolongum, Bifidobacterium gallinarum, Bifidobacterium longum, and Bifidobacterium thermophilum.

Due to the fact that the bifidobacterium species are various, and the bifidobacterium species belonging to the same genus and different species have obvious differences in various aspects such as morphology, physiology, metabolism and physiological functions, so far, few researches find that the bifidobacterium breve can produce conjugated linoleic acid and conjugated linolenic acid, and some researches cannot be carried out, so that the reasons or mechanisms causing the differences are not researched and determined.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a bifidobacterium breve strain which produces conjugated linoleic acid, conjugated linolenic acid and conjugated stearidonic acid, wherein the product mainly comprises c9, t11-CLA or c9, t11, c15-CLNA or c6, c9, t11 and c15-CSA with higher biological activity, and the fatty acid product is remained in fermentation liquor and is easy to separate and purify.

The first purpose of the invention is to provide a Bifidobacterium breve (FBJCP 2M 1) strain which is preserved in Guangdong province collection of microorganisms in 12 months and 30 days in 2019 and has the preservation number of GDMCC No. 60934. The Bifidobacterium breve FBJCP2M1 is also named as CCFM1111 and is preserved in food biotechnology strain collection of south Jiangnan university.

It is a second object of the present invention to provide a microbial preparation containing the above Bifidobacterium breve FBJCP2M 1.

The third purpose of the invention is to provide the application of the bifidobacterium breve FBJCP2M1 or the microbial preparation in preparing conjugated linoleic acid or products containing the conjugated linoleic acid.

In one embodiment of the invention, the conjugated linoleic acid comprises c9, t11-CLA and/or t10, c 12-CLA.

The fourth purpose of the invention is to provide the application of the bifidobacterium breve FBJCP2M1 or the microbial preparation in preparing conjugated linolenic acid or products containing the conjugated linolenic acid.

In one embodiment of the invention, the conjugated linolenic acid comprises c9, t11, c15-CLNA and/or t9, t11, c 15-CLNA.

The fifth purpose of the invention is to provide the application of the bifidobacterium breve FBJCP2M1 in preparing the conjugated stearidonic acid or the product containing the conjugated stearidonic acid.

In one embodiment of the invention, the conjugated stearidonic acid comprises c6, c9, t11, c15-CSA and/or c6, t9, t11, c 15-CSA.

The invention also provides application of the bifidobacterium breve FBJCP2M1 or the microbial preparation in the fields of food, chemistry or pharmacy.

The invention has the beneficial effects that:

the invention provides a Bifidobacterium breve FBJCP2M1 capable of producing conjugated linoleic acid, conjugated linolenic acid and conjugated stearidonic acid, the conversion rate of converting linoleic acid into conjugated linoleic acid to produce conjugated linoleic acid can reach 87.98%, wherein c9, t11-CLA isomer accounts for 98.38%; the conversion rate of converting linolenic acid to produce conjugated linolenic acid can reach 95.6 percent, wherein c9, t11, c15-CLNA accounts for 95.01 percent; the conversion rate of converting the stearidonic acid to produce the conjugated stearidonic acid can reach 55.86%, wherein c6, c9, t11 and c15-CSA account for 90.45%.

Compared with the prior art, the yield of each conjugated fatty acid is higher in the invention, and the total conversion rate, and the yield or the conversion rate of the most physiologically active c9, t11-CLA, c9, t11, c15-CLNA, c6, c9, t11 and c15-CSA isomers are obviously higher than those of the prior art. Particularly, the conversion rate of the conjugated stearidonic acid is improved by 1 to 1.5 times compared with the prior art.

Biological material preservation

A Bifidobacterium breve (FBJCP 2M 1) strain is preserved in Guangdong province microorganism strain preservation center in 2019, 12 and 30 months, and the preservation number is GDMCC No.60934, and the preservation address is No. 59 building 5 building of Michelia Tokyo 100, Guangzhou city.

Drawings

FIG. 1 is a flow chart of the operations of isolating, purifying and preserving Bifidobacterium breve of the present invention.

FIG. 2 shows the conversion of Bifidobacterium breve FBJCP2M1 to conjugated linoleic acid (A) the total concentration of conjugated linoleic acid as a function of the culture time; (B) distribution of the culture medium and intracellular conjugated linoleic acid after 72 hours of culture.

FIG. 3 shows the conversion rate of Bifidobacterium breve FBJCP2M1 for the production of conjugated linolenic acid (A) the total concentration of conjugated linolenic acid as a function of the cultivation time; (B) and after 72 hours of culture, the distribution of the conjugated linolenic acid in the culture solution and cells.

FIG. 4 shows the conversion of Bifidobacterium breve FBJCP2M1 to conjugated stearidonic acid (A) the total concentration of conjugated stearidonic acid as a function of the cultivation time; (B) distribution of intracellular conjugated stearidonic acid in the culture broth after 72 hours of culture.

Detailed Description

The invention is further elucidated with reference to a specific embodiment and a drawing.

In the present invention, "%" or percentages used to indicate concentrations or ratios are weight percentages unless otherwise specified.

The invention relates to the following culture medium and strain culture conditions:

mrss liquid medium: 10g of tryptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 801 mL of tween and 0.5g of cysteine, and adding water to 1000 mL.

The mrss solid medium was obtained by adding 1.5% agar based on the total weight of the liquid medium on the above basis.

The strain culture conditions are as follows: anaerobic culture at 37 ℃.

Example 1: collection of sample and isolation and identification of Bifidobacterium

(1) Collection of samples and isolation of bifidobacteria

The infant feces samples of Chang plain area of Beijing city were collected from the Nanshao Zhenxing Chang Jiayuan of Chang plain area of Beijing city.

Taking 1g of a fecal sample, performing gradient dilution, coating the fecal sample on a mMRS solid culture medium, placing the mMRS solid culture medium in an anaerobic environment, culturing for 72 hours at 37 ℃, observing and recording colony morphology, selecting and streaking and purifying colonies, then culturing for 48 hours at 37 ℃ in a mMRS liquid culture medium, performing gram staining on the obtained colonies, recording strain morphology, discarding gram-negative strains and gram-positive cocci in the colonies, selecting and obtaining gram-positive bacilli, discarding catalase-positive strains after catalase analysis, reserving catalase-negative strains, detecting with fructose-6-phosphokinase to discard negative strains, identifying the obtained strains as bifidobacterium breve through 16S rDNA sequencing, and naming the bifidobacterium breve FBJCP2M1, and meanwhile, naming the bacteria as CCFM, and reserving in the food biotechnology strain preservation center of Jiannan university.

Subculturing Bifidobacterium breve FBJCP2M1, collecting thallus, centrifuging at 3000rpm in a centrifuge tube for 10min, washing, repeating for 3 times, adding the thallus into matrix protectant, and freezing and preserving.

(2) Bacterial colony characteristics and bacterial morphology

Bifidobacterium breve FBJCP2M1 has the following biological properties: the characteristics of the thallus are as follows: is milk white; colony characteristics: bacterial colonies protruding on the mMRS solid plate are smooth, round, milky, semitransparent and 1-2 mm in diameter; growth characteristics: the culture was carried out in MRS medium for about 24h to the end of log under constant temperature anaerobic conditions at 37 ℃.

(3) Physiological and biochemical characteristics

The physiological and biochemical characteristics of Bifidobacterium breve FBJCP2M1 are shown in Table 1.

TABLE 1 physiological and biochemical characteristics of Bifidobacterium breve FBJCP2M1

(4) Molecular biological identification process

16S rDNA amplification conditions: 5min at 95 ℃; 35 cycles (95 ℃ 30s,55 ℃ 30s,72 ℃ 2 min); 10min at 72 ℃.

An amplification primer:

27F：5’-AGAGTTTGATCCTGGCTCAG-3’；

1492R：5’-TACGGCTACCTTGTTACGACT T-3’。

the amplification product purification and sequence alignment procedures were carried out according to the methods described in the literature (Turroni F et al. expanding the diversity of the biochemical amplification in the human endogenous tract [ J ]. Appl Environ Microb.2009; 75(6): 1534-45). The 16S rDNA sequence obtained by sequencing is shown in SEQ ID NO. 1. The identification result shows that the strain is bifidobacterium breve which is named as bifidobacterium breve FBJCP2M 1.

Example 2: application of bifidobacterium breve FBJCP2M1 in preparation of conjugated linoleic acid

1. Activation of bacterial strains

Taking out the glycerol tube with Bifidobacterium breve FBJCP2M1 from-80 deg.C refrigerator, streaking the bacterial liquid on mMRS solid culture medium, and culturing at 37 deg.C for 48 hr under anaerobic environment. And (3) selecting the grown single colony, inoculating the single colony in a mMRS liquid culture medium, culturing for 48h at 37 ℃ in an anaerobic environment, and continuously activating for 3 generations.

2. Preparation of linoleic acid mother liquor

Weighing 300mg of Linoleic Acid (LA) and 200mg of Tween-80, dissolving in water, diluting to a constant volume of 10mL, fully stirring and emulsifying, filtering and sterilizing through a sterile filter membrane of 0.45 mu m, and storing at-20 ℃ in a dark place.

3. Co-culture with linoleic acid

The activated bacterial liquid in step 1 was inoculated into 10mL of MRS liquid medium containing 0.6mg/mL of LA (210. mu.L of the above-mentioned linoleic acid mother liquor) in an inoculum size of 2% (v/v), and cultured at 37 ℃ for 0, 12, 24, 48, and 72 hours in an anaerobic environment, while the medium to which the same amount of the linoleic acid mother liquor was added but no bacterial liquid was added was used as a control. After the culture, transferring the bacterial liquid into a centrifuge tube, centrifuging for 5min at 5000rpm, taking 3mL of fermentation liquid of each fermentation product to a clean centrifuge tube for standby, and centrifuging to obtain thalli for standby.

4. Fatty acid extraction

Extracting fatty acid in the fermentation liquor: adding heptadecanoic acid (C17:0) to 3mL of fermentation broth to a final concentration of 0.075mg/mL as an internal standard, then adding 2mL of isopropanol, and shaking thoroughly for 30 s; then adding 3mL of normal hexane, and fully oscillating for 30 s; centrifuging at 5000rpm for 3min, sucking n-hexane layer into clean fat extraction bottle, and blowing with nitrogen to obtain fatty acid.

Extracting fatty acid in the thallus: step 3 centrifugation of the resulting bacteriaThe mixture was washed with 2mL of a salt solution (0.137mol/L NaCl, 7.0mmol/L K)₂HPO₄，2.5mmoL/L KH₂PO₄) Washing, centrifuging at 4000rpm for 5min, and repeating the washing steps. Then, the thalli is resuspended in 2mL of the salt solution, heptadecanoic acid C17:0 is added until the final concentration is 0.0575mg/mL, and fatty acid extraction and nitrogen blow-drying are carried out according to the same method as the fermentation liquid, so as to obtain the fatty acid in the thalli.

5. Methyl esterification of fatty acids

And (4) drying the fermentation liquid fatty acid and the thallus fatty acid in the step (4) by using nitrogen, respectively adding 400 mu L of methanol, fully oscillating, uniformly mixing, directly performing methyl esterification by using 150 mu L of diazomethane reagent, drying by using nitrogen, redissolving by using 1mL of n-hexane, transferring to a gas phase bottle, and performing GC-MS detection.

6. GC-MS detection

Shimadzu gas chromatograph (GC 2010plus), gas column Rtx-wax (30m × 0.25mm × 0.25 μm), mass spectrometer (Shimadzu Ultra QP 2010).

Temperature programming conditions: the temperature is raised to 200 ℃ at the speed of 5 ℃/min at the beginning of 150 ℃, kept for 10min, then raised to 230 ℃ at the speed of 4 ℃/min, and kept for 18 min. Split-flow sample injection is adopted, the sample injection amount is 1 mu L, the split-flow ratio is 50: 1, and helium is taken as carrier gas. Both the injector temperature and the detector temperature were 240 ℃. The ion source was 220 ℃ and the intensity was 70 eV.

7. Results of the experiment

In the CLA accumulation process, the bifidobacterium breve FBJCP2M1 begins to produce CLA when growing in mrs containing 0.64mg/mL LA for 12 hours, and the concentration of conjugated linoleic acid in the fermentation broth tends to be saturated after the fermentation broth is cultured for 36 hours along with the gradual increase of the content of the conjugated linoleic acid (24 hours and 36 hours) of the thallus growth, as shown in fig. 2-a. After the culture is carried out for about 72 hours, the total content of CLA reaches 0.5631mg/mL, and the total conversion rate of CLA is 87.98 percent based on the total amount of substrate LA.

Through fatty acid analysis, the obtained fermentation product only contains two isomers of CLA1(c9, t11-CLA) and CLA2(t9, t11-CLA) from the viewpoint of the content of each isomer of CLA. The strain started to produce CLA1 after 12h of culture. The isomer CLA1 is rapidly accumulated in 12h to 36h along with the growth of the thallus, the content of CLA1 is saturated after the strain is cultured for 36h, and the content of CLA2 is lower when the strain begins to accumulate CLA (24h), and the concentration of the isomer is further increased along with the prolonging of the culture time. After 72h of culture, the concentration of CLA1 was as high as 0.554mg/mL, accounting for 98.38% of the total CLA production.

From the compositions of the fermentation liquor of the bifidobacterium breve FBJCP2M1 and the thallus fatty acid after 72 hours of culture, the absorption and the conversion rate of the strain to LA are obviously higher than those of the prior art. Analysis showed that only a small amount of substrate LA remained in the fermentation broth, and almost no LA remained in the cells which had not been converted. Most of the converted CLA is in the fermentation liquor, and the amount remained in the bacteria is only 0.068mg/mL, which is obviously less than the CLA concentration in the fermentation liquor. The result also shows that most CLA products are not accumulated in cells, but are transported to the outside of the cells, and the CLA accounts for more than 81.69 percent of the total fatty acids in the fermentation liquor, so the purity is higher, and the later separation and purification of the CLA in the fermentation liquor can be effectively simplified.

Example 3: application of bifidobacterium breve FBJCP2M1 in preparation of conjugated linolenic acid

1. Activation of bacterial strains

2. Preparation of linolenic acid mother liquor

Weighing 300mg of alpha-linolenic acid (alpha-LNA) and 200mg of Tween-80, dissolving in water, diluting to a constant volume of 10mL, fully stirring, emulsifying, filtering with a sterile filter membrane of 0.45 μm, sterilizing, storing at-20 deg.C, and storing in dark place.

3. Co-culture with linolenic acid

Inoculating the activated bacterial liquid in the step 1 into 10mL mMRS liquid culture medium containing 0.4mg/mL of alpha-LNA according to the inoculation amount of 2% (v/v), culturing for 0, 12, 24, 36, 48 and 72h at 37 ℃ in an anaerobic environment, and taking the culture medium added with the same amount of linolenic acid but not with the bacterial liquid as a control. After culturing, transferring the bacterial liquid into a centrifugal tube, centrifuging at 5000rpm for 5 min; 3 parts of 3mL fermentation liquid are taken to a clean centrifuge tube for standby.

4. Fatty acid extraction

Extracting fatty acid in fermentation liquor: adding heptadecanoic acid (C17:0) to 3mL of the fermentation broth to a final concentration of 0.0767mg/mL as an internal standard, adding 2mL of isopropanol, and shaking thoroughly for 30 s; then adding 3mL of normal hexane, and fully oscillating for 30 s; centrifuging at 5000rpm for 3min, absorbing n-hexane layer into clean fat extraction bottle, and blow-drying with nitrogen to obtain fatty acid in the fermentation liquid.

Extracting fatty acid in the thallus: step 3 centrifugation of the resulting mycelia with 2mL of a salt solution (containing 0.137mol/L NaCl, 7.0mmol/L K)₂HPO₄，2.5mmoL/L KH₂PO₄) Washing, centrifuging at 4000rpm for 5min, and repeating the washing steps. The resulting cells were resuspended in 2mL of the above salt solution, heptadecanoic acid (C17:0) was added to a final concentration of 0.0575mg/mL, and fatty acid extraction and nitrogen blow-drying were performed in the same manner as the fermentation broth.

5. Methyl esterification of fatty acids

Adding 400 mu L of methanol into the sample dried by the nitrogen, fully oscillating, uniformly mixing, directly carrying out methyl esterification by using a proper amount of diazomethane reagent, drying by the nitrogen, redissolving by using 1mL of n-hexane, transferring to a gas phase bottle, and carrying out GC-MS detection.

6. GC-MS detection

Shimadzu gas chromatograph (GC 2010plus), gas column Rtx-wax (30m × 0.25mm × 0.25 μm), mass spectrometer (Shimadzu Ultra QP 2010). Temperature programming conditions: the initial temperature is 150 ℃, the temperature is increased to 200 ℃ at the speed of 5 ℃/min, the temperature is maintained for 10min, and then the temperature is increased to 230 ℃ at the speed of 4 ℃/min, and the temperature is maintained for 18 min. Split-flow sample injection is adopted, the sample injection amount is 1 mu L, the split-flow ratio is 50: 1, and helium is taken as carrier gas. Both the injector temperature and the detector temperature were 240 ℃. The ion source was 220 ℃ and the intensity was 70 eV.

7. Results of the experiment

The bifidobacterium breve FBJCP2M1 starts to produce CLNA when growing in mMRS culture medium containing 0.3992mg/mL alpha-LNA for 12h, and the content of the conjugated linolenic acid tends to be saturated after culturing for 36h along with the gradual increase of the content of the conjugated linolenic acid (24h and 36h) in the thallus growth, as shown in figure 3-A. After being cultured for about 72 hours, the total content of CLNA reaches 0.3816mg/mL, and the conversion rate is 95.6 percent based on the total amount of the substrate alpha-LNA.

Through analysis of the content of each isomer of CLNA, only two isomers of CLNA1 and CLNA2 are contained in the obtained product, namely c9, t11, c15-CLNA and t9, t11 and c15-CLNA which have the most biological activity in conjugated linolenic acid. The strain begins to convert the conjugated linolenic acid after being cultured for 12h, the isomer CLNA1 is rapidly accumulated within 12h to 36h along with the growth of thalli, the CLNA1 content is saturated after the strain is cultured for 36h, the CLNA2 content is lower when the strain begins to accumulate CLNA (24h), the isomer concentration is further increased along with the prolonging of the culture time, and finally the CLNA1 concentration is 0.3625mg/mL and accounts for 95.01% of the total CLNA.

From the fermentation liquor of the bifidobacterium breve FBJCP2M1 cultured for 72h and the bacterial body fatty acid composition, the absorption and conversion rate of the strain to alpha-LNA are obviously higher than those of the prior art. And analysis shows that almost no substrate LNA remains in the fermentation liquid, only a very small amount of LNA remains in the bacteria and is not converted, most of the converted CLNA is in the fermentation liquid, and the amount remaining in the bacteria is very small. And the distribution conditions of the CLNA1 and the CLNA2 in the fermentation liquid and the thalli are basically consistent, and the result also shows that most products are not accumulated in the cells, but are transported to the outside of the cells, thereby being beneficial to further separation and purification in the later period.

Example 4: application of bifidobacterium breve FBJCP2M1 in preparation of conjugated stearidonic acid

1. Activation of bacterial strains

2. Preparation of mother liquor of octadecenoic acid

300mg of Stearidonic Acid (SA) and 200mg of Tween-80 are weighed, dissolved in water, and the volume is determined to be 10mL, fully stirred and emulsified, filtered and sterilized by a sterile filter membrane of 0.45 mu m, and then stored at minus 20 ℃ in the dark.

3. Co-culture with linoleic acid

Inoculating the activated bacterial liquid into 10mL of mMRS liquid culture medium containing 0.3mg/mL of SA according to the inoculation amount of 2% (v/v), culturing for 0, 12, 24, 48 and 72h at 37 ℃ in an anaerobic environment, and taking the culture medium added with the same amount of linoleic acid mother liquor without the bacterial liquid as a control. After the culture, transferring the bacterial liquid into a centrifuge tube, centrifuging for 5min at 5000rpm, taking 3mL of fermentation liquid of 3 parts of each fermentation product to a clean centrifuge tube for standby, and centrifuging to obtain thalli for standby.

4. Fatty acid extraction

Extracting fatty acid in the thallus: step 3 centrifugation of the resulting mycelia with 2mL of salt solution (0.137mol/L NaCl, 7.0mmol/L K)₂HPO₄，2.5mmoL/L KH₂PO₄) Washing, centrifuging at 4000rpm for 5min, and repeating the washing steps. Then, the thalli is resuspended in 2mL of the salt solution, heptadecanoic acid C17:0 is added until the final concentration is 0.0575mg/mL, and fatty acid extraction and nitrogen blow-drying are carried out according to the same method as the fermentation liquid, so as to obtain the fatty acid in the thalli.

5. Methyl esterification of fatty acids

And 4, drying the fermentation liquid fatty acid and the thallus fatty acid by using nitrogen, respectively adding 400 mu L of methanol, fully oscillating, uniformly mixing, directly carrying out methyl esterification by using 150 mu L of diazomethane reagent, drying by using nitrogen, redissolving by using 1mL of n-hexane, transferring to a gas phase bottle, and carrying out GC-MS detection.

6. GC-MS detection

7. Results of the experiment

When bifidobacterium breve FBJCP2M1 grows in mMRS containing 0.3022mg/mL SA for 12h, CSA is produced, the content of the conjugated stearidonic acid is gradually increased (24h and 36h) along with the growth of the bacteria, and the content of the conjugated stearidonic acid is saturated after 36h of culture, as shown in figure 3-A. After being cultured for about 72 hours, the total content of CSA reaches 0.1688mg/mL, and the conversion rate is 55.86 percent based on the total amount of the substrate SA.

Through CSA isomer content analysis, the obtained product only has CSA1 and CSA2 isomers, namely c6, c9, t11, c15-CSA and c6, t9, t11 and c15-CSA which have the most biological activity in the conjugated stearidonic acid. The strain begins to convert the conjugated stearidonic acid after 12h of culture, the isomer CSA1 is rapidly accumulated within 12h to 36h along with the growth of thalli, the content of CSA1 is saturated after the strain is cultured for 36h, the content of CSA2 is lower when the strain begins to accumulate CSA (24h), the concentration of the isomer is further increased along with the prolonging of the culture time, and finally the concentration of CSA1 is 0.1526mg/mL and accounts for 90.45 percent of the total CSA.

From the fermentation liquor of the bifidobacterium breve FBJCP2M1 cultured for 72h and the fatty acid composition of the strain, the absorption and the conversion rate of the strain to SA are obviously higher than those of the prior art. Analysis shows that almost no substrate SA remains in the fermentation liquid, only a very small amount of SA remains in the bacteria and is not converted, most of the converted CSA is in the fermentation liquid, and the amount of the converted CSA remaining in the bacteria is very small. And the distribution of CSA1 and CSA2 in the fermentation liquor and thalli is basically consistent, and the results also show that most products are not accumulated in the cells, but are transported to the outside of the cells, thereby being beneficial to further separation and purification in the later period.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

ZHEJIANG LIZIYUAN FOOD Co.,Ltd.

<120> Bifidobacterium breve and application thereof in preparation of conjugated fatty acid

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1390

<212> DNA

<213> Bifidobacterium breve

<400> 1

ctccctcccg caaggggtta ggccaccggc ttcgggtgct gcccactttc atgacttgac 60

gggcggtgtg tacaaggccc gggaacgcat tcaccgcgac gttgctgatt cgcgattact 120

agcgactccg ccttcacgca gtcgagttgc agactgcgat ccgaactgag accggttttc 180

agggatccgc tccagctcgc actgtcgcat cccgttgtac cggccattgt agcatgcgtg 240

aagccctgga cgtaaggggc atgatgatct gacgtcatcc ccaccttcct ccgagttaac 300

cccggcggtc ccccgtgagt tcccggcaca atccgctggc aacacggggc gagggttgcg 360

ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacgacc atgcaccacc 420

tgtgaacccg ccccgaaggg aaaccccatc tctgggatcg tcgggaacat gtcaagccca 480

ggtaaggttc ttcgcgttgc atcgaattaa tccgcatgct ccgccgcttg tgcgggcccc 540

cgtcaatttc tttgagtttt agccttgcgg ccgtactccc caggcgggat gcttaacgcg 600

ttagctccga cacggaaccc gtggaacggg ccccacatcc agcatccacc gtttacggcg 660

tggactacca gggtatctaa tcctgttcgc tccccacgct ttcgctcctc agcgtcagta 720

acggcccaga gacctgcctt cgccattggt gttcttcccg atatctacac attccaccgt 780

tacaccggga attccagtct cccctaccgc actcaagccc gcccgtaccc ggcgcggatc 840

caccgttaag cgatggactt tcacaccgga cgcgacgaac cgcctacgag ccctttacgc 900

ccaataattc cggataacgc ttgcacccta cgtattaccg cggctgctgg cacgtagtta 960

gccggtgctt attcgaaagg tacactcaac acaaagtgcc ttgctcccta acaaaagagg 1020

tttacaaccc gaaggcctcc atccctcacg cggcgtcgct gcatcaggct tgcgcccatt 1080

gtgcaatatt ccccactgct gcctcccgta ggagtctggg ccgtatctca gtcccaatgt 1140

ggccggtcgc cctctcaggc cggctacccg tcgaagccat ggtgggccgt taccccgcca 1200

tcaagctgat aggacgcgac cccatcccat gccgcaaagg ctttcccaac acaccatgcg 1260

gtgtgatgga gcatccggca ttaccacccg tttccaggag ctattccggt gcatggggca 1320

ggtcggtcac gcattactca cccgttcgcc actctcacca ccaagcaaag cccgatggat 1380

cccgttcgac 1390

Claims

1. Bifidobacterium breve (FBJCP 2M 1) is preserved in Guangdong province collection of microorganisms in 12 months and 30 days in 2019, and the preservation number is GDMCC No. 60934.

2. A microbial preparation comprising Bifidobacterium breve FBJCP2M1 as claimed in claim 1.

3. Use of a bifidobacterium breve FBJCP2M1 as claimed in claim 1 or a microbial preparation as claimed in claim 2 in the preparation of conjugated linoleic acid or a product containing conjugated linoleic acid; the conjugated linoleic acid is c9, t11-CLA and/or t9, t 11-CLA.

4. Use of bifidobacterium breve FBJCP2M1 as claimed in claim 1 in the preparation of conjugated linolenic acid or products containing it; the conjugated linolenic acid is c9, t11, c15-CLNA and/or t9, t11, c 15-CLNA.

5. Use of bifidobacterium breve FBJCP2M1 as claimed in claim 1 in the preparation of conjugated stearidonic acid or products containing conjugated stearidonic acid; the conjugated stearidonic acid is c6, c9, t11, c15-CSA and/or c6, t9, t11, c 15-CSA.

6. Use of a bifidobacterium breve as claimed in claim 1 in the preparation of conjugated linoleic acid, or a product containing conjugated linoleic acid, or conjugated linolenic acid, or a product containing conjugated linolenic acid, or conjugated stearidonic acid, or a product containing conjugated stearidonic acid in the food, chemical or pharmaceutical field; the conjugated linoleic acid is c9, t11-CLA and/or t9, t11-CLA, the conjugated linolenic acid is c9, t11, c15-CLNA and/or t9, t11, c15-CLNA, and the conjugated stearidonic acid is c6, c9, t11, c15-CSA and/or c6, t9, t11, c 15-CSA.

7. Use of a microbial preparation according to claim 2 for the preparation of conjugated linoleic acid, or a product comprising conjugated linoleic acid, or conjugated linolenic acid, or a product comprising conjugated linolenic acid, or conjugated stearidonic acid, or a product comprising conjugated stearidonic acid in the food, chemical or pharmaceutical field; the conjugated linoleic acid is c9, t11-CLA and/or t9, t11-CLA, the conjugated linolenic acid is c9, t11, c15-CLNA and/or t9, t11, c15-CLNA, and the conjugated stearidonic acid is c6, c9, t11, c15-CSA and/or c6, t9, t11, c 15-CSA.