CN106148230A - One strain vacation chainlet bacillus bifidus and prepare the application of conjugated linoleic acid or conjugate linolenic acid - Google Patents

Abstract

The invention relates to a strain of pseudo-small chain bifidobacterium CCFM748 and its application. Bifidobacterium pseudosmall chain CCFM748 of the present invention can efficiently convert free linoleic acid and linolenic acid into more biologically active conjugated linoleic acid and conjugated linolenic acid, which can be directly used to prepare conjugated linoleic acid and conjugated linolenic acid. Conjugated linolenic acid can also be used to produce foods rich in conjugated linoleic acid and conjugated linolenic acid.

Description

A strain of pseudo-small-chain bifidobacteria and its method for preparing conjugated linoleic acid or conjugated linolenic acid application

【Technical field】

The invention relates to a strain of Bifidobacterium pseudocatenulatum CCFM748 isolated from feces of newborns, and the application of the strain in preparing conjugated linoleic acid or conjugated linolenic acid.

【Background technique】

Conjugated linoleic acid (CLA) is a general term for octadecadienoic acid containing conjugated double bonds, and is the positional isomer and geometric isomer of linoleic acid (Linoleic acid, 18:2) body. The most abundant and common isomer is cis 9, trans 11-CLA (c9, t11-CLA), also known as rumenic acid. In addition, trans 10, cis 12-CLA (t10, c12-CLA) is also an isomer with relatively high content in nature. Conjugated linoleic acid has attracted attention because of its biological functions, and its physiological activities mainly include: anti-cancer, anti-inflammation, slowing down atherosclerosis, weight loss, alleviating diabetes, promoting bone growth and immune regulation, etc. Different isomers have different physiological functions, among which c9,t11-CLA and t10,c12-CLA are recognized as the most physiologically active conjugated linoleic acid isomers, and the main function of c9,t11-CLA It lies in anti-cancer, anti-inflammation, and immune regulation, while t10, c12-CLA has a very significant effect on weight loss and lipid metabolism. In addition, t9, t11-CLA has also been reported to have anti-inflammatory and other activities.

Conjugated linolenic acid (CLNA) is a general term for various positions and geometric isomers of octadecatrienoic acid derived from linolenic acid (LNA) with conjugated double bonds. Various nutritional and health functions, such as anti-cancer, anti-diabetes, anti-atherosclerosis, reducing body fat content, insulin resistance, regulating body immunity and other nutritional and health functions, have become the research fields of medicine, chemistry, nutrition and so on. hotspot. 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 considered to be the most biologically active isomers.

Natural conjugated linoleic acid mainly exists in milk fat and meat products of rumen animals such as cattle and sheep, and the content of each gram of milk fat varies from 2mg to 25mg, and the content of CLA increases with the age of dairy cows. The non-natural sources of CLA are mainly obtained through artificial synthesis, and the content of each isomer of artificially synthesized CLA varies greatly due to different raw materials and methods. Some plant seeds in nature such as pomegranate seeds, tung seeds, bitter melon seeds, calendula seeds, Trichosanthes and Jacaranda seeds are rich in conjugated linolenic acid. Among the many plant seeds containing conjugated linolenic acid, only Trichosanthes seeds can be eaten directly, and the oil composition in plant seeds is very complex, so it is very difficult to separate and purify conjugated linolenic acid.

In addition, in the prior art, conjugated linolenic acid can also be produced by alkali-treated linolenic acid isomerization method, but the yield of this method is low, and there are reagent residues in the product, which makes it difficult to put into practical application, so the production of conjugated linolenic acid has not been realized so far. Industrial production.

At present, studies have found that microorganisms can transform CLA and CLNA, especially some lactic acid bacteria have the ability to transform conjugated linoleic acid and conjugated linolenic acid. Among the 36 strains of bifidobacteria, 6 were found to produce CLA or CLNA, and among the 6 strains of bifidobacteria, the conversion rates of the two conjugated fatty acids were 53% and 78%, respectively (Gorissen L, et al. Production of conjugated linoleic acid and conjugated linolenic acid isomers by Bifidobacterium species[J].ApplMicrobiol Biotechnol, 2010,87(6):2257-2266.). However, according to its description, the obtained fatty acid isomers are not dominated by the most biologically active c9,t11-CLA and t10,c12-CLA or c9,t11,c15-CLNA and t9,t11,c15-CLNA.

【Content of invention】

The purpose of the present invention is to overcome the defects of the prior art, obtain conjugated linoleic acid, higher yield of conjugated linolenic acid, higher conversion rate, and c9, t11-CLA and t10, c12-CLA or c9, t11 in the product , c15-CLNA and t9, t11, c15-CLNA as the main isomer pseudo-small chain bifidobacteria.

The present invention also provides the application of the above bacterial strain in preparing conjugated linoleic acid or conjugated linolenic acid.

In order to achieve the above object, the present invention provides a strain of Bifidobacterium pseudostenosis CCFM748, which has been preserved on June 12, 2016 in the General Microorganism Center of China Committee for the Collection of Microorganisms, and its preservation number is CGMCCNo.12603.

The pseudosmall chain bifidobacterium CCFM748 of the present invention is newly isolated and screened, and is obtained through bacterial genomic DNA extraction, 16S rDNA specific primer PCR amplification, amplification product purification, DNA sequencing, sequence comparison and other steps Bacterial 16S rDNA sequence sequencing method Species identification was carried out, and it was identified as Bifidobacterium pseudosmall chain, and named as Bifidobacterium pseudosmall chain CCFM748.

Pseudo-small chain bifidobacterium CCFM748 of the present invention has the following biological characteristics:

Bacterial characteristics: The thallus is milky white.

Colony characteristics: On the mMRS solid plate, the colony protrudes, smooth, round, milky white, translucent, and 1-2 mm in diameter.

Growth characteristics: Under constant temperature and anaerobic conditions at 37°C, pseudo small chain Bifidobacterium CCFM748 was cultured in mMRS medium for about 24 hours to reach the end of the logarithm.

Bifidobacterium pseudo small chain is a species in the genus Bifidobacterium. The genus Bifidobacterium contains a total of 35 species, including Bifidobacterium adolescentis, Bifidobacterium animalis subspecies animal, Bifidobacterium animalis subsp. Bifidobacterium breve, Bifidobacterium breve, Bifidobacterium streptoides, Bifidobacterium porpoise, Bifidobacterium coryneform, Bifidobacterium rabbit, Bifidobacterium tooth, Bifidobacterium gallis, Bifidobacterium chicken, Bifidobacterium longum Long subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium large, Bifidobacterium minumum, Bifidobacterium pseudosmall chain, Bifidobacterium pseudolongum subsp. pseudolongum, Bifidobacterium pseudolongum subsp. coccidioides, Chicken Bifidobacterium, Bifidobacterium elongatus, Bifidobacterium thermophile, etc.

Due to the wide variety of Bifidobacteria, there are significant differences in morphology, chemical and physiological properties, metabolism and physiological functions between Bifidobacteria of the same genus and different species. So far, no research has found that pseudosmall-chain Bifidobacteria can transform conjugated Fatty acid, only a few strains of the same genus and different species have a low conversion rate. At present, there is no research in the academic circle to determine the cause or mechanism of this functional difference.

The invention also provides the application of the pseudo-small-chain Bifidobacterium CCFM748 in the preparation of conjugated linoleic acid, especially for converting linoleic acid into conjugated linoleic acid, especially into c9,t11-CLA and t10,c12 -CLA, and has an outstanding conversion rate.

The present invention also provides the application of the pseudo-small chain bifidobacterium CCFM748 in the preparation of conjugated linolenic acid, especially for converting linolenic acid into conjugated linolenic acid, especially into c9, t11, c15-CLNA and t9, t11 ,c15-CLNA, and has an outstanding conversion rate.

The present invention also provides the application of the pseudo-small chain bifidobacterium PA as a food additive.

Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) CCFM748, the strain was preserved on June 12, 2016 in the General Microbiology Center of China Committee for the Collection of Microbial Cultures, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC No.12603.

【Description of drawings】

Fig. 1 is the separation, purification and preservation operation flowchart of the pseudo small chain bifidobacterium in the present invention;

Fig. 2 is the conversion rate of pseudosmall chain bifidobacterium CCFM748 conjugated linoleic acid of the present invention;

(A) The change of the total concentration of conjugated linoleic acid with the culture time;

(B) The distribution of conjugated linoleic acid in the culture medium and cells after 72 hours of culture

Fig. 3 is the conversion rate of the conjugated linolenic acid of pseudosmall chain bifidobacterium CCFM748 of the present invention;

(A) The change of the total concentration of conjugated linolenic acid with the culture time;

(B) The distribution of conjugated linolenic acid in the culture medium and cells after 72 hours of culture

In the figure: SA: stearic acid, VA: vacuolic acid, OA: oleic acid, LA: linoleic acid, CLA1: c9, t11-CLA, CLA2: t9, t11-CLA

【detailed description】

The present invention can be better understood through the following examples, but the technical solutions of the present invention are not limitedly explained.

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

The present invention relates to the following culture medium:

mMRS liquid medium: tryptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, diammonium hydrogen citrate 2g, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.5g, manganese sulfate monohydrate 0.25 g, Tween 80 1mL and 0.5g cysteine, add water to 1000mL.

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

Example 1: Collection of samples and isolation and identification of bifidobacteria

Take 1g of neonatal feces sample, spread it on mMRS solid medium after serial dilution, culture it at 37°C in an anaerobic environment for 72 hours, observe and record the colony shape, pick and purify the colony, and then put it in MMRS liquid medium Cultivate at 37°C for 48 hours, perform Gram staining on the obtained colonies and record the strain morphology, discard the Gram-negative strains and Gram-positive cocci in the colonies, and select the Gram-positive bacilli, which are analyzed by catalase Afterwards, the catalase-positive strains were discarded, and the catalase-negative strains were retained, and the negative strains were detected by fructose-6-phosphate kinase. The obtained were all identified as pseudo small-chain bifidobacteria by 16S rDNA sequencing, and named as pseudobacteria. Small chain Bifidobacterium CCFM748. The obtained pseudosmall chain bifidobacterium was subcultured, and the collected bacteria were placed in a centrifuge tube and centrifuged at 3000r/min for 10 minutes to wash. This was repeated 3 times, and the obtained bacteria were added to a matrix protective agent for freezing.

16S rDNA amplification conditions: 95°C for 5min; 35 cycles (95°C for 30s, 55°C for 30s, 72°C for 2min); 72°C for 10min

Amplification primers:

27F: (5'-AGAGTTTGATCCTGGCTCAG-3')

1492R: (5'-TACGGCTACCTTGTTACGACTT-3'

The process of purification of amplified products and sequence alignment was as described in the literature (Turroni F et al. Exploring the Diversity of the Bifidobacterial Population in the Human Intestinal Tract [J]. Appl Environ Microb. 2009; 75(6): 1534-45.) method to proceed.

Identification of bifidobacteria: The isolated and purified bifidobacteria were identified as pseudo-small-chain bifidobacteria by 16S rDNA sequence analysis, and their basic characteristics are shown in Table 1.

Table 1 Basic characteristics of Bifidobacterium pseudocatenulatum CCFM748

Example 2: Biotransformation of conjugated linoleic acid by Pseudomonas Bifidobacterium CCFM748

The specific experiment is as follows:

1. Strain activation

Take out the glycerol tube holding Bifidobacterium Pseudomonas CCFM748 from the -80°C refrigerator, take out the bacterial liquid and streak it on the mMRS solid medium, and culture it at 37°C for 48h in an anaerobic environment. The grown single colonies were picked and inoculated in mMRS liquid medium, cultured at 37°C for 48 hours under anaerobic environment, and continuously activated for 3 generations.

2. Preparation of linoleic acid mother liquor

Weigh 300mg of linoleic acid (LA) and 200mg of Tween-80, dissolve in water and dilute to 10mL, stir and emulsify thoroughly, filter and sterilize through a 0.45μm sterile filter membrane, and store at -20°C in the dark.

3. Co-culture with linoleic acid

Inoculate the activated bacterial solution in step 1 into 10mL mMRS liquid medium containing 0.528mg/mL LA according to the inoculum amount of 2% (v/v), and culture at 37°C under anaerobic environment for 0, 12, 24, 48, 72h , at the same time, the medium with the addition of equal amount of linoleic acid without the addition of bacterial solution was used as the control. After culturing, transfer the bacterial solution to centrifuge tubes, and centrifuge at 5000rpm for 5min. Take 3 parts of 3mL fermentation broth for each culture and transfer them to clean centrifuge tubes for later use.

4. Fatty acid extraction

Extraction of fatty acids in fermentation broth: add heptadecanoic acid (C17:0) to 3 mL of fermentation broth to a final concentration of 0.075 mg/mL as an internal standard, then add 2 mL of isopropanol, shake fully for 30 seconds; then add 3 mL of n-hexane , shake fully for 30s; centrifuge at 5000rpm for 3min. Absorb the n-hexane layer into a clean fat extraction bottle and dry it with nitrogen.

Extraction of fatty acids in the bacteria: the bacteria obtained by the aforementioned centrifugation were washed with 2 mL of salt solution (containing 0.137mol/L NaCl, 7.0mmol/L K ₂ HPO ₄ and 2.5mmoL/L KH ₂ PO ₄ ), centrifuged at 4000rpm for 5min, and the washing steps were repeated . Resuspend the bacteria in 2 mL of the aforementioned salt solution, add heptadecanoic acid (C17:0) to a final concentration of 0.0575 mg/mL, perform fatty acid extraction and nitrogen blow-drying in the same way as the fermentation broth, and extract of fatty acids.

5. Fatty acid methylation

Add 400 μL of methanol to the fatty acid in the fermentation liquid and the fatty acid in the bacteria respectively after being blown dry with nitrogen gas, shake and mix well, then use 150 μL diazomethane reagent to carry out methylation directly, blow dry with nitrogen gas, redissolve with 1 mL of n-hexane, and transfer to Gas phase bottle, pending GC-MS detection.

6. GC-MS detection

Shimadzu gas chromatograph (GC 2010plus), gas column Rtx-wax (30m×0.25mm×0.25μm), mass spectrometer (Shimadzu Ultra QP2010). Temperature programming conditions: Initially 150°C, the temperature was raised to 200°C at a rate of 5°C/min, and kept for 10 minutes, then raised to 230°C at a rate of 4°C/min, and kept for 18 minutes. Split injection was adopted, the injection volume was 1 μL, the split ratio was 50:1, and helium was used as the carrier gas. Both the injector temperature and the detector temperature were 240 °C. The ion source is 220°C and the intensity is 70eV.

7. Experimental results

The obtained fatty acid contains: stearic acid 0.0144 mg/mL, oleic acid 0.0682 mg/mL, linoleic acid 0.1649 mg/mL, and CLA 0.3118 mg/mL.

During the accumulation process of CLA, Bifidobacterium pseudospermia CCFM748 began to produce CLA when it was grown in mMRS containing 0.528mg/mL LA for 12h, and the content of conjugated linoleic acid gradually increased with the growth of the bacteria (24h, 36h) , the concentration of conjugated linoleic acid in the fermentation broth tends to be saturated after 36 hours of cultivation, as shown in Figure 2. The maximum conversion rate of the bacteria to LA was cultured for about 72 hours, and the total concentration of CLA reached 0.3118 mg/mL. The total conversion rate of CLA was 59.05% calculated by the total amount of substrate LA.

According to the analysis of fatty acid components, the product contains only two isomers of CLA1 (c9, t11-CLA) and CLA2 (t10, c12-CLA) from the content of each isomer of CLA. The strain began to transform CLA1 after 12 hours of culture. With the growth of the bacteria, the isoform CLA1 quickly accumulated within 12h to 36h, and its content tended to be saturated after the strain was cultivated for 36h, while the content of CLA2 was low when the strain began to accumulate CLA (24h). , the concentration of CLA2 also increased further. After culturing for 72h, the concentration of CLA1 was as high as 0.223mg/mL, accounting for 72.02% of the total CLA.

After 72 hours of culturing, the analysis of the fermentation broth and the fatty acid composition of Bifidobacterium pseudosystolicum CCFM748 shows that the absorption and conversion rate of LA by the bacteria are extremely high, which are higher than those of the prior art. Only a small amount of substrate LA remained in the fermentation broth and almost no unconverted LA remained in the bacteria. Most of the obtained CLA is in the fermented liquid, and the residual amount in the bacteria is also very small. The results show that most of the products do not accumulate in the cell, but are transported to the outside of the cell, and the proportion of CLA in the total fatty acid in the fermentation broth reaches 55.7%, and the purity is significantly higher than that of the prior art, because it can simplify the later stage of fermentation. Separation and purification of CLA in liquid.

Example 3: Biotransformation of conjugated linolenic acid by Pseudomonas Bifidobacterium CCFM748

1. Strain activation

With embodiment 2.

2. Preparation of linolenic acid mother liquor

Weigh 300mg of α-linolenic acid (α-LNA) and 200mg of Tween-80, dissolve in water and make up to 10mL, stir and emulsify thoroughly, filter and sterilize through a 0.45μm sterile filter membrane, and store at -20°C in the dark.

3. Co-culture with linolenic acid

The activated bacterial solution was inoculated into 10 mL mMRS liquid medium containing 0.3759 mg/mL α-LNA according to the inoculum amount of 2% (v/v), and cultured at 37°C under anaerobic environment for 0, 12, 24, 36, 48, 72h, and the culture medium added with equal amount of linolenic acid without adding bacterial solution was used as the control. After culturing, transfer the bacterial solution to a centrifuge tube and centrifuge at 5000rpm for 5 minutes; take 3 parts of 3mL fermentation broth for each culture and transfer it to a clean centrifuge tube for use.

4. Fatty acid extraction

Extraction of fatty acids in fermentation broth: add heptadecanoic acid (C17:0) to 3 mL of fermentation broth to a final concentration of 0.0767 mg/mL as an internal standard, then add 2 mL of isopropanol, shake fully for 30 seconds; then add 3 mL of n-hexane , shake fully for 30s; centrifuge at 5000rpm for 3min, absorb the n-hexane layer into a clean fat extraction bottle; blow dry with nitrogen to obtain the fatty acid in the fermentation broth.

Extraction of fatty acids in bacteria: the bacteria obtained from the aforementioned centrifugation were washed with 2 mL of salt solution (containing 0.137mol/L NaCl, 7.0mmol/L K ₂ HPO ₄ and 2.5mmoL/L KH ₂ PO ₄ ), centrifuged at 4000rpm for 5min, and washed repeatedly . Resuspend the bacteria in 2 mL of salt solution, add heptadecanoic acid (C17:0) to a final concentration of 0.0575 mg/mL, perform fatty acid extraction and nitrogen blow-drying in the same way as the fermentation broth, and obtain fatty acid.

5. Fatty acid methylation

Add 400 μL of methanol to the aforementioned obtained fatty acids after blowing dry with nitrogen gas, oscillate and mix well, and directly carry out methylation with 150 μL diazomethane reagent, after blowing dry with nitrogen gas, redissolve with 1 mL of n-hexane, transfer to a gas phase bottle, and wait for GC- MS detection.

6. GC-MS detection

Shimadzu gas chromatograph (GC 2010plus), gas column Rtx-wax (30m×0.25mm×0.25μm), mass spectrometer (Shimadzu Ultra QP2010). Temperature programming conditions: Initially 150°C, the temperature was raised to 200°C at a rate of 5°C/min, kept for 10 minutes, then raised to 230°C at a rate of 4°C/min, and kept for 18 minutes. Split injection was adopted, the injection volume was 1 μL, the split ratio was 50:1, and helium was used as the carrier gas. Both the injector temperature and the detector temperature were 240 °C. The ion source is 220°C and the intensity is 70eV.

7. Experimental results

Bifidobacterium pseudosmatae CCFM748 began to produce CLNA when it was grown in mMRS containing 0.3759mg/mLα-LNA for 12h, and the content of conjugated linolenic acid gradually increased with the growth of the bacteria (24h, 36h). After 36h of cultivation, the conjugated flax The acid content increases gradually. The maximum conversion rate of the bacteria to LNA was about 72 hours after cultivation, and the total content of CLNA reached 0.3402 mg/mL (in addition, the product also contained a very small amount of vacantoleic acid, oleic acid, linoleic acid and the substrate α-LNA). The conversion rate of the total amount of substrate α-LNA is 90.5%, which is significantly better than the prior art.

According to fatty acid analysis, from the perspective of CLNA isomer content, there are only two isomers, CLNA1 and CLNA2, in the fermentation product of this bacteria, which are recognized as the two isomers with the highest biological activity. The strain began to convert conjugated linolenic acid after 12 hours of culture. With the growth of the bacteria, the isomer CLNA1 quickly accumulated within 12 hours to 36 hours, and its content tended to be saturated after the strain was cultivated for 36 hours, while CLNA2 was accumulated when the strain began to accumulate CLNA ( 24h) content is low, with the extension of culture time, the concentration of this isomer further increased. Among the converted CLNA isomers, CLNA1 was absolutely dominant, with a final concentration of 0.3267 mg/mL, accounting for 96% of the total CLNA content.

It can be found from the composition of bifidobacterium CCFM748 fermentation broth and cell fatty acid after 72 hours of cultivation that the absorption and conversion rate of this bacterial strain to LNA are extremely high, which is significantly better than that of the prior art. There is almost no substrate LNA remaining in the fermentation broth, and There is also only a very small amount of LNA left in the bacteria that has not been transformed. In addition, as far as the transformed CLNA is concerned, most of it is in the fermentation broth, and the amount remaining in the bacteria is also very small. In addition, the distribution of CLNA1 and CLNA2 in the fermentation broth and in the bacteria was basically the same. This result also indicated that most of the products were not accumulated in the cell, but were transported to the outside of the cell, which was beneficial for further separation and purification in the later stage.

Claims (8)

1. A strain of Bifidobacterium pseudocatenulatum CCFM748, which was deposited on June 12, 2016 in the General Microorganism Culture Collection Center of China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC No.12603. 2. the application of the pseudo-small chain bifidobacterium CCFM748 described in claim 1 in the preparation of conjugated linoleic acid. 3. The application according to claim 2, characterized in that the pseudo-small-chain Bifidobacterium CCFM748 converts linoleic acid into conjugated linoleic acid. 4. The application according to claim 2, characterized in that the conjugated linoleic acid is c9, t11-CLA and t10, c12-CLA. 5. The application of the pseudo-small chain bifidobacterium CCFM748 according to claim 1 in the preparation of conjugated linolenic acid. 6. The application according to claim 5, characterized in that the pseudo-small-chain Bifidobacterium CCFM748 converts linolenic acid into conjugated linolenic acid. 7. The application according to claim 2, characterized in that the conjugated linolenic acid is c9, t11, c15-CLNA and t9, t11, c15-CLNA. 8. The application of the pseudo small chain bifidobacterium CCFM748 described in claim 1 in food additives.