CN110846241B

CN110846241B - Bifidobacterium animalis capable of decomposing and utilizing human milk oligosaccharide, culture method thereof and food or medicine

Info

Publication number: CN110846241B
Application number: CN201810949886.2A
Authority: CN
Inventors: 郭慧媛; 任发政; 赵亮; 张明; 陈剑亮
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-08-20
Filing date: 2018-08-20
Publication date: 2022-02-11
Anticipated expiration: 2038-08-20
Also published as: CN110846241A

Abstract

The invention provides a microorganism, a method for culturing the microorganism and a food or a medicament. The microorganism is Bifidobacterium bifidum (Bifidobacterium animalis subsp. lactis A6), is preserved in China general microbiological culture collection center (CGMCC NO. 9273) in 6-5 th month in 2014, and has a preservation number of CGMCC NO. 9273. The microorganism of the invention has the capability of decomposing and utilizing the human milk oligosaccharide, can grow by taking the human milk oligosaccharide as a carbon source, and particularly can grow by taking the human milk oligosaccharide as a unique carbon source. Therefore, theoretical basis is provided for the addition of the human milk oligosaccharide, the animal bifidobacterium, the probiotics and the human milk oligosaccharide in the food or the medicine, and the like.

Description

Bifidobacterium animalis capable of decomposing and utilizing human milk oligosaccharide, culture method thereof and food or medicine

Technical Field

The present invention relates to the field of biology. In particular, the invention relates to animal bifidobacterium capable of decomposing and utilizing human milk oligosaccharide, a culture method thereof and food or medicine. More particularly, the present invention relates to a microorganism, a method of culturing a microorganism, and a food or pharmaceutical.

Background

At present, people pay more and more attention to health, and the demand for functional foods is also larger and larger. Wherein the probiotic food and health product are rapidly developed. Currently, the most widely used probiotic species in the market are three types, namely, Bifidobacterium (Bifidobacterium), Lactobacillus casei/paracasei (Lactobacillus paracasei) and Lactobacillus acidophilus (Lactobacillus acidophilus). Among them, Bifidobacterium is isolated from human intestinal tract, and many experiments verify its safety, and Bifidobacterium can make human intestinal tract more stable, which all have been widely accepted. The Bifidobacterium has 46 species and subspecies, and closely related to human body, such as Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium apis, Bifidobacterium catenulatum, and Bifidobacterium odontois.

Human Milk Oligosaccharides (HMOs) are a class of water-soluble compounds that cannot be digested and absorbed by the Human body, and are mainly formed by the combination of five basic monosaccharides, galactose (Gal), glucose (Glc), N-acetylglucosamine (GlcNAc), sialic acid (Sia), and fucose (Fuc), with various glycosidic bonds. The human milk oligosaccharide is a main carbon source for growth of bifidobacteria in infant intestinal tracts and plays an important role in the construction process of infant intestinal flora. The bifidobacterium is a dominant microorganism in the intestinal tract of the infants, the abundance of the bifidobacterium can reach more than 90 percent of that of the intestinal microorganism, and the bifidobacterium plays an important role in the growth and development of the infants. The bifidobacterium animalis has strong survival capability, is a probiotic supplement commonly used for infants, screens bifidobacterium animalis strains capable of decomposing and utilizing human milk oligosaccharides, is beneficial to improving the survival and colonization of the strains in intestinal tracts of infants, and has important significance.

However, the use of bifidobacterium animalis for human milk oligosaccharides is still under investigation.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.

To this end, in one aspect of the invention, the invention proposes a microorganism. According to the embodiment of the invention, the microorganism is Bifidobacterium (Bifidobacterium animalis subsp.lactis A6) which is preserved in China general microbiological culture collection center (CGMCC NO. 9273) at 6-5 months in 2014. The preservation address is No. 3 Xilu No. 1 of Beijing, Chaoyang, China.

When the infants only take breast milk as a nutrient source, lactose is absorbed by human bodies, and human milk oligosaccharide is retained in intestinal tracts because of being incapable of being absorbed by the human bodies, so that the human milk oligosaccharide becomes a main carbon source of intestinal microorganisms. Most of intestinal microorganisms such as cocci, clostridia, etc. cannot utilize human milk oligosaccharides to provide energy for their growth, and thus do not grow well. And part of bifidobacteria contain genes for encoding human milk oligosaccharide hydrolase, so that human milk oligosaccharide can be decomposed and utilized to supply energy for self growth, and a probiotic effect is exerted. For this reason, bifidobacteria can be the predominant microorganism in the infant gut, with an abundance of more than 90% of gut microorganisms. However, the inventors found that not all bifidobacteria have the ability to break down and utilize human milk oligosaccharides. Furthermore, the inventor screens out a strain of Bifidobacterium animalis subsp.lactis a6 (the invention also becomes b.animalis a6) through a large number of experiments, and the Bifidobacterium animalis subsp.lactis a6 has the capability of decomposing and utilizing human milk oligosaccharides, can grow by taking the human milk oligosaccharides as a carbon source, and particularly can grow by taking the human milk oligosaccharides as a unique carbon source. Therefore, theoretical basis is provided for the addition of the human milk oligosaccharide, the animal bifidobacterium, the probiotics and the human milk oligosaccharide in the food or the medicine, and the like.

According to an embodiment of the invention, the microorganism may also have the following additional technical features:

according to an embodiment of the invention, the microorganism has the ability to break down and utilize human milk oligosaccharides. According to a specific embodiment of the invention, the microorganism uses human milk oligosaccharides as a carbon source. Animallis a6 has the ability to break down and utilize human milk oligosaccharides, and thus can grow on human milk oligosaccharides as a carbon source, and particularly can grow on human milk oligosaccharides as a sole carbon source.

According to an embodiment of the invention, the human milk oligosaccharides comprise at least one of 2 '-fucosyllactose, 3-fucosyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-difucotetraose, lacto-N-tetraose, lacto-N-neotetraose, 6' -sialyllactose and 3 '-sialyllactose, wherein the utilization ratio of the 2' -fucosyllactose and the 3-fucosyllactose is 35-50%, the utilization ratio of the lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-pentaose V is 15-30%, the utilization rate of the lactose-N-tetrasaccharide and the lactose-N-neotetrasaccharide is 20-30%; the utilization rate of the 6 '-sialyllactose and the 3' -sialyllactose is 10-20%.

The inventors found that b.animallis a6 had a higher availability of 2 '-fucosyllactose (also referred to herein as "2' -FL"), 3-fucosyllactose (also referred to herein as "3-FL"), lacto-N-tetraose (also referred to herein as "LNT"), lacto-N-neotetraose (also referred to herein as "LNnT"), lacto-N-fucopentaose (also referred to herein as "LNFP") I/II/III/V, 6 '-sialyllactose (also referred to herein as "6' -SL"), and 3 '-sialyllactose (also referred to herein as "3' -SL"), which could be used to maintain the self-growth metabolism.

According to an embodiment of the invention, the microorganism has human milk oligosaccharides as sole carbon source. Thus, the microorganisms of the present invention are particularly capable of acting as a prebiotic for infants who only consume breast milk.

According to the embodiment of the invention, the microorganism takes 2 '-fucose lactose as a sole carbon source, and the utilization rate of the 2' -fucose lactose is 20-30%. The inventors found that b.animalis a6 has a high utilization of 2' -fucosyllactose and can utilize it to maintain its own growth metabolism.

According to the embodiment of the invention, the microorganism takes 3-fucosyllactose as a sole carbon source, and the utilization rate of the 3-fucosyllactose is 20-30%. The inventor finds that the utilization rate of the B.animalis A6 on the 3-fucose lactose is high, and the self-growth metabolism can be maintained by the utilization rate.

According to the embodiment of the invention, the microorganism can degrade 3-fucose lactose, degradation products comprise lactate and acetic acid, and the mass ratio of the lactate to the acetic acid is 1: 1-3. The inventor finds that B.animalis A6 is cultured by using 3-fucosyllactose as a sole carbon source, and the cultured product contains lactate and acetic acid, and the ratio of the lactate to the acetic acid is 1: 1-3.

According to an embodiment of the invention, the microorganism has genes encoding a fucosidase or a functional analogue thereof, an L-fucose dehydrogenase or a functional analogue thereof, a fucose permease or a functional analogue thereof, an L-fucoidan dehydratase or a functional analogue thereof, an L-lactaldehyde reductase or a functional analogue thereof, and/or a propylene glycol oxidoreductase or a functional analogue thereof. Thus, b.animalis a6 has the ability to break down and utilize human milk oligosaccharides, and can grow using human milk oligosaccharides as a carbon source, and in particular can grow using human milk oligosaccharides as a sole carbon source.

In another aspect of the invention, the invention provides a food or pharmaceutical product. According to an embodiment of the invention, the food or pharmaceutical product contains the aforementioned microorganisms. As described above, the b.animalis a6 of the present invention has the ability to break down and utilize human milk oligosaccharides, and can grow using human milk oligosaccharides as a carbon source, and particularly using human milk oligosaccharides as a sole carbon source. Therefore, theoretical basis is provided for the addition of the human milk oligosaccharide, the animal bifidobacterium, the probiotics and the human milk oligosaccharide in the food or the medicine, and the like.

It will be appreciated by those skilled in the art that the features and advantages described above for the microorganism are equally applicable to the food or pharmaceutical product and will not be described in further detail herein.

In a further aspect of the invention, the invention provides a method of cultivating a microorganism as hereinbefore described. According to an embodiment of the invention, the method comprises: subjecting the microorganism to a fermentation culture in a medium comprising human milk oligosaccharides. As described above, the b.animalis a6 of the present invention has the ability to break down and utilize human milk oligosaccharides, and can grow using human milk oligosaccharides as a carbon source, and particularly using human milk oligosaccharides as a sole carbon source. Therefore, theoretical basis is provided for the addition of the human milk oligosaccharide, the animal bifidobacterium, the probiotics and the human milk oligosaccharide in the food or the medicine, and the like.

According to an embodiment of the invention, the culture medium comprises 10-20 g/L of human milk oligosaccharide. The inventor finds that B.animalis A6 has a good growth condition in a culture medium containing 10-20 g/L of human milk oligosaccharide, and the utilization rate of the human milk oligosaccharide is high. According to a specific embodiment of the present invention, the human milk oligosaccharide comprises: 1.52-2.48 g/L of at least one of 2' -fucosyllactose and 3-fucosyllactose; 0.66-1.32 g/L lactotetraose; 1.75-4.80 g/L of at least one of lactoyl-N-fucopentaose I, lactoyl-N-fucopentaose II, lactoyl-N-fucopentaose III and lactoyl-N-fucopentaose V; 1.44-1.92 g/L of at least one of 6 '-sialyllactose and 3' -sialyllactose; and 2.26 to 4.52g/L of at least one of lactoyl-N-neotetraose and lactoyl-N-tetraose. Thus, b.animalis a6 could maintain self-growing metabolism in the superior medium described above.

According to an embodiment of the invention, the medium further comprises: 0.01 to 0.05g/L xanthine, 0.1 to 0.5g/L aspartic acid, 0.1 to 0.5g/L proline, 0.0001 to 0.001g/L p-aminobenzoic acid, 1 to 5g/L sodium acetate, 0.1 to 0.5g/L glutamic acid, 0.1 to 0.5g/L serine, 0.001 to 0.005g/L nicotinic acid, 0.5 to 1.5g/L triammonium citrate, 0.1 to 0.5g/L alanine, 0.1 to 0.5g/L threonine, 0.0003 to 0.008g/L folic acid, 1 to 5g/L potassium dihydrogen phosphate, 0.1 to 0.5g/L arginine, 0.1 to 0.5g/L tryptophan, 0.1 to 0.5g/L pantothenate, 0.001 to 0.005g/L dipotassium hydrogen phosphate, 1 to 5g/L glycine, 0.1 to 0.5g/L tyrosine, 0.0005 to 0.5g/L biotin, 0.1-0.5 g/L histidine, 0.1-0.5 g/L, VB 60.001.001-0.005 g/L valine, 0.01-0.1 g/L manganese sulfate, 0.1-0.5 g/L isoleucine, 0.2-0.8 g/L, VB 120.0005-0.0015 g/L cysteine hydrochloride, 0.01-0.05 g/L ferrous sulfate, 0.1-0.5 g/L leucine, 0.1-0.5 g/L methionine, 0.001-0.005 g/L riboflavin, 800.5-1.5 mL Tween, 0.1-0.5 g/L lysine, 0.1-0.5 g/L phenylalanine, and 0.01-0.05 g/L adenine. Therefore, the B.animalis A6 can be normally metabolized and fully utilize human milk oligosaccharide to maintain the metabolism.

According to an embodiment of the invention, the fermentation culture is anaerobic culture at 37 ℃ for 48-72 hours, preferably 72 hours. After 72 hours of culture, the utilization rates of 2' -fucosyllactose and 3-fucosyllactose are 35-50%, the utilization rates of lactoyl-N-fucopentaose I, lactoyl-N-fucopentaose II, lactoyl-N-fucopentaose III and lactoyl-N-fucopentaose V are 15-30%, the utilization rates of 6' -sialyllactose and 3' -sialyllactose are 10-20%, and the utilization rates of lacto-N-tetraose and lacto-N-neotetraose are 20-30%.

It will be appreciated by those skilled in the art that the features and advantages described above for the microorganism apply equally to the method of culturing the microorganism and will not be described in detail here.

In a further aspect of the invention, the invention provides a method of culturing a microorganism as hereinbefore described. According to an embodiment of the invention, the method comprises: and (2) carrying out fermentation culture on the microorganism in a culture medium, wherein the culture medium comprises 12-20 g/L of 2' -fucose lactose and/or 3-fucose lactose. The inventors have found that the b.animallis a6 of the present invention is capable of growing on 2 '-fucosyllactose and/or 3-fucosyllactose as carbon source, in particular on 2' -fucosyllactose or 3-fucosyllactose as sole carbon source. Therefore, theoretical basis is provided for adding the human milk oligosaccharide, the animal bifidobacterium, the probiotics and the human milk oligosaccharide in the infant food or the medicine, and the like.

According to an embodiment of the invention, the medium further comprises: 0.01 to 0.05g/L xanthine, 0.1 to 0.5g/L aspartic acid, 0.1 to 0.5g/L proline, 0.0001 to 0.001g/L p-aminobenzoic acid, 1 to 5g/L sodium acetate, 0.1 to 0.5g/L glutamic acid, 0.1 to 0.5g/L serine, 0.001 to 0.005g/L nicotinic acid, 0.5 to 1.5g/L triammonium citrate, 0.1 to 0.5g/L alanine, 0.1 to 0.5g/L threonine, 0.0003 to 0.008g/L folic acid, 1 to 5g/L potassium dihydrogen phosphate, 0.1 to 0.5g/L arginine, 0.1 to 0.5g/L tryptophan, 0.1 to 0.5g/L pantothenate, 0.001 to 0.005g/L dipotassium hydrogen phosphate, 1 to 5g/L glycine, 0.1 to 0.5g/L tyrosine, 0.0005 to 0.5g/L biotin, 0.1-0.5 g/L histidine, 0.1-0.5 g/L, VB 60.001.001-0.005 g/L valine, 0.01-0.1 g/L manganese sulfate, 0.1-0.5 g/L isoleucine, 0.2-0.8 g/L, VB 120.0005-0.0015 g/L cysteine hydrochloride, 0.01-0.05 g/L ferrous sulfate, 0.1-0.5 g/L leucine, 0.1-0.5 g/L methionine, 0.001-0.005 g/L riboflavin, 800.5-1.5 mL Tween, 0.1-0.5 g/L lysine, 0.1-0.5 g/L phenylalanine, and 0.01-0.05 g/L adenine. Thus, the B.animalis A6 can be normally metabolized and fully utilize 2' -fucosyllactose, 3-fucosyllactose, lacto-difucotetraose, 6' -sialyllactose, 3' -sialyllactose and/or lacto-N-neotetraose.

According to an embodiment of the invention, the fermentation culture is anaerobic culture at 37 ℃ for 48-72 hours, preferably 72 hours. After culturing for 72 hours, the utilization rate of 2' -fucose lactose as the only carbon source is 20-30%, and the utilization rate of 3-fucose lactose as the only carbon source is 20-30%.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a graph of the growth of microorganisms in a chemically defined medium containing 16g/L HMO according to one embodiment of the invention; and

FIG. 2 shows a L-fucose metabolism map according to an embodiment of the present invention.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

In this example, the present inventors evaluated the degree of utilization of human milk oligosaccharides by each strain by using the microorganism b.animalis a6 of the present invention as a positive control strain and another 9 strains of bifidobacterium (table 1) as study subjects, using human milk oligosaccharides as a sole carbon source, microwell-culturing in a chemically defined medium, and analyzing the composition and content of oligosaccharides in the supernatant of the culture broth using LC-QTOF-MS.

TABLE 112 basic information on Bifidobacterium strains

Bacterial strains	Numbering
		Bifidobacterium animalis	CICC	6250
Bifidobacterium animalis	CICC				6167
		Bifidobacterium animalis	CICC	21715
Bifidobacterium animalis	CICC				6174
		Bifidobacterium animalis	CGMCC 1.3003
Bifidobacterium animalis	CGMCC 1.2226
		Bifidobacterium animalis	CICC	6165
Bifidobacterium animalis	A6 (the invention)
		Bifidobacterium longum	CICC	6069
Bifidobacterium bifidum	CICC				6071

1. Culture medium

Preparing an MRS liquid culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of beef extract, 20.0g of glucose, 5.0g of anhydrous sodium acetate, 2.0g of anhydrous dipotassium phosphate, 2.0g of diammonium hydrogen citrate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate tetrahydrate and 800.1 mL of tween-10, adding distilled water to 1000mL, adjusting the pH value to 6.2-6.4, subpackaging 10mL of culture solution in an anaerobic tube, filling sufficient nitrogen, covering a plug to isolate air, and sterilizing at 121 ℃ for 15 minutes. And adding 15g of agar powder into the MRS solid culture medium on the basis of the liquid culture medium.

The formula of the chemically defined medium is shown in table 1, wherein the human milk oligosaccharide is a mixed oligosaccharide obtained by removing protein and most lactose from breast milk by an alcohol precipitation method. In order to avoid loss of nutrient elements, mineral salts and amino acids in the culture medium are sterilized at 121 deg.C for 15min, and carbon source, vitamins and nucleic acid base are sterilized by filtration with 0.22 μm microporous membrane, and mixed before use.

TABLE 2 chemically defined Medium formulation

The human milk oligosaccharide is a mixed oligosaccharide after deproteinization by an alcohol precipitation method and most of lactose is removed, and the composition is shown in the following table, wherein the content of 2' -FL and 3-FL is 1.52-2.48 g/L, LDFT and is 0.66-1.32 g/L, the content of lactyl-N-fucopentaose I/II/III/V is 1.75-4.80 g/L, the content of 6' -SL and 3' -SL is 1.44-1.92 g/L, LNT and the content of LNnT is 2.26-4.52 g/L.

TABLE 2 composition of human milk oligosaccharides

Note: h: six carbon sugar (glucose or galactose) N: acetylglucosamine F: fucose A: sialic acid

2. Culture method

Inoculating bifidobacterium strain powder preserved in an environment of-80 ℃ into an MRS liquid culture medium anaerobic tube for activation, then sequentially transferring the powder into the MRS liquid culture medium anaerobic tube by using the inoculation amount of 1 percent, utilizing the activated strain of the third passage, taking 9ml of bacterial liquid in a 10ml sterile centrifuge tube in an ultra-clean bench, centrifuging for 15min at 4500rpm and 4 ℃, pouring off the supernatant, adding 9ml of sterile physiological saline, and uniformly mixing to be used as the inoculation liquid. Respectively transferring the inoculation liquid into a chemically defined culture medium according to the inoculation amount of 1%, transferring the inoculated culture liquid into a 96-well cell culture plate of corning 3599 under an aseptic environment, forming a 100-microliter system in each well, performing parallel culture of 3 wells at each time point, and performing constant-temperature culture at 37 ℃ under an anaerobic environment.

3. Growth of the Strain

And (3) completing the measurement of the bifidobacterium growth curve by adopting a multifunctional microplate reader. As shown in FIG. 1, most of the bifidobacteria grew slowly in the chemically defined medium with 16.0g/L HMO as the sole carbon source and reached the maximum OD after 54h₆₀₀The value is obtained. Among them, b.bifidum cic 6071, b.animallis a6, and b.infarnatis cic 6069 grow better than other strains, and among them, b.animallis a6 strain grows better.

It is thus shown that not all microorganisms, in particular not all bifidobacteria from animals, have the ability to break down and utilize human milk oligosaccharides, which can be used as a carbon source, even the sole carbon source. The B.animalis A6 of the invention has better capability of decomposing and utilizing human milk oligosaccharide, and can perform better growth and metabolism in a culture medium only taking the human milk oligosaccharide as a carbon source.

4. Utilization of fucosylated neutral oligosaccharides

The 2' -FL and the 3-FL in the culture medium are significantly reduced (P <0.05) compared with 0h after 72h of culture of B.animallis A6, wherein the relative content of the 2' -FL and the 3-FL is reduced to 58.32% after 72h of culture of B.animallis A6, which shows that the utilization degree of the 2' -FL and the 3-FL is higher in the growth and propagation processes. It is presumed that a gene fragment encoding fucosidase may be present in the b. animalis a6 gene, so that the strain can decompose 2' -FL and 3-FL. However, after the b.animalis cic count 6250 and the b.animalis CGMCC 1.3003 are cultured for 0, 24, 48 and 72 hours, the 2'-FL and the 3-FL in the culture medium are not significantly reduced, which indicates that the b.animalis cic count 6250 and the b.animalis CGMCC 1.3003 have low utilization degree of the 2' -FL and the 3-FL, and this may be that the gene sequences of the two strains do not have a gene segment encoding fucosidase or unique ABC transporter thereof.

Animallis a6 there was substantially no major change in absolute peak intensity of lactotetraose (also referred to herein as "LDFT") in the medium after 72h of culture, indicating that these four bifidobacteria strains did not utilize LDFT substantially.

The absolute peak intensity of LNFP I/II/III/V did not change significantly (P >0.05) in the media after 72h of culture (B.animalis CICC6250 and B.bifidum CICC 6071) compared to 0h, indicating that it does not utilize LNFP I/II/III/V substantially. After the strain A6 is cultured for 72 hours, the absolute peak intensity of LNFP I/II/III/V in a culture medium is slightly reduced, and the relative content of LNFP I/II/III/V is reduced to 79.73 percent, which shows that the strain can utilize LNFP I/II/III/V, but the utilization degree is not high.

From the above data, it is shown that b.animalis a6 favors the use of shorter chain HMOs such as 2' -FL, 3-FL for bathosylated neutral oligosaccharides. B. animalis a6 was used to a lesser extent for bathosylated neutral oligosaccharides such as LDFT, LNFP I/II/III/V, etc.

5. Utilization of non-fucosylated neutral oligosaccharides

Non-fucosylated neutral oligosaccharides account for about 25% of HMOs, the vast majority of which are Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT). Animallis a6 the relative amount of non-bathosylated neutral oligosaccharides in the medium decreased with increasing culture time, and the relative amount of LNT, LNnT decreased to 71.86% after 72h of culture, indicating that the bacterium was able to utilize LNT and LNnT.

6. Utilization of sialylated oligosaccharides

The relative content of 3'-SL, 6' -SL in the culture medium after 72h of culture of B.animalis CICC6250 and B.animalis CGMCC 1.3003 is not changed significantly (P >0.05), which indicates that the 3'-SL, 6' -SL cannot be basically utilized. However, the relative content of 3'-SL and 6' -SL in the culture medium was significantly reduced after 72h of culture (P <0.05) by b. animalis a6, and the relative content was reduced to 85.82%, respectively, indicating that it can utilize 3'-SL and 6' -SL.

Example 2

In this example, the effect of lactose, glucose, 2' -FL, 3-FL, LNnT, 3' -SL or 6' -SL as sole carbon source on the growth metabolism of different microorganisms was investigated:

the bacterial species, culture medium and culture mode were the same as in example 1, except that the human milk oligosaccharides were replaced with lactose, glucose, 2' -FL, 3-FL, LNnT, 3' -SL or 6' -SL in the culture medium.

1. Growth of the Strain

Three strains B.animallis A6, B.animallis CGMCC 1.3003 and B.animallis CICC6250 grew best in a chemical medium with glucose as the sole carbon source and relatively better with lactose as the carbon source. However, when 3-FL, 2' -FL, LNnT, 3' -SL and 6' -SL are respectively used as unique carbon sources, the two strains B.animallis CGMCC 1.3003 and B.animallis CICC6250 do not grow. In contrast, B.animalis A6 grew in chemically defined media with 3-FL and 2' -FL as the sole carbon source, respectively, and did not grow substantially in chemically defined media with LNnT, 3' -SL, and 6' -SL as the sole carbon source.

2. Utilization of 2' -FL and 3-FL

Animalis a6 the percentage content of the remaining 2'-FL and 3-FL in the culture medium changed significantly (P <0.05) after culturing for 0, 24, 54, 72h, etc., wherein the peak area of the total scanning peak of the remaining 2' -FL and 3-FL in the m/z-487.35-488.85 range gradually decreased with the increase of the culturing time. The remaining percentage content of 2'-FL in the medium of B.animalis A6 was 72.69% after 72h of culture in chemically defined medium with 16.0 g/L2' -FL as sole carbon source; the remaining percentage content of 3-FL in the medium of B.animalis A6 was 77.43% after 72h of culture in chemically defined medium with 16.0 g/L3-FL as the sole carbon source. And in different time points such as 0, 24, 54, 72h and the like after culture, the B.animalis A6 always provides energy for the growth and the propagation of the strain by utilizing 2' -FL and 3-FL along with the increase of time. From this, it was concluded that enzymes involved in metabolizing 2' -FL and 3-FL were present in the genome of B.animalis A6 strain.

3. Utilization of LNnT

Animallis a6 the percentage content of LNnT remaining in the medium did not change significantly (P >0.05) after incubation for various times of 0, 24, 54, 72h, etc. in chemically defined media containing LNnT as sole carbon source, which did not take up and decompose LNnT.

4. Utilization of 3'-SL and 6' -SL

The percentage content of the remaining 3'-SL and 6' -SL in the culture medium is not significantly changed (P >0.05) after the animallis A6 is cultured in a chemically limited culture medium containing 16.0g/L (w/v) of 3'-SL and 6' -SL as the only carbon source for 0, 24, 54, 72h and the like, which indicates that the B.animallis A6 cannot decompose and utilize the 3'-SL and 6' -SL to a certain extent.

Example 3

In this example, the metabolism of fucosylated oligosaccharides by bifidobacteria was studied:

the strain is harvested at the end of log after activated culture in MRS medium. Centrifuging at 8000 Xg and 4 deg.C for 10min to obtain bacterial sludge, and determining fucosidase and lacto-N-diglycosidase by conventional method.

1. Fucosidase Activity

Fucosidase is a metabolic enzyme required by microorganisms to sialylate human milk oligosaccharides, can cleave fucose connected to glucose or galactose in the form of alpha 1-2, alpha 1-3 and alpha 1-4 glycosidic bonds, provides the initial enzyme digestion for strains to utilize such oligosaccharides, and is an enzyme which is more critical for microorganisms to utilize fucosylated human milk oligosaccharides.

Fucosidase activity detected in B.animallis A6 is 6.86 +/-0.57 mU/mg protein, while no fucosidase activity is detected in B.animallis CGMCC 1.3003 and B.animallis CICC6250, which shows that the B.animallis A6 strain has the capability of enzyme-cutting fucosidase with a modified end, and shows that the strain can utilize fucosylated human milk oligosaccharide to a certain extent. Therefore, the fucosidase is preliminarily determined to have an important effect on the utilization of fucosylated human lacto-oligosaccharides by bifidobacterium animalis strains.

2. lacto-N-diglycosidase Activity

lacto-N-diglycosidase, which was first discovered by Jun Wada et al from Bifidobacterium bifidum JCM1254 strain, is encoded by the lnbB gene, belongs to the GH20 family, encodes a protein containing 1,112 amino acid residues of a signal, and is a type of catabolic enzyme capable of cleaving lacto-N-biose (Gal β 1,3GlcNAc) from the major component lacto-N-tetrase (Gal β 1,3GlcNAc β 1,3Gal β 1,4Glc) of human milk oligosaccharides.

The enzyme activity of lacto-N-diglycosidase was not detected in animallis a6, and thus non-fucosylated human lactooligosaccharides such as LNnT and LNT could not be used well.

3. End product of Bifidobacterium animalis on 3-FL metabolism

In this experiment, 16.0g/L (w/v)3-FL was used as the sole carbon source, and after culturing in chemically defined medium for 48 hours, the supernatant was taken for determination of the metabolic end products. The results show that the metabolites of degradation of 3-FL by b.animalis a6 are mainly lactate and acetate, and the ratio is 1: 2. Wherein the content of lactate in the culture solution of B.animalis A6 is 221.8 + -29.60 μ g/mL, and the content of acetic acid is 516.43 + -42.03 μ g/mL.

4. Bifidobacterium L-fucose metabolic pathway analysis

Research has shown that b.longum subsp.infinantis can metabolize L-fucose to 1, 2-propanediol, while the pathway of bifidobacterium to metabolize L-fucose is not clear, and the pathway of e.coli to metabolize L-fucose is now clear compared to the pathway of xanthomas campestis pv. The inventors consult the metabolic enzymes necessary for the two metabolic pathways, and search similar homologous proteins through NCBI to obtain the whole protein sequence group of B.animalis A6, and locally blastp the strain protein sequence and the metabolic enzyme homologous protein to identify L-fucose metabolism related genes of the bifidobacteria. The results are shown in Table 3.

TABLE 3 genes associated with L-fucose metabolism by Bifidobacterium strains

Note: the bit scores and e values for hits obtained in the gene ID and brackets are shown in the table above.

As can be seen from the above table, b.animalalis a6 has a putative L-fucose dehydrogenase, fucose permease, L-fucoidan dehydratase, L-lactaldehyde reductase, and propylene glycol oxidoreductase, but homologous proteins of metabolic enzymes such as L-2-keto-3-deoxyfucoidan-4-dehydrogenase, L-2, 4-dione-3-deoxyfucoidan hydrolase, and L-fucoidan hydrolase have not been detected. The possible metabolic pathways of L-fucose by b.animalis a6 are thus shown in figure 2.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. Use of a microorganism in vitro decomposition and utilization of human milk oligosaccharides, wherein the microorganism is bifidobacterium (bifidobacterium: (bifidobacterium))Bifidobacterium animalis subsp.lactis) A6, deposited in China general microbiological culture Collection center on 5 th 6 th 2014 with the deposit number of CGMCC NO. 9273.

2. Use according to claim 1, characterized in that the human milk oligosaccharide is selected from at least one of 2' -fucosyllactose, 3-fucosyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-difucotetraose, lacto-N-tetraose, lacto-N-neotetraose, 6' -sialyllactose and 3' -sialyllactose.

3. Use according to claim 1, wherein the microorganism has human milk oligosaccharides as sole carbon source.