CN111728976A - Application of fucooligosaccharide in preparation of intestinal prebiotics - Google Patents
Application of fucooligosaccharide in preparation of intestinal prebiotics Download PDFInfo
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- CN111728976A CN111728976A CN202010680173.8A CN202010680173A CN111728976A CN 111728976 A CN111728976 A CN 111728976A CN 202010680173 A CN202010680173 A CN 202010680173A CN 111728976 A CN111728976 A CN 111728976A
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- Prior art keywords
- fucooligosaccharide
- intestinal
- acid
- level
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- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 229940005605 valeric acid Drugs 0.000 description 1
- 238000009834 vaporization Methods 0.000 description 1
- 230000008016 vaporization Effects 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
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Abstract
The invention discloses application of fucoidan oligosaccharide in preparation of intestinal prebiotics, wherein the fucoidan oligosaccharide has the functions of promoting carbohydrate metabolism, biosynthesis and metabolism of sugar, reducing membrane transport function, enhancing digestive system function or reducing disease infection, is used for promoting the generation of short-chain fatty acid in intestinal tracts and improving the level of intestinal microbial flora, is used for preparing the intestinal prebiotics, is used for human bodies or livestock and poultry animals, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of fucooligosaccharide in preparation of intestinal prebiotics.
Background
Fucose is a rare 6-deoxyhexose in the L-configuration, commonly found in microbial exopolysaccharides, brown algae, and mammals. Fucose modifications have been identified as being associated with a number of biological functions, including immunomodulation and cancer. Sulfate-containing fucoidans rich in brown algae have anticoagulant, antithrombotic, immunomodulatory, anticancer, and antiproliferative activities. However, the yield of fucoidan in plants or algae is low, and its composition varies with climate and season. The fucose-rich exopolysaccharides produced by the microorganisms are considered to be a better alternative in view of the advantages of microorganisms having higher growth rates and easier control of production conditions. In recent decades, other fucose-containing exopolysaccharides with the highest yield have been reported to be produced by Enterobacter (Enterobacter A47) at a yield of 13.23 g/L. The various physicochemical properties of the fucose-rich exopolysaccharide, such as rheological properties, adhesive properties, emulsifying capacity, and the use of biodegradable films, show important commercial value.
In previous studies, we isolated a Kosakonia strain and identified it as Kosakonia sp.cctcc M2018092, elucidating the genome-wide sequence and genetic characteristics of this strain (Complete genome sequence of Kosakonia sp.strain CCTCC 2018092, a fusase-Rich exopolysporac producer. singfengfengfeng Niu, 2019). The function of the capsular exopolysaccharide hydrolyzed fucooligosaccharides produced by Kosakonia sp was not studied.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an application of fucooligosaccharide in preparation of intestinal prebiotics; the invention also aims to provide the application of the fucooligosaccharide in promoting the generation of short-chain fatty acids in intestinal tracts; the invention also aims to provide the application of the fucooligosaccharide in improving the level of the intestinal microbial flora.
In order to achieve the purpose, the invention provides the following technical scheme:
1. application of fucooligosaccharide in preparing intestinal prebiotics is provided.
The fucoidin has the functions of promoting carbohydrate metabolism, sugar biosynthesis and metabolism, reducing membrane transport function, enhancing the function of a digestive system or reducing disease infection; the application of the prebiotics in preparing the prebiotics for promoting carbohydrate metabolism, sugar biosynthesis and metabolism, reducing membrane transport function, enhancing digestive system function or reducing disease infection.
In the invention, the fucooligosaccharide is prepared by hydrolyzing extracellular polysaccharide generated by fermentation of Kosakonia sp.CCTCC M2018092 by trifluoroacetic acid or enzymolysis and the like.
Preferably, the trifluoroacetic acid is hydrolyzed by adding 5% of exopolysaccharide by mass fraction into trifluoroacetic acid to make the final concentration of the trifluoroacetic acid 0.1M, hydrolyzing at 100 ℃ for at least 1 hour, and then removing the trifluoroacetic acid by using a 200Da nanofiltration membrane to obtain the fucooligosaccharide.
2. Application of fucooligosaccharide in promoting production of intestinal tract short chain fatty acid is provided.
Preferably, the short chain fatty acid is acetic acid or propionic acid.
3. Use of fucoidan for improving intestinal microbial flora level is provided.
Preferably, the improving gut microflora levels are increasing bacteroidetes levels, decreasing firmicutes and proteobacteria levels.
Preferably, the improving the level of the intestinal microflora is increasing the level of parabacteroides, norcolobacterium and prevotella, and decreasing the level of lactobacillus and bacteroides.
The invention has the beneficial effects that: the invention provides a fucoidin oligosaccharide with a probiotic function, and discloses the fucoidin oligosaccharide with the functions of promoting carbohydrate metabolism, biosynthesis and metabolism of sugar, reducing a membrane transportation function, enhancing a digestive system function or reducing disease infection for the first time, and the fucoidin oligosaccharide can promote the generation of short-chain fatty acid in an intestinal tract and improve the level of intestinal microbial flora, so that the fucoidin oligosaccharide can be prepared and can be applied to the fields of human health, livestock and poultry animal culture and the like.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a GC-MS total ion chromatogram of a monosaccharide standard.
FIG. 2 is a GC-MS total ion flow chromatogram of the oligosaccharide complete acid hydrolysis product.
FIG. 3 is a graph of the effect of fucooligosaccharides on pH of the fermentation broth.
Figure 4 is the effect of fucooligosaccharides on short chain fatty acids.
FIG. 5 shows the levels of the phyla of test tube fermentation intestinal flora (SGCON is a control group, SGFOP is a group added with fucooligosaccharides).
FIG. 6 shows the levels of test tube fermentation gut flora (SGCON for control group, SGFOP for fucoidan-added group).
FIG. 7 is the preparation of mucosal pellets.
FIG. 8 shows the results of mucosal globule simulation (A: lumen; B: mucosa; S, H, J resolution represents ascending, transverse, descending colon, the latter numbers represent days of fermentation, w represents stop of sample addition).
FIG. 9 shows the results of the simulated intestinal tract (A: level of phylum of intestine; B: level of genus of intestine; C: level of phylum of mucosa; D: level of genus of mucosa; S: ascending colon; H: transverse colon; J: descending colon; 0,3, w respectively represent the control group, and fucoidan addition was stopped on day 3 of fucoidan addition).
Fig. 10 is the effect of fucoidan on the metabolic activity of the intestinal flora (left histogram is the proportion of the metabolic pathway in all secondary metabolic pathways, right is the significance value of the two groups comparison, blue represents the strong metabolic pathway of the control group, orange represents the strong metabolic pathway of the 0.1% fucoidan experimental group, and the metabolic pathways are arranged from small to large according to the significance value).
FIG. 11 is the results of evaluation of safety of fucoidan oligosaccharide (A: the effect of fucoidan oligosaccharide on weight; B: the effect of fucoidan oligosaccharide on brain and heart; C: the effect of fucoidan oligosaccharide on colon length; D: the effect of fucoidan oligosaccharide on liver weight; E: the effect of fucoidan oligosaccharide on liver function and kidney function).
FIG. 12 shows the results of measurement of short-chain fatty acids in mouse feces (A: HPLC; B: GC; C: GC-MS; D: statistical results of short-chain fatty acid content).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 preparation of microbial Extracellular Polysaccharide (EPS)
EPS is produced by fermentation of Kosakonia sp.CCTCC M2018092 strain under fed-batch conditions, and the specific steps are as follows: culturing Kosakonia sp.CCTCC M2018092 strain in 30mL 250mL shake flask containing 30mL culture medium at 30 deg.C and 200rpm for 20 hr, transferring 30mL bacterial culture solution into 15L fermentation tank, and performing pre-growth culture at 30 deg.C and 300rpm for 13 hr (aeration amount of 1.5M)3H). Thereafter, 3L of the pre-grown bacterial broth was transferred to a 50L fermentor (containing 30L of medium) for fed-batch fermentation. A200 g/L glucose solution was fed in portions starting at a rate of 0.9-3.8rpm 13h after the start of the fermentation using a peristaltic pump according to the residual sugar amount. The ventilation of the fermentation tank is 1.5m3And/h, controlling the dissolved oxygen concentration to be more than 10% by automatically adjusting the rotating speed (300-550rpm) through the linkage of the rotating speed. The pH of the 50L fermenter was controlled at 7.0 by feeding sodium hydroxide and the temperature was controlled at 30 ℃. The composition of each medium during the cultivation is shown in Table 1.
TABLE 1 culture medium composition for extracellular polysaccharide production by Kosakonia sp.CCTCC M2018092 fermentation
And (3) extraction of extracellular polysaccharide:
the method comprises the following steps: the first route is that after the fermentation is finished, the pH value of the fermentation liquor is adjusted to 2.0 by using sulfuric acid, and then the fermentation liquor is subjected to high-speed centrifugation at 12000rpm for 20min to remove thalli and calcium sulfate; after the protein of the supernatant is removed by a Sevage method, deionized water is dialyzed (the cut-off molecular weight is 8000-14000Mw) and then freeze-dried to obtain the original fermentation polysaccharide (EPS), and the yield is 13.5 g/L. However, the small size of the cells requires high-speed centrifugation, which is not favorable for the industrial large-scale preparation of the polysaccharide.
The method 2 comprises the following steps: the extraction route is that after the fermentation is finished, the pH value of the fermentation liquor is adjusted to 2.0 by sulfuric acid, and the fermentation liquor is hydrolyzed for 4 hours at the temperature of 80 ℃. Then filtering through a 0.22 mu m ceramic membrane to remove thalli and calcium sulfate, and filtering through an ultrafiltration membrane with 10kDa cut-off quantity to remove micromolecules such as pigments and the like. After filtration and protein removal, dialysis (molecular weight cut-off 8000- < 14000 > Mw) and freeze-drying are carried out to obtain the partially hydrolyzed polysaccharide (AH-EPS) with the yield of 12.6 g/L.
Example 2 preparation of Low molecular weight Fucosaccharides
Dissolving the hydrolyzed polysaccharide prepared in example 1 in deionized water to prepare polysaccharide aqueous solution with mass fraction of 5%, adding trifluoroacetic acid to make the final concentration 0.1M, hydrolyzing in 100 deg.C oil bath for 0.5h, 1h, 1.5h, 2h, and 3h, removing trifluoroacetic acid from the obtained polysaccharide hydrolysate with 200Da nanofiltration membrane, concentrating to small volume at 40 deg.C by rotary evaporation, lyophilizing to obtain oligosaccharide sample, and measuring molecular weight by high performance liquid chromatography with chromatographic column G3000PWXL, column temperature 40 deg.C, and mobile phase 0.1M NaNO3The injection volume was 15. mu.l, and the results are shown in Table 1. The results show that the extraction rate of the fucoidan oligosaccharide except trifluoroacetic acid is 93 percent by using a 200Da nanofiltration membrane, the higher extraction rate of the fucoidan oligosaccharide can be realized under the condition of removing trifluoroacetic acid, the molecular weight of the fucoidan oligosaccharide subjected to primary hydrolysis by sulfuric acid is 47916Da, the PDI (polymer dispersity) is higher, the molecular weight of a sample is in a very inhomogeneous state, the molecular weight of the fucoidan oligosaccharide obtained after hydrolysis by trifluoroacetic acid for 0.5h, 1h, 1.5h, 2h and 3h is 14813Da, 1702Da, 2650Da, 7528Da and 1875Da respectively, the molecular weight is not reduced after hydrolysis for 1h, the glycosidic bond of the polysaccharide molecule which can be broken by trifluoroacetic acid at the concentration reaches the upper limit, and the molecular weight is abnormally increased during hydrolysis for 1872 h, so that the hydrolyzed polysaccharide has instability.
TABLE 1 trifluoroacetic acid hydrolysis of polysaccharides
| Name of component | Retention volume (mL) | Number average molecular weight (Mn)) | Weight average molecular weight (Mw) | PDI |
| Primary hydrolysis | 7.42 | 3940.89 | 47916.31 | 12.16 |
| 0.1M TFA,0.5h,100℃ | 8.08 | 10932.02 | 14813.61 | 1.36 |
| 0.1M TFA,1h,100℃ | 8.77 | 1613.3 | 1702.95 | 1.06 |
| 0.1M TFA,1.5h,100℃ | 8.78 | 2042.87 | 2650.55 | 1.3 |
| 0.1M TFA,2h,100℃ | 8.73 | 2600.63 | 7528.55 | 2.89 |
| 0.1M TFA,3h,100℃ | 8.83 | 1683.18 | 1875.48 | 1.11 |
GC-MS analysis of the monosaccharide composition in fucoidan oligosaccharides:
and (3) taking xylose as an internal standard substance, and determining quantitative correction factors of fucose, glucose, galactose and glucuronic acid in the fucoidan by adopting an internal standard method. 14.6mg of fucooligosaccharide was accurately weighed and dissolved in 0.5mL of xylose solution (8g/L), 3mL of TFA (2mol/L) was added, capping was performed, and the mixture was heated in a 120 ℃ oil bath for 2 hours and dried with nitrogen at 55 ℃. 2mL of ethiol and 1mL of TFA were added and magnetically stirred in a water bath at 25 ℃ for 25 min. Blowing to dry at 55 ℃ with nitrogen, adding 4mL of acetic anhydride-pyridine mixture (1:1, V/V), magnetically stirring in 55 ℃ water bath for 5h, blowing to dry with 0.5mL of sample nitrogen, redissolving in methanol, and injecting for GC-MS analysis. Transferring 8g/L fucose, glucose, galactose, xylose and glucuronic acid standard samples of 0.5mL respectively into the same glass tube, and mixing. After nitrogen blow-drying, derivatization is carried out according to the method, and then GC-MS analysis is carried out. GC-MS conditions: shimadzu (GCMS-QP2010, Japan), Rtx-5 capillary column (0.25mm × 30m), vaporization chamber temperature of 280 deg.C, high purity helium as carrier gas, flow rate of 1 mL/min; the amount of sample was 1. mu.L. Column temperature program: the initial temperature is 80 deg.C, holding for 2min, heating to 200 deg.C at a rate of 15 deg.C/min, heating to 210 deg.C at a rate of 1 deg.C, heating to 280 deg.C at a rate of 25 deg.C/min, and holding for 6 min. The interface temperature is 260 ℃; positive ion ionization mode, mass range: m/z is 35-600. The GC-MS total ion flow chromatogram of the standard product is shown in figure 1, and the GC-MS total ion flow chromatogram of the oligosaccharide complete acid hydrolysis product is shown in figure 2.
GC-MS analysis with xylose as an internal standard shows that the fucoidan consists of fucose, glucose, glucuronic acid and galactose, and the molar ratio of the fucose, the glucose, the galactose and the glucuronic acid in the fucoidan is 1.58:1.00:1.00: 1.46.
Example 3 test tube simulated intestinal fermentation of fucooligosaccharides
Taking 1L of fermentation medium, wherein the medium comprises the following components: peptone 2 g/L; 2g/L of yeast extract; NaCl 0.1 g/L; k2HPO40.04g/L；KH2PO40.04g/L；MgSO4·7H2O 0.01g/L；CaCl2·6H2O 0.01g/L；NaHCO32 g/L; tween 802 mL/L, and heme is added according to 0.02g/L after sterilization; 10mL/L vitamin K1(ii) a 0.5g/L bile salt; 0.5g/L cysteine hydrochloride, and adding fucooligosaccharide according to the mass fraction of 0.1%.
Firstly, inoculating 6 test tubes into 25ml culture medium, inoculating 10% fecal bacteria liquid, introducing anaerobic gas, and keeping for 5min (85% N)2， 10％CO2，5％H2) And sealed with paraffin, sampling every 12h, measuring the pH and gas of the fermentation liquor, and the result is shown in figure 3. The results show that the pH drop rate of the fermentation broth added with 0.1% of fucooligosaccharide is higher than that of the control group.
After 36 hours, sampling and sending to 16s rRNA measurement, taking 1mL of fermentation liquor to measure short-chain fatty acid, wherein the method for measuring the short-chain fatty acid in the fermentation liquor is as follows:
short-chain fatty acid extraction: collecting 1mL supernatant, centrifuging at 6000rpm/min for 10min, sucking out supernatant, adding 100 μ L concentrated hydrochloric acid and 5mL diethyl ether, mixing, extracting at room temperature for 20min, centrifuging at 4 deg.C for 10min at 5000r/min, and collecting supernatant. Transferring the supernatant to another tube, adding 500 μ L of 1M NaOH, mixing, extracting at room temperature for 20min, centrifuging at 4 deg.C for 10min at 5000r/min, and collecting the lower aqueous phase. Transferring the lower layer water phase into another tube, adding 100 μ L concentrated hydrochloric acid, mixing, filtering the obtained sample with 0.22 μm filter membrane, and analyzing by high performance liquid chromatography.
Chromatographic conditions are as follows: analysis was performed using a ZORBAX SB-Aq (4.6X 250mm 5-Micron) column with a mobile phase of 0.025% aqueous phosphoric acid (pH 2.8): acetonitrile 95: 5, eluting at 1.0mL/min, wherein the sample injection amount of each needle is 20 mu L; detection wavelength: 210 nm; column temperature: at 30 ℃.
Short chain fatty acid labeling assay: preparing standard sample solutions of acetic acid, propionic acid, n-butyric acid and n-valeric acid with the concentration of 1mL/L respectively, and performing high performance liquid chromatography analysis under the detection conditions to determine the retention time of each acid. According to the concentration of short-chain fatty acid in the fermentation liquor estimated by the pre-experiment, a mixed standard solution with the acetic acid concentration of 2mL/L, the propionic acid concentration of 4mL/L, the n-butyric acid concentration of 2mL/L and the n-valeric acid concentration of 1mL/L is prepared. Then diluting with ultrapure water according to the proportion. The mixed standard solution was subjected to high performance liquid chromatography under the above-mentioned detection conditions, peak areas at the respective concentrations were measured, and a short-chain fatty acid standard curve was plotted, with the results shown in table 2.
TABLE 2 Standard Curve for short chain fatty acids
The calculation results are shown in fig. 4. The results show that the yield of propionic acid is 2 times of that of the control group after adding the fucooligosaccharide of 0.1 percent, and the fucooligosaccharide can be preliminarily proved to promote intestinal bacteria to produce propionic acid so as to promote the health of human bodies.
Test tube fermentation 16s rRNA sequencing
1) DNA extraction and PCR amplification
The microbial genome is extracted from human excrement and fermented human excrement genome by using a Tiangen kit. The V4-V5 region of the 16S rRNA gene of the bacteria is amplified by PCR, and amplification primers are as follows: 338F 5 '-barcode-ACTCCTACGGGAGGCAGCA-3'; 806R 5 '-GGACTACHVGGGTWTCTAAT-3' wherein the barcode is an eight base sequence unique to each sample. The amplification system was (20 μ L): 4 μ L of 5 XFastPFu Buffer, 2 μ L of 2.5mM dNTPs, 0.8 μ L of positive and negative primers (5 μ M), 0.4 μ L of FastPFu polymerase, 10ng of DNA template; the amplification procedure is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, and annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; extending for 5min at 72 ℃; the cycle was 25 times.
2) Illumina MiSeq high throughput sequencing
The amplicons were gel recovered after electrophoresis in 2% agarose gel and extracted using AxyPrep DNA gel extraction kit according to manufacturer's protocolPurification was performed using QuantiFluorTMPurified amplicons were pooled in an equimolecular sequencing pool and paired-sequenced on Illumina MiSeq, Dow Corning Biotech Ltd (2 × 300).
3) Sequencing data processing and analysis
The original fastq file was cut into libraries and quality filtered using QIIME (version 1.17) according to the following criteria: truncate 250 bp reads at any site with an average quality score of less than 20 over a 10bp sliding window, and discard truncated reads shorter than 50 bp. The barcode sequence, the sequence with 2 nucleotide mismatches in the primer, the reads containing ambiguous bases were removed. Only reads with overlap region larger than 10bp are assembled, and reads which cannot be assembled are discarded. Sequences with 97% similarity were clustered by UPARSE into one operational unit (OTUs) and used UCHIME to identify and remove chimeric sequences. Phylogenetic relatedness of each 16S rRNA gene sequence in the silva (SSU115)16S rRNA database with a 70% confidence threshold was analyzed and annotated using an RDP classifier (http:// RDP. cme. msu. edu /).
The results are shown in FIGS. 5 and 6, and show that levels of Bacteroides (p < 0.05) are significantly increased, while levels of Proteobacteria (p < 0.05) comprising a plurality of gram-negative pathogenic or conditional pathogenic bacteria are significantly decreased.
Example 4 CDMN simulated intestinal fermentation
Preparing 2L of intestinal fermentation medium: (g/L): 8.0g/L of corn starch; peptone 3.0 g/L; 4.5g/L of yeast extract; tryptone 3.0 g/L; mucin 0.5 g/L; 0.8g/L of L-cysteine hydrochloride; no. 3 bile salt 0.4 g/L; 0.05 g/L of heme; 4.5g/L of sodium chloride; tween 801.0 mL/L; 2.5g/L of potassium chloride; 0.4g/L of monopotassium phosphate; 4.5g/L of magnesium chloride hexahydrate; 0.2g/L of calcium chloride hexahydrate; trace elements 2 mL/L.
Microelement stock solution (g/L): magnesium sulfate heptahydrate 3.0; ferrous sulfate heptahydrate 0.1; 0.1 part of calcium chloride dihydrate; 0.32 parts of manganese chloride tetrahydrate; 0.18 parts of cobalt sulfate heptahydrate; 0.01 of blue vitriol; 0.18 parts of zinc sulfate heptahydrate; nickel chloride hexahydrate 0.092.
Dissolving corn starch in distilled water at 100 deg.C for 5min, adding intestinal fermentation medium (containing no mucin, hemoglobin, 3 # bile salt, cysteine, and microelements), sterilizing at 121 deg.C for 15min, and adding mucin, hemoglobin, 3 # bile salt, cysteine, and microelements.
Preparation of mucosal pellets: weighing 2g of agar, heating and dissolving in 100mL of distilled water, cooling to 60 +/-5 ℃ after the agar solution is clear and transparent, weighing 0.5g of mucin, dissolving while hot, adjusting the pH to about 6.8, transferring to a spherical grinding tool after ultraviolet sterilization for 30min in a super clean bench to obtain mucosa gel beads with the diameter of 7-8mm, bagging and hanging the gel beads in three fermentation tanks to simulate intestinal mucosa (figure 7), wherein the parameters are shown in Table 3.
TABLE 3 colon segment parameters
| Parameter(s) | Ascending colon | Transverse colon | Descending colon |
| Fermentation volume (ml) | 300 | 400 | 300 |
| Temperature (. degree.C.) | 37 | 37 | 37 |
| Rotational speed | 150 | 130 | 170 |
| pH | 5.8 | 6.2 | 6.8 |
Intestinal bacteria liquid: 20g of excrement of two normal young (24 years old) males is collected and dissolved into 160ml of PBS solution, three layers of gauze are used for filtering, solid insoluble substances are removed, excrement bacterial liquid is obtained, excrement microorganisms are respectively inoculated into 3 fermentation tanks according to the inoculation amount of 10%, and 15 mucous membrane pellets are added into each tank. The pH automatic control system is supplemented with 0.5mol/L NaOH solution and 0.5mol/L HCl to adjust the fermentation pH, and the fermentation temperature is kept constant at 37 ℃ by means of a heating and condensing system. In order to control the anaerobic environment in which fermentation is strict, nitrogen is introduced into each fermenter every morning, noon and evening to exhaust the air in the fermenter. After 24h of inoculation culture, to maintain the normal growth of the microorganisms, the nutrients were replenished and 300mL were drained daily to maintain the fermentation volume constant, and the mucosal pellet in the jar was replaced with 3 new ones daily to simulate mucosal regeneration.
The results are shown in fig. 8, and show that the fucooligosaccharide promotes the production of acetic acid and propionic acid in the intestinal lumen, while butyric acid and acetic acid are mainly distributed in the mucosa, the fucooligosaccharide promotes the production of acetic acid, and after the addition of the sample is stopped, the level of short-chain fatty acid produced by the intestinal bacteria is not greatly changed, which can indicate that the fucooligosaccharide has the effect similar to the intestinal flora remodeling.
16s rRNA sequencing: the microbial genome was extracted from human feces using a Tiangen kit. The V4-V5 region of the 16S rRNA gene of the bacteria is amplified by PCR, and amplification primers are as follows: 338F: 5 '-barcode-ACTCCTACGGGAGGCAGCA-3'; 806R: 5 '-GGACTACHVGGGTWTCTAAT-3' wherein barcode is an eight base sequence unique to each sample. The amplification system was (20 μ L): 4 μ L of 5 XFastPFu Buffer, 2 μ L of 2.5mM dNTPs, 0.8 μ L of positive and negative primers (5 μ M), 0.4 μ L of FastPFu polymerase, 10ng of DNA template. The amplification procedure is as follows: 2min at 95 ℃; 30s at 95 ℃; 30s at 55 ℃; 30s at 72 ℃; 5min at 72 ℃; the cycle was 25 times.
Illumina MiSeq high throughput sequencing: amplicons were gel recovered after 2% agarose gel electrophoresis, purified using AxyPrep DNA gel extraction reagent kit according to manufacturer's instructions, and QuantiFluorTMPurified amplicons were pooled in an equimolecular sequencing pool and pair-wise sequenced on Illumina Miseq, Dow-Ning Biotechnology Ltd (2 × 300).
Processing and analyzing sequencing data: the original fastq file was cut into libraries and quality filtered using QIIME (version 1.17) according to the following criteria: truncate 250 bp reads at any site with an average quality score of less than 20 over a 10bp sliding window, and discard truncated reads shorter than 50 bp. The barcode sequence, the sequence with 2 nucleotide mismatches in the primer, the reads containing ambiguous bases were removed. Only reads with an overlapping region larger than 10bp are assembled, and reads which cannot be assembled are discarded. Sequences with 97% similarity were clustered by UPARSE into one operational unit (OTUs) and the chimera sequences were identified and removed using UCHIME. Phylogenetic relatedness of each 16S rRNA gene sequence in the silva (SSU115)16S rRNA database with a 70% confidence threshold was analyzed and annotated using an RDP classifier (http:// RDP. cme. msu. edu /).
As shown in FIG. 9, A in FIG. 9 is at the level of the phylum Enteromorpha, and it can be seen that the levels of Bacteroides (Bacteroides) and Rhabdominobacteria (Firmicutes) decrease, the levels of Bacteroides (Bacteroides) increase, and the levels of Bacteroides (Bacteroides) and Rhabdominoidis (Colostrich) remain at relatively stable levels when the addition of fucooligosaccharides is stopped. B in FIG. 9 is at the level of enterobacteria, and it can be seen that the level of Lactobacillus transversus (Lactobacillus) is significantly decreased, Bacteroides (Bacteroides) is slightly decreased, Parabacteroides (Parabacteroides) is significantly increased (p < 0.05), Bacteroides norcolatoides (Bacteroides) is significantly increased (p < 0.05), Parabacteroides (Parabacteroides) is significantly increased (p < 0.05), and the level of Parabacteroides transversus (Parabacteroides) is increased (p < 0.05) after the addition of fucoidan-oligosaccharide is stopped compared to the level without fucoidan-oligosaccharide addition. In fig. 9C is the level of the intestinal mucoportis, it can be seen that Bacteroidetes (Bacteroidetes) significantly rises in the ascending colon, the transverse colon and the descending colon 3 days after the addition of fucooligosaccharide, bacteroides (bacteroides) rises significantly in the ascending colon, and wherein Proteobacteria (Proteobacteria) level significantly falls and Firmicutes (Firmicutes) level falls. Increased levels of Firmicutes and Proteobacteria (Proteobacteria) were obtained after the addition of fucooligosaccharides was stopped. FIG. 9D shows that Prevotella (p < 0.05) increased most in the ascending colon, Bacteroides (p < 0.05) increased in the transverse colon, and Lactobacillus (p < 0.05) decreased in each intestinal segment after fucoidan was added to the mucosa; after the addition of fucooligosaccharides was stopped, the levels of colonic and colonic mucosa-descending lactic acid bacteria (Lactobacillus) were increased again.
The effect of fucooligosaccharides on the metabolic activity of the intestinal flora is shown in fig. 10. The results show that the oligosaccharide has the functions of promoting carbohydrate metabolism, sugar biosynthesis and metabolism, reducing membrane transport function, enhancing the function of a digestive system, reducing disease infection and the like.
Example 5 evaluation of fucooligosaccharide safety in vivo
Male wild-type mice of 8 weeks old were randomly divided into 3 groups of 6 mice each, and then subjected to intragastric administration of physiological saline (0.9% sodium chloride), 0.1% fucooligosaccharide (79.2mg/d/kg), and 0.5% fucooligosaccharide (396mg/d/kg), respectively, to collect feces once a week, and after 1 month, the organs of the mice were collected and the weights thereof were measured, and the results are shown in A in FIG. 11.
Collecting excrement: fixing the mouse, lifting the tail of the mouse, lightly pressing the lower abdomen of the mouse with fingers, collecting fresh excrement in a plastic tube with a cover and a corresponding number, immediately sealing, storing the small tube in an ice box, and storing all sample tubes in a low-temperature refrigerator at-80 ℃ for later use.
Each physiological index can preliminarily reflect whether the fucooligosaccharide has adverse effect on mouse body, and the weight, brain, colon, pancreas, liver weight and colon length of the mouse are measured after 1 month, and the results are shown as B-D in figure 11.
At the end of the experiment, the eyes were removed and blood was collected, serum samples were analyzed in triplicate, Gamma Glutamyl Transferase (GGT), alanine Aminotransferase (ALT), alkaline phosphatase (ALP) were used as the evaluation indices for liver function, processed according to the method of the kit instructions and tested with a full automatic enzyme labeling machine, and the results are shown in fig. 11E.
Blood was collected from the eye, serum samples were analyzed in triplicate, urea nitrogen (BUN), Creatinine (CRE), etc. were used as the evaluation indices for renal function, processed according to the methods described in the kit instructions and tested with a full-automatic enzyme calibrator, with the results shown in fig. 11E.
The result shows that the physiological indexes and the liver and kidney functions of the mice treated by the fucoidin have no significant difference (P is more than 0.05) compared with the control group, and the fucoidin is safer oligosaccharide and can be used for being eaten by organisms.
The physiological indexes and liver and kidney functions of mice treated by the fucoidin have no significant difference (P is more than 0.05) compared with those of a control group, which indicates that the fucoidin is a safer oligosaccharide and can be used for being eaten by organisms.
Determination of short-chain fatty acids in mouse feces:
weighing a small amount of fresh excrement of a mouse, dissolving the weighed excrement in an EP tube containing 500 mu L of methanol solution, standing for 5-10 min, and uniformly mixing the excrement and the methanol solution by oscillation to prepare excrement suspension. Adjusting the pH value of the suspension to 2-3 with sulfuric acid, standing for 5min, and shaking and uniformly mixing for several times. Centrifuging the EP tube at 5000r/min for 20min, centrifuging the supernatant at 5000r/min for 5min, and analyzing the supernatant by gas chromatography-mass spectrometry. The results of initial experiments using HPLC, GC-MS and GC to measure short chain fatty acids in mouse feces are shown in FIG. 12.
The results show that the spectra measured by the three measurement methods show that GC-MS peak separation and peak shape are good, the peaks are obtained according to the standard, and acetic acid, propionic acid, butyric acid and valeric acid which are sequentially arranged from left to right can be used for measuring short-chain fatty acid in mouse excrement. The determination result shows that the mice added with 0.1 percent of fucoidin oligosaccharide generate acetic acid and propionic acid which are increased, and the rock algae oligosaccharide sample is utilized by special intestinal flora in the intestinal tracts of the mice and stimulates the special intestinal flora to generate short chain fatty acid, so that the human body shows a probiotic effect.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or the change made by the person skilled in the art on the basis of the present invention are within the protection scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (9)
1. Application of fucooligosaccharide in preparing intestinal prebiotics is provided.
2. Use according to claim 1, characterized in that: the fucooligosaccharide is applied to the preparation of prebiotics for promoting carbohydrate metabolism, biosynthesis and metabolism of sugar, reducing membrane transport function, enhancing digestive system function or reducing disease infection.
3. Use according to claim 1 or 2, characterized in that: the fucooligosaccharide is prepared by hydrolyzing exopolysaccharide produced by fermentation of Kosakonia sp.CCTCCMM2018092.
4. Use according to claim 3, characterized in that: and the trifluoroacetic acid hydrolysis comprises the steps of adding extracellular polysaccharide with the mass fraction of 5% into trifluoroacetic acid to enable the final concentration of the trifluoroacetic acid to be 0.1M, hydrolyzing for at least 1 hour at 100 ℃, and then removing the trifluoroacetic acid by using a 200Da nanofiltration membrane to obtain the fucooligosaccharide.
5. Application of fucooligosaccharide in promoting production of intestinal tract short chain fatty acid is provided.
6. Use according to claim 3, characterized in that: the short-chain fatty acid is acetic acid or propionic acid.
7. Use of fucoidan for improving intestinal microbial flora level is provided.
8. Use according to claim 7, characterized in that: the improving intestinal microbial flora level comprises increasing bacteroidetes level and reducing firmicutes and proteobacteria level.
9. Use according to claim 7, characterized in that: the improving intestinal microflora level is to increase the levels of Parabacteroides, Bacteroides norcolmanii and Prevotella, and reduce the levels of Lactobacillus and Bacteroides.
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