CN113826900A - Gellan gum oligosaccharide and application thereof in prebiotics - Google Patents
Gellan gum oligosaccharide and application thereof in prebiotics Download PDFInfo
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- CN113826900A CN113826900A CN202111149559.7A CN202111149559A CN113826900A CN 113826900 A CN113826900 A CN 113826900A CN 202111149559 A CN202111149559 A CN 202111149559A CN 113826900 A CN113826900 A CN 113826900A
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- China
- Prior art keywords
- oligosaccharide
- gellan gum
- product
- gellan
- butyric acid
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 150000002482 oligosaccharides Chemical class 0.000 title claims abstract description 93
- 229920002148 Gellan gum Polymers 0.000 title claims abstract description 90
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- 235000010492 gellan gum Nutrition 0.000 title claims abstract description 77
- 235000013406 prebiotics Nutrition 0.000 title abstract description 14
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims abstract description 70
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 41
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- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims abstract description 23
- 235000021391 short chain fatty acids Nutrition 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 20
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- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims abstract description 12
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses gellan gum oligosaccharide and application thereof in prebiotics, belonging to the technical field of biology. The invention provides a gellan gum oligosaccharide, wherein the basic main chain structure of the gellan gum oligosaccharide comprises tetrasaccharide units, the tetrasaccharide units are D-glucose, D-glucuronic acid and L-rhamnose, and the molar ratio of the D-glucose, the D-glucuronic acid and the L-rhamnose is (4-6): (1.2-1.9): (2.4-3.6), and the polymerization degree range of the gellan gum oligosaccharide is 2-6. According to the invention, through an in vitro static and dynamic fecal strain fermentation method, the novel gellan gum oligosaccharide is found to remarkably increase the yield of acetic acid, propionic acid, butyric acid and total SCFAs, improve the structure of fecal flora and remarkably improve the relative abundance of butyric acid producing bacteria.
Description
Technical Field
The invention relates to gellan gum oligosaccharide and application thereof in prebiotics, belonging to the technical field of biology.
Background
Gellan gum is a safe microbial polysaccharide approved by the united states Food and Drug Administration (FDA), produced in large quantities at low cost by pseudomonas elodea, and widely used in the food industry, agriculture, chemical industry, and life sciences. Gellan gum is difficult to digest by bifidobacteria and lactobacilli, limiting their potential as prebiotics. Oligosaccharides derived from gellan gum have the potential to be commercially safe functional oligosaccharides.
Oligosaccharides are a class of indigestible carbohydrates, typically consisting of less than 10 monosaccharides. Increasingly oligosaccharides are used as prebiotics to benefit health by modulating the activity of the gut flora, stimulating the growth of beneficial bacteria, and accumulating the terminal metabolite Short Chain Fatty Acids (SCFAs). SCFAs are composed primarily of acetate, propionate, and butyrate, which account for many physiological interactions between the host and the gut flora, including energy homeostasis, metabolism of lipids and carbohydrates, and inhibition of inflammatory signals. For example, arabinoxylo-oligosaccharides stimulate the growth of bifidobacteria and the production of Short Chain Fatty Acids (SCFAs) in human fecal cultures. Supplementation with galactooligosaccharides may reverse malnutrition of the intestinal flora of rats with alcohol withdrawal syndrome and restore normal intestinal permeability. However, gellan oligosaccharides are currently less studied as prebiotics.
At present, the research on the fermentation probiotic effect of oligosaccharides mostly focuses on animal in-vivo experiments, the animal experiment period is long, the cost is high, and the change in animal intestinal tracts cannot be monitored in real time in the experiment process. If the intestinal tract reactor can be used for replacing animal experiments, a large amount of time can be saved, the cost is reduced, the fermentation process can be accurately monitored in real time, and better repeatability is achieved. Researchers in domestic and foreign countries have developed many different types of in vitro digestion reactors, and foreign countries already have gastrointestinal tract reactors put on the market. Molly et al designed a five-tank human gastrointestinal microecological Simulator (SHIME) with human mouth, stomach and intestines as prototype. Minekus et al developed TIM-1 which mimics gastric and small intestinal digestion and TIM-2 which mimics large intestinal fermentation, respectively. SHIME and TIM are currently the most common simulated human digestion bioreactors. The inventor subject group develops a simulated gastrointestinal tract reactor (GSR) combining functions of TIM-1 and TIM-2 in the early period, namely the GSR can simulate the digestion process of the stomach and small intestine of a human body and the fermentation process of the colon of the large intestine; how to obtain a novel gellan gum oligosaccharide, and applying the gastrointestinal tract reactor to simulate the fermentation process of the gellan gum oligosaccharide in colon; to explore their potential for use as prebiotics, and become a hotspot in research.
Disclosure of Invention
In order to obtain a novel gellan gum oligosaccharide and use the novel gellan gum oligosaccharide as a prebiotic, the invention provides a gellan gum oligosaccharide, wherein the basic main chain structure of the gellan gum oligosaccharide comprises tetrasaccharide units, the tetrasaccharide units are D-glucose, D-glucuronic acid and L-rhamnose, and the molar ratio of the D-glucose, the D-glucuronic acid and the L-rhamnose is (4-6): (1.2-1.9): (2.4-3.6).
In one embodiment of the invention, the D-glucose and D-glucuronic acid are linked by a beta- (1-4) bond.
In one embodiment of the invention, the D-glucuronic acid and the D-glucose are linked by a beta- (1-4) bond.
In one embodiment of the invention, the D-glucose and the L-rhamnose are linked by an alpha- (1-4) bond.
In one embodiment of the invention, the L-rhamnose and the D-glucose are linked by a beta- (1-3) bond.
In one embodiment of the present invention, the gellan gum oligosaccharide has a polymerization degree in the range of 2 to 6.
The invention also provides a preparation method of the gellan gum oligosaccharide, which comprises the following steps:
(1) adding gellan gum into water, stirring, uniformly mixing, adding an HCl solution to enable the final concentration of HCl to be 0.4-0.5M, obtaining a reaction system, and stirring and hydrolyzing the reaction system in a water bath at 70-80 ℃ for at least 36 hours;
(2) adjusting the pH value of the reaction system after hydrolysis in the step (1) to 7.0, passing the solution through a microporous filter membrane, and concentrating the supernatant to 1/10-1/15 of the original volume by using a rotary evaporator to obtain a concentrated solution;
(3) adding ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 70-75% to obtain a mixture; standing the mixture for 6-10 hours, centrifuging the solution, and removing the precipitate;
(4) adding ethanol into the supernatant obtained in the step (3) to enable the final concentration of the ethanol to reach 85-90%, and standing for 6-10 hours to obtain oligosaccharide precipitate;
(5) the oligosaccharide precipitate is dialyzed by a dialysis bag of 100-500Da to remove the salt and dried to obtain the gellan gum oligosaccharide.
In one embodiment of the invention, the step (1) is that 5-10 g of gellan gum is added into 1L of deionized water, stirring is continuously carried out to uniformly mix the gellan gum and the deionized water, then HCl solution is added to ensure that the final concentration of HCl is 0.4-0.5M, a reaction system is obtained, and the reaction system is stirred and hydrolyzed in a water bath at 70-80 ℃ for 36 hours;
in one embodiment of the invention, the system after hydrolysis in step (1) is adjusted to pH 7.0 with 5-10M NaOH.
In one embodiment of the invention, the concentrated solution in the step (2) is precipitated by 70-75% (v/v) ethanol to remove macromolecules.
In one embodiment of the invention, the supernatant obtained in the step (3) is precipitated by 85-90% (v/v) ethanol to obtain an oligosaccharide precipitate.
In one embodiment of the present invention, the cut-off molecular weight of the dialysis bag is in the range of 100-500 Da.
The invention also provides a product for improving the content of short-chain fatty acids in metabolites of intestinal flora, wherein the product contains the gellan gum oligosaccharide.
In one embodiment of the present invention, the short chain fatty acid is one or more of acetic acid, propionic acid and butyric acid, or the content of the short chain fatty acid refers to the total amount of the short chain fatty acids.
In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical product.
The invention also provides a product for improving the abundance of butyric acid bacteria in intestinal tracts, and the product contains the gellan gum oligosaccharide.
In one embodiment of the invention, the butyric acid producing bacteria include Ruminococcus (Ruminococcus), Lachnospiraceae (Lachnospiraceae), Lachnospridium and Lachnospira (Lachnospira).
In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical product.
The invention also provides the application of the gellan gum oligosaccharide or the method in improving the content of short-chain fatty acids in metabolites of intestinal flora or improving the abundance of butyric acid bacteria in intestinal tracts.
The invention also provides application of the gellan gum oligosaccharide in preparation of a product for improving the abundance of butyric acid bacteria in intestinal flora or a product for improving the abundance of butyric acid bacteria in intestinal tracts.
The invention also provides a method for improving the abundance of butyric acid bacteria in intestinal flora, which comprises the step of inoculating the extracted intestinal flora into a culture medium containing gellan gum oligosaccharide for culture.
The invention also claims the use of gellan gum oligosaccharides in the preparation of prebiotic-related products.
Advantageous effects
(1) The invention obtains gellan gum oligosaccharide with polymerization degree of 2-6, wherein monosaccharide comprises glucose, rhamnose and glucuronic acid, and the molar ratio is (4-6) - (1.2-1.9) - (2.4-3.6). In static fecal bacteria fermentation in vitro, the novel gellan gum oligosaccharides significantly increase the yield of acetic acid, butyric acid and total SCFAs, improve the structure of fecal flora and significantly increase the relative abundance of butyric acid producing bacteria, especially in the genera Ruminococcaceae (Ruminococcaceae), Lachnospiraceae (Lachnospiraceae) and lachnocristium, compared to inulin.
(2) In an in vitro gastrointestinal tract reactor, the novel gellan gum oligosaccharide is degraded in a fermentation process of 24-48 h, the yields of acetic acid, propionic acid, butyric acid and total SCFAs are significantly increased, the structure of fecal flora is improved and the relative abundance of butyric acid producing bacteria is significantly increased, especially Ruminococcaceae (Ruminococcaceae), Lachnospiraceae (lachnospiaceae) and Lachnospira (Lachnospira), which corresponds to a significantly increased yield of butyric acid.
Drawings
FIG. 1: a real object diagram of novel gellan gum oligosaccharide.
FIG. 2: is the molecular weight distribution of the novel gellan gum oligosaccharide.
FIG. 3: the relative abundance of the flora changes in the fermentation of fecal bacteria in vitro; wherein (A) is family level, (B) is genus level; inulin 1, inulin 2 and inulin 3 refer to fecal fermentations of healthy human donors 1, 2 and 3, respectively; gellan gum oligosaccharide 1, gellan gum oligosaccharide 2 and gellan gum oligosaccharide 3 refer to fecal fermentation from healthy human donor 1, donor 2 and donor 3, respectively.
FIG. 4: a flora that significantly changes in inulin and gellan oligosaccharide fermentation in vitro fecal bacteria fermentation.
FIG. 5: the relative abundance of butyric acid producing bacteria changes during in vitro fecal bacteria fermentation; wherein inulin 1, inulin 2 and inulin 3 refer to fecal fermentation of healthy human donor 1, donor 2 and donor 3, respectively; gellan gum oligosaccharide 1, gellan gum oligosaccharide 2 and gellan gum oligosaccharide 3 refer to fecal fermentation from healthy human donor 1, donor 2 and donor 3, respectively.
FIG. 6: consumption of gellan gum oligosaccharides during the gastrointestinal tract reactor process.
FIG. 7: changes in pH and NaOH consumption during the gastrointestinal tract reactor.
FIG. 8: a change in the relative abundance of the flora in the gastrointestinal tract reactor; wherein (A) is at the family level and (B) is at the genus level.
FIG. 9: the relative abundance of the butyric acid producing bacteria in the gastrointestinal tract reactor varies.
Detailed Description
The gellan gum referred to in the following examples was purchased from imperial lunbang (inner mongolia) biotechnology limited.
The media involved in the following examples are as follows:
basic nutrient medium (g.L)-1)：NaCl 0.1，K2HPO4 0.04，KH2PO4 0.04，NaHCO32.0, 0.5 of L-cysteine hydrochloride, 0.5 of bile salt, MgSO4 0.01，CaCl20.01, heme 0.025, vitamin K0.002, peptone 2.0, yeast extract 2.0, resazurin 0.001, Tween 802 mL.
Basal fermentation Medium (g.L)-1): starch 5, arabinogalactan 1.0, pectin 2.0, xylan 1.0, yeast extract 3.0, tryptone 1.0, casein 2.0, L-cysteine 0.5, KCl 1.0, NaCl 0.5, K2HPO4 0.5，KH2PO4 0.5，CaCl2·6H2O 0.15，MgSO4·7H2O0.01, hemin 0.025, bile salt 0.4, FeSO4·7H2O0.005, Tween 801 mL, vitamin mixed solution 1mL, and pH value of 5.8.
Wherein the vitamin mixed solution (g.L)-1) The components are as follows: menadione 1.0, D-biotin 2.0, vitamin B-120.5, pantothenic acid 10.0, nicotinamide 5.0, para aminobenzoic acid 5.0, thiamine 4.0.
The detection methods referred to in the following examples are as follows:
analysis of monosaccharide composition:
the monosaccharide composition of the novel gellan gum oligosaccharides was analyzed by using High Performance Liquid Chromatography (HPLC). The specific method comprises the following steps: approximately 5mg of the sample was dissolved in 300. mu.L of 2M trifluoroacetic acid and then hydrolyzed at 110 ℃ for 1 h. The hydrolyzed sample was blow dried with nitrogen, 300 μ L of methanol was added, and then blow dried three times to remove trifluoroacetic acid. The final hydrolysate was dissolved in 25mL of distilled water for analysis. Detection of the hydrolysate and 1 g.L in a Dionex ion chromatography System (ICS 5000; Dionex Corp.) by means of a CarboPac PA20 column-1Monosaccharide standards (glucose, galactose, fructose, mannose, xylose, rock)Trehalose, rhamnose, arabinose and glucuronic acid) and finally the monosaccharide composition and content of gellan gum oligosaccharide.
Molecular weight analysis of gellan gum oligosaccharides:
the molecular weight of the novel gellan gum oligosaccharide was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The specific method comprises the following steps: 1mL of DHB matrix solution was prepared by dissolving 10mg of 2, 5-dihydroxybenzoic acid in 1mL of 50% acetonitrile/0.1% trifluoroacetic acid/water (v/v/v) and then adding 10. mu.L of 2.84M NaCl solution. mu.L of oligosaccharide sample (2g/L) was spotted onto an MTP AnchorChip 400/384TF target and dried at ambient temperature. Add 1. mu.L of DHB matrix solution to the same location and dry at ambient temperature. The analysis was performed in the mass range of m/z 700-.
Method for detecting changes in flora composition
Bacterial genomic DNA was extracted by using the QIAamp DNA pool Mini Kit. The V3-V4 region of the 16S rRNA gene was amplified using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3'). Sequencing libraries were generated using the TruSeq-DNAPCR-Free sample preparation kit and pyrosequencing was performed by MiSeq PE250 platform. Assembly and quality control of paired end reads were performed using FLASH (version 1.2.7) and Qiime (version 1.9.1), respectively. Upearse (v7.0.1001) assigns sequence prunes with > 97% similarity to the same Operational Taxon (OTU). The Silva database (heep:// www.arb-Silva. de /) is used to annotate the bio-taxonomy information based on the mothur algorithm. Alpha and Beta diversity analysis was performed using Qiime and displayed using R software (version 2.15.3). Redundancy analysis was performed using R software.
Method for detecting yield of SCFAs
SCFAs yields, including acetic acid, propionic acid and butyric acid, were analyzed by using Gas Chromatography (GC). The supernatant of 1mL of fermentation broth was transferred to a new Eppendorf tube. About 250 μ L HCl and 1mL ether were added to the supernatant, 1mM (final concentration) 2-methylbutyrate was used as an internal standard, and the tube was vortexed for 3 minutes. The organic component of the upper layer was collected and dehydrated with anhydrous sodium sulfate, and the supernatant was collected and passed through a filter of 0.22 μm pore size. SCFAs were analyzed by GC (Agilent-7890A, Santa Clara, Calif., USA) equipped with HP-INNOWAX chromatography columns. The oven temperature was 60 ℃ and increased to 190 ℃ over 4 minutes. The injector temperature was set at 220 deg.C and the detector temperature was set at 250 deg.C. mu.L of the sample was injected into the GC instrument at a split ratio of 1:20, with nitrogen as the carrier gas and a flow rate of 1.5 mL/min. The acetic, propionic and butyric acid contents were calculated according to the internal standard method.
Method for detecting degradation of gellan gum oligosaccharide
Thin Layer Chromatography (TLC) was used to assess degradation of gellan oligosaccharides. The supernatant of the fermentation broth (2. mu.L) was spotted onto a 10X 10cm thin-layer chromatography plate (Silica gel 60F254, Merck KGaA), which was then transferred to a forced air drying oven and dried at 60 ℃ for 3 minutes. The thin layer chromatography plate was developed in a developing solvent of n-propanol/water (7:3, v/v) for 100 min. The thin layer chromatography plate was then transferred to a forced air drying oven and dried at 60 ℃ for 3 minutes. The dried thin layer chromatography plate was immersed in a rehmannia phenol reagent (900mg of rehmannia phenol, 25mL of water, 375mL of ethanol, 50mL of concentrated sulfuric acid) for staining and immediately heated at 105 ℃ for 4 minutes.
Example 1: preparation of gellan gum oligosaccharide
The method comprises the following specific steps:
(1) adding 10g of gellan gum into 1L of deionized water, and continuously stirring to uniformly mix the gellan gum; adding HCl into the reaction system to make the final HCl concentration reach 0.4M; the hydrolysis system was hydrolyzed in a water bath at 80 ℃ for 36 hours with stirring.
(2) And (3) adjusting the pH value of the system hydrolyzed in the step (1) to 7.0 by using 5-10M NaOH, and enabling the solution to pass through a microporous filter membrane (the diameter is 0.45 mu M). The supernatant was concentrated to 1/12 of the original volume by using a rotary evaporator to give a concentrated solution.
(3) Adding 95% ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 70-75%, and standing the mixture for more than 6 hours; the solution was centrifuged at 8000r/min for 20 minutes to discard the precipitate.
(4) Adding absolute ethyl alcohol into the supernatant to enable the final concentration of the ethyl alcohol to reach 85-90%, standing for more than 6 hours, and centrifuging the solution at 8000r/min for 20 minutes to obtain a precipitate.
(5) The oligosaccharide precipitate was dialyzed through a 100-500Da dialysis bag to remove the salt and lyophilized in a vacuum freeze-dryer (see FIG. 1) to prepare gellan gum oligosaccharides.
Example 2: monosaccharide composition analysis and molecular weight analysis of novel gellan gum oligosaccharide
The method comprises the following specific steps:
(1) the gellan gum oligosaccharide prepared in example 1 was subjected to monosaccharide composition analysis, and the results were as follows:
the result shows that the monosaccharide composition of the novel gellan gum oligosaccharide is glucose, rhamnose and glucuronic acid, and the molar ratio is (4-6) - (1.2-1.9) - (2.4-3.6).
(2) The molecular weight of the novel gellan gum oligosaccharide was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and the results were as follows:
the results showed that the gellan gum oligosaccharides contained peaks (see FIG. 2) having M/z of 401,547,563,709,925,1071([ M +2Na ] +) and 379,525,541,687([ M + Na ] +) corresponding to the gellan gum oligosaccharides having a molecular weight of 357,503,665,881,1027, respectively, corresponding to the polymerization Degrees (DP)2,3,4,5,6 of the gellan gum oligosaccharides, respectively.
The results show that the DP of the gellan gum oligosaccharide is 2-6.
Example 3: application of novel gellan gum oligosaccharide as prebiotics
The method comprises the following specific steps:
1. in-vitro static fecal strain fermentation method of gellan gum oligosaccharide
(1) Fresh human feces are obtained from three healthy donors (one male and two females, age 35-50 years, BMI 24.60-29.24 kg/m2) Collecting with sterile feces collecting tube, placing into anaerobic gas generating bag, and placing on ice. Healthy donors do not take probiotics or prebiotics for at least two months prior to the donation of the stool sample;
(2) the fresh stools of the three persons were each diluted with sterilized 0.1M PBS buffer to obtain 10% (w/v) stool dilutions, and were filtered through four layers of gauze sponges under aseptic conditions to remove food residues, to obtain stool slurry;
(3) three human feces slurries were inoculated into the basal nutrient medium at a ratio of 10% (v/v) each as a blank.
(4) Adding the gellan gum oligosaccharide prepared in example 1 to a basic nutrient medium at a concentration of 0.3-0.5% (w/v), and inoculating a fecal slurry to the medium at a ratio of 10% (v/v);
as a control, inulin is added into a basic nutrient medium at a concentration of 0.3-0.5% (w/v), and excrement serous fluid is inoculated into the medium according to a proportion of 10% (v/v) to form a positive control group;
(5) each group is subjected to anaerobic incubation for 48 hours at 37 ℃ in three parallel containers to respectively prepare fermentation liquor; fermenting at 12000 r.min-1Centrifuging for 3 minutes, detecting flora changes in the bacterial sediment, and simultaneously detecting acetic acid, propionic acid and butyric acid changes in the supernatant; the results are shown in Table 1 and FIGS. 3 to 5; wherein total SCFAs means the total amount of acetic acid, propionic acid and butyric acid.
TABLE 1 yield of acetic acid, propionic acid, butyric acid and total SCFAs in vitro fecal bacteria fermentation
Wherein the different letters (a, b) indicate a statistically significant difference between inulin and gellan oligosaccharides (p < 0.05).
The results show that the novel gellan oligosaccharides significantly increased the production of acetic acid, butyric acid and total SCFAs compared to inulin in static fecal strain fermentation in vitro.
By analysis of the flora structure, see fig. 3 and 4; compared to inulin, the novel gellan oligosaccharides significantly increase the relative abundance of Lachnospiraceae (Lachnospiraceae), tannophilaceae (tannorellanaceae), clostridiaceae (fusobateriaceae), veillonellaceae (Erysipelotrichaceae) at the family level; meanwhile, the novel gellan gum oligosaccharide remarkably increases the relative abundance of the genus level of porphyromonas (paramacteroides) and lachnocristium, thereby improving the structure of fecal flora.
Notably, the novel gellan oligosaccharides significantly increased the relative abundance of butyric acid producing bacteria (see fig. 5), particularly of the genera Lachnospiraceae (Lachnospiraceae), Ruminococcaceae (Ruminococcaceae), lachnocristium, which corresponds to significantly increased butyric acid production.
Example 4: application of novel gellan gum oligosaccharide as prebiotics
The fermentation method of the gellan gum oligosaccharide in an in vitro gastrointestinal tract reactor comprises the following steps: the gastrointestinal tract reactor device adopts a novel simulated gastrointestinal tract reactor (the simulated gastrointestinal tract reactor is described in a patent No. ZL 201810069220.8) which is independently developed by a laboratory, and the structure condition, the control system and the functional characteristics of the gastrointestinal tract reactor verify the feasibility of dynamic fermentation.
The method comprises the following specific steps:
(1) a healthy donor (a male, age 45 years, BMI 27.86 kg/m) was collected using a sterile fecal collection tube2) Then putting the excrement into an anaerobic gas generating bag and putting on ice. Healthy donors do not take probiotics or prebiotics for at least two months prior to the donation of the stool sample;
(2) diluting fresh feces with sterilized 0.1M PBS to obtain 10% (w/v) feces, and filtering through four layers of gauze sponge under aseptic conditions to remove food residues to obtain feces slurry;
(3) preparing a gellan gum oligosaccharide culture medium: changing starch in basic fermentation medium to 5 g.L-1Gellan gum oligosaccharide (prepared in example 1);
(4) 160mL of basic fermentation medium (the total volume of the reactor is 230mL) is filled into an intestinal tract reactor (GSR), a pH electrode is calibrated by using pH calibration solutions of 4.0 and 6.8, then the intestinal tract reactor is sterilized for 20min at the temperature of 115 ℃, and when the intestinal tract reactor is naturally cooled to the room temperature, the excrement slurry prepared in the step (2) is inoculated into a super clean workbench in an inoculation amount of 10% (v/v);
(5) after inoculation, GSR is transferred to a fixed position, then a gastrointestinal tract reactor is communicated with a circulating water device, warm water is pumped into a space between a glass jacket and a flexible hose at regular time intervals, and intestinal tract wriggle simulation is realizedWarm water is added to keep the inner cavity of the model at the body temperature (37 ℃); the pH was set to 5.8 (simulating the pH of the proximal colon) and NaOH solution (0.5 mol. L.) was pumped in via a pH sensor in combination with-1) The pH value is automatically adjusted to about 5.8; the GSR was slowly aerated every 8h for 10min N2 to remove oxygen from the GSR to maintain the anaerobic state of the system.
(6) The fecal flora in GSR reached a stable stage after 16h fermentation, and then starved for 2h to deplete the carbon source (starch) in the medium, at which time the initial fermentation time (0h) was recorded; then at 2mL min-1The medium in the GSR was discharged at 40mL by a pump at a rate of 2 mL/min-1Pumping 40mL of the gellan gum oligosaccharide culture medium prepared in the step (3) into an experimental period;
every 12h, 40mL gellan gum oligosaccharide medium was fed and every 12h samples were taken, so that fermentation was continued for 48 h.
After the fermentation was completed, each of the obtained samples was 12000 r.min-1Centrifugation was carried out for 3 minutes, the bacterial pellet was used for detection of changes in flora, and the supernatant was used for detection of changes in acetate, propionate and butyrate. TLC was used to detect degradation of gellan gum oligosaccharides in the supernatant of the fermentation broth, and the results are shown in Table 2 and FIGS. 6-9. Wherein total SCFAs means the total amount of acetic acid, propionic acid and butyric acid.
TABLE 2 production of gellan gum oligosaccharides in gastrointestinal tract reactor for acetic acid, propionic acid, butyric acid and total SCFAs
Wherein different letters (a, b, c) indicate statistically significant differences between different fermentation times (p < 0.05).
The results show that in the in vitro gastrointestinal reactor, the novel gellan gum oligosaccharides are mainly degraded during 24h to 48h fermentation (see fig. 6), producing large amounts of acetic acid, propionic acid, butyric acid and total SCFAs (table 2), and significantly increasing NaOH consumption (see fig. 7).
In the fermentation process of 24 h-48 h, the novel gellan gum oligosaccharide obviously increases the relative abundance of Lachnospiraceae (Lachnospiraceae), tannaceae (tannorellaneae) and Ruminococcaceae (Ruminococcaceae) at the family level and the relative abundance of porphyromonas (Parabacteroides), UBA1819(Ruminococcaceae UBA1819) and Lachnospira (Lachnospira) at the genus level (see figure 8), thereby improving the structure of the fecal flora.
Notably, the novel gellan oligosaccharides significantly increased the relative abundance of butyric acid producing bacteria (see fig. 9), particularly the Ruminococcaceae (Ruminococcaceae), Lachnospiraceae (Lachnospiraceae) and Lachnospira (Lachnospira), which corresponds to significantly increased butyric acid production.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The gellan gum oligosaccharide is characterized in that a basic main chain structure of the gellan gum oligosaccharide comprises tetrasaccharide units, wherein the tetrasaccharide units are D-glucose, D-glucuronic acid and L-rhamnose, and the molar ratio of the D-glucose, the D-glucuronic acid and the L-rhamnose is (4-6): (1.2-1.9): (2.4-3.6).
2. The gellan oligosaccharide of claim 1, wherein the gellan oligosaccharide has a degree of polymerization in the range of 2 to 6.
3. A process for the preparation of gellan oligosaccharide according to claim 1 or 2, wherein the process comprises the steps of:
(1) adding gellan gum into water, stirring, uniformly mixing, adding an HCl solution to enable the final concentration of HCl to be 0.4-0.5M, obtaining a reaction system, and stirring and hydrolyzing the reaction system in a water bath at 70-80 ℃ for at least 36 hours;
(2) adjusting the pH value of the reaction system after hydrolysis in the step (1) to 7.0, passing the solution through a microporous filter membrane, and concentrating the supernatant to 1/10-1/15 of the original volume by using a rotary evaporator to obtain a concentrated solution;
(3) adding ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 70-75% to obtain a mixture; standing the mixture for 6-10 hours, centrifuging the solution, and removing the precipitate;
(4) adding ethanol into the supernatant obtained in the step (3) to enable the final concentration of the ethanol to reach 85-90%, and standing for 6-10 hours to obtain oligosaccharide precipitate;
(5) the oligosaccharide precipitate is dialyzed by a dialysis bag of 100-500Da to remove the salt and dried to obtain the gellan gum oligosaccharide.
4. A product for increasing the content of short chain fatty acids in metabolites of the gut flora, comprising gellan gum oligosaccharides according to claim 1 or 2.
5. The product of claim 4, wherein the short chain fatty acid is one or more of acetic acid, propionic acid, butyric acid.
6. The product of claim 4 or 5, wherein the product comprises a food, pharmaceutical or nutraceutical product.
7. A product for increasing the abundance of butyric acid producing bacteria in the gut, wherein the product comprises gellan oligosaccharide according to claim 1 or 2.
8. The product of claim 7, wherein said butyric acid producing bacteria comprise the Ruminococcaceae family (Ruminococcaceae), Lachnospiraceae family (Lachnospiraceae), lachnocristium genus and Lachnospira genus (Lachnospira).
9. The product of claim 7 or 8, wherein the product comprises a food, pharmaceutical or nutraceutical product.
10. Use of a gellan oligosaccharide according to claim 1 or claim 2 or the method according to claim 3 for increasing the content of short chain fatty acids in gut flora metabolites, or for increasing the abundance of butyric acid producing bacteria in the gut, or for the manufacture of a product capable of increasing the content of short chain fatty acids in gut flora metabolites, or for the manufacture of a product capable of increasing the abundance of butyric acid producing bacteria in the gut.
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