CN114128889A - Konjak oligosaccharide, preparation method and application thereof - Google Patents

Konjak oligosaccharide, preparation method and application thereof Download PDF

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CN114128889A
CN114128889A CN202110068327.2A CN202110068327A CN114128889A CN 114128889 A CN114128889 A CN 114128889A CN 202110068327 A CN202110068327 A CN 202110068327A CN 114128889 A CN114128889 A CN 114128889A
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kgmos
konjac
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方建平
董群
杜敏刚
刘路
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Shanghai Xingtang Biotechnology Co ltd
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Abstract

The invention relates to konjac oligosaccharide, a preparation method and application thereof. The weight average molecular weight of the konjac oligosaccharide KGMOS is 2200-4000Da, the molecular weight distribution is 1.01-1.4, and the polymerization degree dp ranges from 18-24. The konjac oligosaccharide is prepared by degrading konjac polysaccharide and purifying the konjac polysaccharide through centrifugal dialysis. Experimental results show that the konjac oligosaccharide KGMOS has the effect of improving the health state of intestinal tracts. Compared with the konjac polysaccharide with high molecular weight and the konjac oligosaccharide with ultra-low molecular weight, the konjac oligosaccharide KGMOS with low molecular weight has the function of more remarkably improving the structure of intestinal flora. In addition, the experimental result also shows that the konjac oligosaccharide KGMOS can enhance acetylation of histones H3 and H4 by promoting the production of the colonic butyric acid, improve the expression of the Muc2 gene, finally increase the content of colonic mucosa barrier protection molecule mucin and improve the mucosa barrier function.

Description

Konjak oligosaccharide, preparation method and application thereof
Technical Field
The invention relates to the technical field of konjac oligosaccharides and application thereof, in particular to konjac oligosaccharide KGMOS obtained by degrading, separating and purifying konjac polysaccharide, a preparation method and application thereof.
Background
The intestinal tract is extremely important for maintaining human health. More and more studies have shown that the intestinal flora has a crucial role in health and disease, including the regulation of immune and inflammatory responses and the promotion of nutrient digestion (massowski, k.m., & Mackay, c.r. nat Immunol 2011,12, 5-9; Cardarelli, h.r. et al, Benef Microbes 2016,7, 271-285.). First, the intestinal epithelial cells serve as the primary defense barrier against invading pathogens (Garrett, w.s., et al, Cell 2010,140, 859-870). With the increasing incidence of metabolic diseases such as obesity and diabetes, the improvement effect of dietary fibers (such as KGM) on intestinal health has attracted more and more research attention in recent years (Johansson, M.E., et al, Cell Mol Life Sci 2011,68, 3635-.
Konjac Glycan (KGM) is a neutral polysaccharide extracted from the bulb of Amorphophallus Konjac (Amorphophalus Konjac), and its repeating unit is composed of- (1 → 4) -linked D-glucose and D-mannose. The ratio of monosaccharides in KGM, the molecular weight, and the modification of the acetylation of sugar residues are related to the production area, species and mode (Gomez, B., et al., J Agr Food Chem 2017,65, 2019-2031). KGM has found widespread use in the food, chemical, cosmetic and pharmaceutical industries over the last two decades.
Research shows that KGM is beneficial to human health, and mainly plays a role in regulating intestinal flora, losing weight, managing diabetes, reducing cancer risk and the like. Also, applications in drug delivery and tissue engineering are receiving attention. However, the characteristics of high molecular weight (ranging from 200 to 2000kDa), high viscosity and low solubility of natural KGM limit its application (Tester, R.F., Al-Ghazzewi, F.H., J Sci Food Agr 2016,96, 3283-3291).
KGM is degraded enzymatically or by physicochemical methods to give low molecular weight oligosaccharides, thereby significantly reducing its viscosity and increasing water solubility. KGM, and in particular KGM oligosaccharides, have been reported to have significant promoting effects on intestinal health, mainly including prebiotic effects, antioxidant activity and enhancing immune function (Jiang, m., et al, J Zhejiang Univ-Sc B2018, 19, 505-514). KGM also has therapeutic potential for the treatment of inflammatory bowel disease and ulcerative colitis (Zhang, L., et al., Food Funct 2019,10, 1928-.
Research shows that the health promoting effect of KGM oligosaccharide is closely related to its molecular structure and purity. It has been reported that ultra-low molecular weight KGM oligosaccharides (dp 2-4) can improve the intestinal environment by enhancing probiotics (Jiang, M., et al, J Zhejiang Univ-Sc B2018, 19, 505-514.).
However, the literature has not conducted extensive studies on KGM oligosaccharides with dp18 to 24. At the same time, there is very little information in the current literature about the production process and purity of KGM oligosaccharides. In addition, there is increasing evidence that the molecular weight of KGM used in previous studies is more or less heterogeneous and less than 90% pure. This severely affected KGM oligosaccharide-related functional studies (Jiang, M., et al, J Zhejiang Univ-Sc B2018, 19, 505-.
Therefore, the KGM oligosaccharide with dp18-24 and the application thereof need to be studied in depth.
Disclosure of Invention
The invention carries out controlled degradation on natural KGM to obtain the konjac oligosaccharide with a specific molecular weight range, thereby overcoming the problems encountered in application.
The invention firstly degrades konjak polysaccharide by acid to obtain konjak oligosaccharide KGMOS (dp 18-24) with molecular weight of 2200-4000Da, which is analyzed into uniform oligosaccharide by HPGPC and analyzed by monosaccharide composition,13C NMR、1h NMR analysis shows that the composition of the repeating units of the oligosaccharide is consistent with that reported in the prior literature, and the oligosaccharide consists of mannose and glucose in a molar ratio of about 1.5: 1.
The invention firstly researches the effect of konjac oligosaccharide KGMOS (dp 18-24) with the molecular weight of 2200-.
The invention firstly obtains the konjac oligosaccharide KGMOS with uniform structure, and further evaluates the improvement effect on colon function in C57BL/6 mice. Meanwhile, the invention discloses a new action mechanism of the konjac oligosaccharide, enhances acetylation of histones H3 and H4 by promoting the production of the colonic butyric acid, increases the expression of the Muc2 gene, and finally improves the colonic mucin content so as to enhance the colonic mucosa barrier protection function. The invention is beneficial to designing and developing health care products and medicines for treating intestinal diseases based on KGM.
The invention provides a konjac oligosaccharide KGMOS, wherein the weight average molecular weight is 2200-4000Da, the molecular weight distribution is 1.01-1.4, and the polymerization degree (dp) range is 18-24.
In another aspect, the present invention provides a method for preparing the konjac oligosaccharide KGMOS, comprising the steps of:
a) degrading konjac polysaccharide KGM in the presence of acid;
b) centrifuging the solution obtained in the step a), taking supernatant, and evaporating to dryness;
c) dissolving the product obtained in the step b) in deionized water, and centrifuging;
d) collecting the supernatant obtained after the centrifugation in the step c), and dialyzing the supernatant in a dialysis bag with the molecular weight cut-off of 3500Da by using ultrapure water to obtain a final product KGMOS.
Fig. 6 shows a process flow diagram for preparing the konjac oligosaccharide KGMOS of the present invention according to one embodiment of the present invention, but the present invention is not limited thereto. For example, the residue after centrifugation may be recycled for further degradation.
In one embodiment of the present invention, the step a) is performed as follows: dissolving konjac polysaccharide KGM in an acid solution, and degrading at 60-100 ℃.
The konjac polysaccharide KGM may be prepared according to a method known in the art without particular limitation. For example, it can be prepared as follows: (1) dispersing food-grade konjac flour in 40% ethanol water to dissolve impurities, and then carrying out solid-liquid separation to obtain a first precipitate; (2) re-dispersing the first precipitate with water to obtain hydrosol, and performing solid-liquid separation to obtain a first supernatant; (3) adding ethanol into the first supernatant until the final concentration of the ethanol is 30%, and then carrying out solid-liquid separation to obtain a second precipitate; (4) dispersing the second precipitate in 70% ethanol water solution, and performing solid-liquid separation to obtain a third precipitate; (4) and dispersing the third precipitate in 100% ethanol, performing solid-liquid separation to obtain a fourth precipitate, and drying to obtain the konjac polysaccharide KGM. The present invention is not limited thereto.
In one embodiment of the present invention, the step b) is performed as follows: centrifuging the solution obtained in the step a) at the rotating speed of 6000-10000rpm, and taking the supernatant to evaporate to dryness.
In one embodiment of the present invention, said step c) is carried out by dissolving the residue obtained in step b) by evaporation with deionized water and centrifuging at 6000-.
In one embodiment of the present invention, said step d) is performed as follows: collecting the supernatant obtained after the centrifugation in the step c), putting into a dialysis bag with the molecular weight cut-off of 3500Da, dialyzing in ultrapure water, and freeze-drying to obtain the final product of the konjac oligosaccharide KGMOS.
In one embodiment of the invention, the acid may be selected from: trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, in particular trifluoroacetic acid.
In one embodiment of the present invention, in the step a), the temperature of the degradation reaction may be 60 to 100 ℃, preferably 70 to 98 ℃, more preferably 80 to 95 ℃, for example 85 ℃ and 90 ℃. The time for the degradation reaction may be 1 hour or more, 1.5 hours or more, 2 hours or more, for example, 2 to 4 hours. In particular, degradation may be carried out at 90 ℃ for 2 to 4 hours.
In one embodiment of the present invention, in the steps b) and c), the number of centrifugal revolutions may be 6000 to 10000 revolutions, for example 7000 to 9800 revolutions.
In one embodiment of the present invention, in the steps b) and c), the centrifugation time may be 10 minutes or more, for example, 10 to 40 minutes.
In one embodiment of the present invention, in the step d), the dialysis time may be 1 day or more, for example, 1 to 5 days.
The present invention evaluated the effect of the konjac oligosaccharide KGMOS of the present invention on improvement of colon function in C57BL/6 mice. Experimental results show that the konjac oligosaccharide KGMOS has the effects of improving intestinal flora and maintaining intestinal health. The experimental result also shows that the konjac oligosaccharide can enhance acetylation of histones H3 and H4 by promoting the production of the colonic butyric acid, increase the expression of the Muc2 gene and finally improve the colonic mucin content so as to enhance the colonic mucosa barrier protection function. Therefore, the invention is helpful to design and develop health products and medicines for treating intestinal diseases based on KGM.
Therefore, a further aspect of the present invention relates to a pharmaceutical or food composition comprising the konjac oligosaccharide KGMOS according to the present invention. The medicament may also comprise other pharmaceutically active ingredients and/or pharmaceutically acceptable carriers. The food composition may also comprise other edible ingredients. The pharmaceutical or food composition is prepared by adding the konjac oligosaccharide KGMOS of the present invention.
In still another aspect of the present invention, there is provided a use of the konjac oligosaccharide KGMOS of the present invention, the use being selected from the group consisting of: the application of the composition in preparing medicines or foods for promoting the synthesis of butyric acid in colon, the application in preparing medicines or foods for enhancing the acetylation of histones H3 and H4 of mucosal epithelial cells, the application in preparing medicines or foods for increasing the content of colonic mucin, the application in preparing medicines or foods for improving the barrier function of mucosa, and the application in preparing medicines or foods for improving intestinal flora and maintaining intestinal health.
All features or conditions defined herein as numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have covered and specifically disclosed all possible subranges and individual numerical values within the ranges, particularly integer numerical values. For example, a description of a range of "1 to 8" should be considered to have specifically disclosed all subranges such as 1 to 7,2 to 8, 2 to 6,3 to 6,4 to 8,3 to 8, and so on, particularly subranges bounded by all integer values, and should be considered to have specifically disclosed individual values such as 1, 2, 3, 4, 5, 6,7, 8, and so on, within the range. Unless otherwise indicated, the foregoing explanatory methods apply to all matters contained in the entire disclosure, whether broad or not.
If an amount or other value or parameter is expressed as a range, preferred range, or a list of upper and lower limits, then it is to be understood that all ranges subsumed therein as either the upper or preferred value for that range and the lower or preferred value for that range are specifically disclosed herein, regardless of whether ranges are separately disclosed. Further, when a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range.
In this context, numerical values should be understood to have the precision of the number of significant digits of the value, provided that the object of the invention is achieved. For example, the number 40.0 should be understood to cover a range from 39.50 to 40.49.
Unless expressly stated otherwise, numerical values throughout this specification represent approximate measures or limitations to the extent that such deviations from the given values, as well as embodiments having approximately the stated values and having the exact values stated, are included. Other than in the operating examples provided at the end of the detailed description, all numbers expressing quantities or conditions of parameters (e.g., quantities or conditions) used in the specification (including the appended claims) are to be understood as being modified in all instances by the term "about" whether or not "about" actually appears before the number. "about" means that the numerical value so stated is allowed to be somewhat imprecise (with some approach to exactness in that value; about or reasonably close to that value; approximately). As used herein, "about" refers to at least variations that can be produced by ordinary methods of measuring and using such parameters, provided that the imprecision provided by "about" is not otherwise understood in the art with this ordinary meaning. For example, "about" can include less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5% variation, and in some aspects, less than or equal to 0.1% variation.
The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.
Drawings
In FIG. 1, A is an HPLC chromatogram of the konjac oligosaccharide KGMOS of example 1 of the present invention; b is a monosaccharide composition analysis HPLC (high Performance liquid chromatography) map of the konjac oligosaccharide KGMOS in the embodiment 1 of the invention; c is Konjac oligosaccharide KGMOS of the invention example 11H NMR chart; d is Konjac oligosaccharide KGMOS of the invention example 113C NMR chart.
FIG. 2 shows the effect of the konjac oligosaccharide KGMOS of example 1 of the invention on mouse body weight, feeding and drinking volume and colon length.
FIG. 3 shows the effect of the konjac oligosaccharide KGMOS of example 1 of the present invention on the intestinal flora of mice.
FIG. 4 shows the effect of the konjac oligosaccharide KGMOS of example 1 of the present invention on the mouse colonic mucus barrier.
FIG. 5 shows the effect of the konjac oligosaccharide KGMOS of example 1 of the invention on the acetylation of short-chain fatty acids and histones H3 and H4 in mouse feces.
Fig. 6 is a process flow diagram for preparing konjac oligosaccharide KGMOS according to an embodiment of the present invention.
Detailed Description
The invention is further illustrated below with reference to specific examples. However, these examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the manufacturer.
The instrument used in the examples of the present invention is as follows:
high performance liquid chromatography (GPC-MALLS-RI): US Waters 2695 connects an octagonal laser detector (MALLS, US Wyatt) and a differential refraction detector (RI, US Waters)
Nuclear magnetic resonance spectrometer: bruker AVANCEIII USA
Gas chromatograph: japan Shimadzu GC-2014
A picture scanner: hungary Pannoramic Scanner
A sequencer: illumina Miseq platform of USA
Real-time quantitative (RT-qPCR) instrument: applied Biosystems, USA
Food-grade konjac flour (purified super grade, 90%, cat # 03-TK) was purchased from qingjiang konjac products limited, wuhan.
Example 1: preparation of konjac oligosaccharide KGMOS
Dispersing food-grade rhizoma Amorphophalli powder in 40% ethanol water solution at 10% w/v, heating to 70 deg.C, and stirring to dissolve impurities. After 4 hours, filtering by using a No. 3 sand core funnel, re-suspending and dispersing filter residue precipitate by using 10 times volume of water, heating to 70 ℃ overnight (about 16 hours) to obtain hydrosol, cooling to room temperature, centrifuging for 30 minutes at 9000 r/min, discarding the precipitate, taking supernatant, adding ethanol to the final concentration of ethanol of 30%, stirring, standing for 4 hours at room temperature, centrifuging for 30 minutes at 9000 r/min again to obtain precipitate, then respectively washing and centrifuging once by using 70% ethanol aqueous solution and 100% ethanol, and finally obtaining a precipitate sample to obtain the konjac glucomannan KGM by vacuum drying.
KGM was dispersed in 0.2M aqueous trifluoroacetic acid (TFA) at a concentration of 1% w/w and incubated for 2 hours at 90 ℃ with stirring. The solution was centrifuged at 9000g for 20 minutes and the supernatant was dried at 60 ℃ under reduced pressure. The resulting product was dissolved in distilled water and centrifuged at 9000g for 20 minutes. And (3) selecting a dialysis bag with the molecular weight cut-off of 3500Da, dialyzing the supernatant in Milli-Q water for 3 days, and freeze-drying to obtain a KGM degradation product KGMOS.
And (3) measuring physicochemical properties:
chromatographic conditions are as follows: waters e2695, chromatography column, Japan Tosoh TSK-GEL G2500PWXL(7 μm, 7.8mm x 30cm), column temperature 35 ℃; a detector: an octagonal laser detector (LS), a refractive index detector (RI, 35 ℃). The wavelength of the light source of the laser detector is selected from 623.8 nm. The refractive index increment (dn/dc) of the polysaccharide in solution was calculated as 0.146 mL/g.
Sample preparation: weighing 5mg of sample, dissolving in 1mL of mobile phase, wherein the mobile phase is 0.05mol/L NaH2PO4And 0.15mol/L of NaNO3Solution (pH 7, 0.02 wt% sodium azide) was prepared as a 5mg/mL solution. Centrifuging at 12000 Xg for 20min, collecting supernatant, filtering with 0.22 μm water phase microporous membrane, and analyzing by HPGPC-MALLS-RI. The GPC chromatogram obtained is shown in A of FIG. 1.
Data processing: light scattering data was collected and analyzed using Astra (version 6.1.1, Wyatt Technology, Santa Barbara, CA) data analysis software to calculate molecular weight.
Through calculation, the konjac oligosaccharide KGMOS is a uniform oligosaccharide, the molecular weight is 2200-4000Da, the weight average molecular weight Mw is 3200Da, the number average molecular weight Mn is 2900Da, the molecular weight distribution (PDI) ═ Mw/Mn) is about 1.1, and the polymerization degree dp is 18-24.
Analysis of monosaccharide composition:
1) a1 mg sample of KGMOS oligosaccharide was dissolved in 0.5ml of water and 0.5ml of 4mol/L aqueous TFA was added, hydrolyzed at 110 ℃ for 4h, cooled and then methanol was added and the TFA was evaporated under reduced pressure.
2) 100mL of 0.3mol/L NaOH aqueous solution was added to dissolve the hydrolysate, and 100mL of 0.5 mol/L1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution (0.4355g/5mL) was added thereto and mixed by vortexing.
4) Reacting in 70 deg.C water bath for 100min, taking out, standing for 10min, and cooling to room temperature.
5) Adding 100mL of 0.3mol/L HCl aqueous solution for neutralization, adding water to 1mL, adding an equal volume of chloroform, vortexing, and standing. The upper aqueous phase was extracted and the extraction was repeated 2 more times.
6) The solution was filtered through a 0.22mm microporous membrane and then filled into a liquid bottle for HPLC analysis.
Liquid chromatography conditions: a linear gradient elution procedure was used (buffer A: 15 vol.% acetonitrile and 85 vol.% 0.05M KH)2PO4An aqueous solution; and (3) buffer solution B: 40% by volume acetonitrile and 60% by volume 0.05M KH2PO4Aqueous solution, pH 6.7), detection wavelength 254 nm. And determining the monosaccharide composition and content of the KGMOS by comparing the retention time and peak area of the KGMOS with the monosaccharide standard.
The HPLC profile of sugar composition analysis is shown in B of FIG. 1. As can be seen, the KGMOS repeat unit consists of mannose and glucose in a molar ratio of about 1.5: 1.
And (3) identifying a chemical structure:
analysis was performed using nuclear magnetic resonance.
Taking a certain amount of KGMOS at room temperature, dissolving the KGMOS with 500 mu l of heavy water, and obtaining the KGMOS by using a Bruker nuclear magnetic resonance instrument with the frequency of 500MHz1H NMR and13c NMR spectra are shown in C and D of FIG. 1, respectively.
Through the analysis of the composition of the monosaccharide,13C NMR、1h NMR analysis shows that the composition of the repeating unit of the oligosaccharide is consistent with the report in the prior art.
In the field of carbohydrate chemistry, even if the repeating units are the same, their steric structure may be different due to the difference in molecular weight, and such a change in steric structure may result in a significant change in activity. KGMOS is different from the reported konjac oligosaccharides with the molecular weight range of 100-6000Da (polymerization degree of 1-37) and the polymerization degree of 2-4 (Gomez, B., et al, J Agr Food Chem 2017,65, 2019-Sc 2031; Jiang, M., et al, J ZHEjiang Univ-Sc B2018, 19,505-514), and the konjac oligosaccharide KGMOS in the invention is considered to be a novel oligosaccharide compound. Research shows that the molecular weight of oligosaccharide can affect the viscosity, solution conformation and other physical and chemical properties, and further cause the difference of biological activity.
Example 2: effect of konjak oligosaccharide KGMOS on Colon health status of C57BL/6 mice
The following experiment was performed using the konjac oligosaccharide KGMOS prepared in example 1.
2.1 animal feeding
C57BL/6 mice (6 to 8 weeks) were randomized into three groups (n-15/group) and administered orally daily. Physiological saline (control group), low dose (10 mg/kg/day, KGMOS10 group) and high dose (100 mg/kg/day, KGMOS100 group) were administered separately for eight weeks. Food and water intake was recorded every 5 days and mice were weighed weekly.
After the end of the dosing period, all mice were passed over CO2Suffocation and sacrifice. Blood samples were collected and separated by centrifugation (4000g, 5 min) to obtain serum. Serum samples were stored at-80 ℃ for further analysis. SmallAfter sacrifice, colon tissue was rapidly dissected. After measuring colon length, tissues were snap frozen in liquid nitrogen and stored at-80 ℃ for biochemical examination or histological analysis after fixation with Carnoy fixative. At the same time, feces were collected for intestinal flora analysis.
The results of the experiment are shown in fig. 2, where a shows the effect of administration of KGMOS on feeding, B shows the effect of administration of KGMOS on water intake, C shows the effect of administration of KGMOS on body weight, and D shows the effect of administration of KGMOS on intestinal length.
As can be seen from fig. 2, the food consumption of mice in both administration groups was significantly reduced from 3 weeks after the administration (a in fig. 2), but the water intake was significantly increased (B in fig. 2) compared to the control group. During the administration period, the body weight of mice was gradually increased in all treatment groups, but oral KGMOS decreased the body weight gain rate of mice and exhibited dose dependence compared to the control group (C of fig. 2). Meanwhile, compared with the control group, the length of the colon of the mice in the KGMOS100 group is remarkably increased (D in fig. 2), which indicates that the KGMOS has the functions of promoting the repair of the intestinal tract tissue and maintaining the health of the intestinal tract tissue.
2.2 analysis of microbial composition
Bacterial DNA was extracted from control, KGMOS low and high dose group treated colonic fecal samples using the QIAGEN QIAamp DNA pool Mini Kit (QIAGEN, germany) to analyze the effect of KGMOS on intestinal flora.
The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3').
The amplification steps are as follows: at 95 ℃ for 2 min, followed by denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, with 25 cycles, and final extension at 72 ℃ for 5 min. Amplicons were purified on the Illumina MiSeq platform, quantified and paired-end sequenced. The resulting original fastq file was de-compiled and quality screened using QIIME (version 1.17). Operational Taxonomic Units (OTUs) were clustered using UQIIME (version 7.1, http:// drive5.com/uparse /) (97% similarity cut-off). The chimeric sequences were identified and removed using UCHIME. OTUs were taxonomically classified by 70% sequence homology using Ribosol Database Project (RDP, http:// RDP. cme. msu. edu /) in combination with the silva (SSU123)16S rRNA Database.
The results are shown in FIG. 3, wherein A shows a Venn diagram of OTU taxonomic classification, B shows PCA analysis results, C shows portal relative abundance analysis results, and D shows genus relative abundance analysis results.
The Venn plot shows that 805 OTUs were shared among the three groups, while the number of unique OTUs in the control, low dose and high dose groups were 830, 728 and 510 (a of fig. 3), respectively.
The results of the PCA analysis showed that different doses of KGMOS treatment resulted in significant changes in the overall floral structural composition (FIG. 3B).
As can be seen from C of FIG. 3, at the phylum level, the dominant phyla are Bacteroides, Mycobacteria, Proteobacteria and Microbacterium verruculoides (Verrucomicrobia). KGMOS significantly reduced the abundance of bacteroides, and was dose-dependent compared to the control group. However, both the low-dose and high-dose KGMOS groups significantly increased the abundance of firmicutes from 18.6% to 28.0% and 28.8%, respectively, compared to the control group. In addition, the KGMOS dose at 10mg/kg significantly increased the abundance of proteus and apobacter (deferobacteres) compared to the control group, and the relative level of verrucomicrobia in the intestinal tract of mice of this dose group was significantly higher than that of the control group and the high dose group (C of fig. 3).
As can be seen from D of fig. 3, at the genus level, the high dose group significantly increased the relative abundance of helicobacter pylori, bordetella, and klebsiella, compared to the control and KGMOS10 groups. Both treatment concentrations significantly reduced the relative levels of non-culturable bacteroides (unsaluted _ bacteroides), bacteroides (Alloprevotella) and paracasella (parasitella) bacteria, increasing the abundance of Desulfovibrio (Desulfovibrio), dithibacterium (Oscillibacter) and anaerobacter (anaerobotryuncus) bacteria, but did not affect the abundance of xenobacter (Alistipes) and rikogaceae _ RC9_ gut group (rikennellaceae _ RC9_ gut _ group) bacteria. In addition, low-dose KGMOS treatment increased the relative proportion of alcanha (Akkermansia) bacteria.
The literature reports show that ultra-low molecular weight KGM oligosaccharides (dp 2-6) increase the relative abundance of Akkermansia, Bacteroides, [ Prevotella ] and Bifidobacterium, but decrease the relative abundance of Helicobacterae _ unclassified and S24-7_ unclassified (Zheng, J., et al., J agricultural Food Chem 2018,66, 5821-. High molecular weight konjac polysaccharides can significantly increase the relative abundance of the beneficial bacteria Megasphaera elsdenii, but decrease the relative abundance of the deleterious bacteria populations Alistipes, Alloprevotella, Bacteroides acidifaciens and Parabacteroides goldsteini (Kang, y., et al., Int J Obes (Lond)2019,43, 1631-. In addition, it has been shown that feeding high molecular weight konjac polysaccharide increases the relative proportions of Bateroides and Akkermansia in the mouse gut, but decreases the abundance of Firmicutes and mucirillum (Zhai, x., et al., J Agric Food Chem 2018,66, 12706-. Compared with the literature report, the KGMOS can also increase the proportion of the intestinal protective bacteria Akkermansia. Different from the effects of the konjak oligosaccharide and the konjak polysaccharide with ultra-low molecular weight reported in the literature, the KGMOS treatment can reduce the abundance of bacteroides in intestinal microorganisms. Bacteroides enterobacter is involved in carbohydrate metabolism and thereby increases the release of energy from dietary fiber available to the host. Given that KGMOS treatment can significantly reduce mouse body weight, KGMOS is presumed to reduce energy gain in the host by reducing the abundance of bacteroides intestinalis, suggesting that KGMOS has potential as an anti-obesity drug.
Unlike the efficacy of the high molecular weight konjac polysaccharides reported in the literature, the above data indicate that KGMOS with a dp18-24 significantly increases the abundance of bacteria in Firmicutes of the phylum Firmicutes that produce butyric acid, such as the family Ruminoccaceae. This will help the gut micro-organisms to metabolize dietary fibre (including KGMOS itself) to produce butyric acid. In addition, KGMOS can increase the relative abundance of the bacteria Mucispirillum with gut protective effect (preventing salmonella infection).
The above data indicate that KGMOS treatment can reduce the abundance of bacteria from the phylum Spirochaetes, such as the genus Treponema _2, an important class of pathogens in the family spirochaeteaceae, and can lead to many diseases with global epidemics. This suggests that KGMOS may play a positive role in reducing pathogen susceptibility of the host.
In conclusion, compared with the ultra-low molecular weight konjac oligosaccharides and the high molecular weight konjac polysaccharides, the KGMOS with the polymerization degree of dp18-24 can further optimize the intestinal flora structure, increase the abundance of beneficial flora and reduce the relative proportion of harmful flora, thereby remarkably improving the health state of intestinal tracts, particularly colon.
2.3 histopathological analysis
The intestinal mucus layer has the functions of shielding intestinal wall and preventing harmful components and pathogenic microorganisms from damaging organisms. While the mucus layer is mainly formed by mucin continuously secreted by goblet cells. To evaluate the effect of KGMOS on intestinal function, colon tissues were fixed (n-8 per group), dehydrated with ethanol, and embedded in paraffin. Tissue sections (10 μm) were stained with alcian blue to specifically recognize mucin particles, and then scanned for photographs and goblet cell counts recorded. Three different positions 100 μm apart were selected, goblet cells were counted and the average was calculated.
The results are shown in A-C of FIG. 4, in which A shows the results of Alisin blue staining, B shows the results of mucin particle area quantification, and C shows the results of goblet cell number quantification.
Colonic mucus staining results indicated that mucin levels were significantly higher in the high dose group than in the low dose group and control mice (fig. 4 a and B), indicating that high dose KGMOS promoted mucin secretion, whereas goblet cell numbers were similar in the three treatment groups (fig. 4C). Since mucin is the major component of colonic mucus, it isolates intestinal contents (including food, bacteria, fungi, viruses, etc.) and host intestinal epithelial cells. Plays an important role in maintaining intestinal mucosa homeostasis. Our data indicate that KGMOS treatment can significantly increase colonic mucin content, thereby increasing colonic barrier function and improving intestinal health.
2.4 Real-Time quantitative polymerase chain reaction (Real-Time qPCR) detection of the Effect of KGMOS on the transcriptional level of Key proteins that control the secretion of intestinal mucus in mice
Mouse colon tissue blocks of control group and KGMOS-treated group were subjected to total RNA extraction according to the Trizol reagent (Invitrogen USA) protocol, then reverse-transcribed into cDNA using PrimeScript RT Master Mix (Takara, Japan), and then used TB GreenTM Premix Ex TaqTM(Takara, Japan) according toInstructions Real-Time qPCR assays were run on ABI 7500 (Applied Biosystems, usa). The brief steps are as follows:
primers were designed using Primer-BLAST from NCBI, as shown in the following table.
Figure BDA0002905023300000101
Figure BDA0002905023300000111
The reaction mixture (volume 20. mu.l) contained 10. mu.l of 2 XTB Green Premix Ex Taq, 200nM each of the forward and reverse primers, and 0.2. mu.l of ROX reference dye II.
After the PCR reaction components were added sequentially, the reaction was performed in triplicate using the following protocol: 95 ℃ for 30s, then 95 ℃ for 3s and 60 ℃ for 30s, 40 cycles.
Dissociation curve analysis was performed after each reaction to determine target specificity, and data was analyzed using 7500System SDS Software, and quantified using the relative Threshold cycle (Ct) method: delta CT＝CT target–CT reference，ΔΔCT＝ΔCT test sample–ΔCTcalibretor sample. The mRNA level of the target gene in the test sample (test sample) relative to that in the reference sample (calibretor sample) was 2-ΔΔC TWherein beta-actin (beta-actin) is an internal reference (reference), and the control group is calibretor sample. The relative expression level of the target gene was calculated by the above method, and all the data were the relative expression amount of the mRNA of the target gene.
The results are shown in D-E of FIG. 4, in which D shows the results of quantifying the gene involved in mucin production and E shows the results of quantifying the gene involved in intestinal barrier function.
The results show that high-dose KGMOS treatment significantly increased transcription of the colonic mucin gene Muc2, while other mucin-encoding genes Muc3 and Muc5ac were not significantly altered. Meanwhile, in both administration groups, the transcription of goblet cell proteins Tff1 and Tff3 involved in mucosal protection and repair was not significantly changed (D of fig. 4). In addition, administration of KGMOS had no effect on transcription of claudin ZO1, ZO2, occiudin and choice in colon (E of fig. 4).
2.5 quantitative analysis of Short Chain Fatty Acids (SCFA) in mouse feces
After the feces were prepared into a suspension, short-chain fatty acids were extracted from the colonic fecal sample using ethyl acetate, and then measured using a gas chromatograph (shimadzu GC-2014, japan) connected to a glass capillary column and a flame ionization detector.
The results are shown in A of FIG. 5, which shows the results of quantitative analysis of the content of short-chain fatty acids in colonic feces. The results show that KGMOS treatment increased butyrate levels in the colon and exhibited dose dependence with no significant change in the mean acetic and propionic acid levels in colon tissue. The KGMOS of different dosages has no obvious influence on the total short-chain fatty acid level. KGM has been shown to have the effect of increasing SCFA in vitro (Connolly, M., et al., J Funct Foods 2010,2,219-224), whereas the KGMOS of the present invention increases SCFA butyrate content in the colon of mice in vivo.
2.6KGMOS on the regulation of mouse colon histone acetylation level
Short chain fatty acids, including butyrate, have the ability to inhibit histone deacetylase activity and thus histone deacetylation, thereby enhancing gene expression, including Muc2 (Hatayama, h., et al. biochem Bioph Res co2007,356, 599-603).
For the collected colon samples, the change of histone acetylation degree in the mouse colon after KGMOS treatment was determined by western blotting. Briefly, colon tissue blocks were lysed with RIPA lysate (chinese cloudband sky) on ice and protein concentration was determined with BCA kit (usa Invitrogen) and after boiling denaturation with loading buffer, semi-dried (usa Bio-rad) to nitrocellulose (usa Pall) after SDS-PAGE, blocked with skim milk, incubated with anti-mouse acetylated histone primary antibody (usa CST), washed, incubated with horseradish peroxidase-coupled secondary antibody, and finally developed with substrate ECL. The results are shown in B-D of fig. 5, where B shows western blot results of acetylated histones H3 and H4 and histones H3 and H4 in colon tissue, and C and D show quantitative analysis results of acetylated histones H3 and H4 and histones H3 and H4 in colon tissue.
The results show that administration of KGMOS significantly increased the levels of acetylated histones H3 and H4 (B, C and D of fig. 5) compared to the control group.
The research on the biological function of the KGMOS researches the influence of oligosaccharide KGMOS on the intestinal function from the three aspects of the influence on intestinal flora, the expression of colon mucin genes and the analysis on the content of intestinal short-chain fatty acids. The results show that the KGMOS oligosaccharide improves the proportion of intestinal flora and the content of probiotics; in addition, KGMOS promotes the expression of mucin by improving the transcription of colon Muc2 mucin gene, and the increase of mucin secretion can effectively shield the intestinal wall and prevent the entry of harmful components and pathogenic microorganisms; butyric acid as a short-chain fatty acid mainly comes from intestinal flora, and can act on corresponding receptors after entering the body through intestinal tracts, so as to regulate appetite, energy intake and utilization, and further have the regulation effect on metabolic abnormalities such as obesity, hyperglycemia and the like. These results show that KGMOS can produce a variety of healthful activities by regulating intestinal function.
The results of the above study show that the colon length of mice is significantly increased after KGMOS100 treatment. Given that KGMOS treatment dose-dependently increases production of butyric acid in the colon, which is also one of the energy sources of colonic epithelial cells, it has been reported to promote cell growth. Therefore, it is believed that ingestion of KGMOS promotes the growth of colon tissue, thereby contributing to the health of the colon or the repair of intestinal tract wound tissue.
Sequence listing
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Claims (10)

1. The konjac oligosaccharide KGMOS is characterized in that the weight average molecular weight is 2200-4000Da, the molecular weight distribution is 1.01-1.4, and the polymerization degree dp ranges from 18-24.
2. A method for preparing konjac oligosaccharide KGMOS is characterized by comprising the following steps:
a) degrading konjac polysaccharide KGM in the presence of acid;
b) centrifuging the solution obtained in the step a), taking supernatant, and evaporating to dryness;
c) dissolving the product obtained in the step b) in deionized water, and centrifuging;
d) collecting the supernatant obtained after the centrifugation in the step c), and dialyzing the supernatant in a dialysis bag with the molecular weight cut-off of 3500Da by using ultrapure water to obtain a final product KGMOS.
3. The method according to claim 2, characterized in that said step a) is carried out as follows: dissolving konjac polysaccharide KGM in an acid solution, and degrading at 60-100 ℃;
particularly, the temperature of the degradation reaction is 70-98 ℃, and preferably 80-95 ℃;
in particular, the time of the degradation reaction is more than 1 hour, for example 2 to 4 hours;
in particular, the acid is selected from: trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, in particular trifluoroacetic acid.
4. The method according to claim 2, wherein step b) is performed as follows: centrifuging the solution obtained in the step a) at the rotating speed of 6000-10000rpm, and taking the supernatant to evaporate to dryness.
5. The method as claimed in claim 2, wherein step c) is carried out by dissolving the residue from step b) with deionized water and centrifuging at 6000-10000 rpm.
6. The method according to claim 4 or 5, wherein the number of centrifugal revolutions is 7000-9800 revolutions; and/or the centrifugation time is 10 minutes or more, for example 10 to 40 minutes.
7. The method according to claim 2, wherein said step d) is carried out as follows: collecting the supernatant obtained after the centrifugation in the step c), putting into a dialysis bag with the molecular weight cut-off of 3500Da, dialyzing in ultrapure water, and freeze-drying to obtain the final product of the konjac oligosaccharide KGMOS.
8. The method according to claim 7, wherein in step d) the dialysis time is more than 1 day, such as 1 to 5 days.
9. A pharmaceutical or food composition comprising a konjac oligosaccharide KGMOS according to claim 1 or prepared by the method of any one of claims 2 to 8.
10. Use of a konjac oligosaccharide KGMOS according to claim 1 or prepared according to the method of any one of claims 2-8, selected from the group consisting of: the application of the polypeptide in preparing the medicine or food for promoting the synthesis of butyric acid in colon, the application in preparing the medicine or food for enhancing the acetylation of the mucosal epithelial cell histones H3 and H4, the application in preparing the medicine or food for increasing the expression of Muc2 gene, the application in preparing the medicine or food for improving the mucosal barrier protection function and the application in preparing the medicine or food for improving the intestinal flora structure and maintaining the intestinal health.
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