阅读说明：本技术 一种具有促戊糖片球菌生长的党参果聚糖复合物及制备方法和应用 (Codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus and preparation method and application thereof ) 是由 李建宽 高建平 董丽娜 王妍 于 2020-12-29 设计创作，主要内容包括：本发明公开了一种具有促戊糖片球菌生长的党参果聚糖复合物及制备方法和应用。该党参果聚糖复合物主要由一种呋喃型果聚糖和一种吡喃型果聚糖组成。呋喃果聚糖为β-D-呋喃果糖基-(2→1)-连接而成的果聚糖,吡喃果聚糖为β-D-吡喃果糖基-(2→1)-连接而成的果聚糖,其分子量分别为2000～2500Da和1000～1500Da。该党参果聚糖复合物制备方法包括如下步骤：(1)党参总多糖的提取；(2)党参总多糖提取液乙醇醇沉；(3)党参多糖的分离。该党参果聚糖复合物在药品、保健品、功能食品及饲料添加剂方面的应用。(The invention discloses a codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus, and a preparation method and application thereof. The radix Codonopsis fructan compound mainly comprises furan type fructan and pyran type fructan. The fructofuranose is a fructan formed by connecting beta-D-fructofuranose- (2 → 1) -and the fructopyranose is a fructan formed by connecting beta-D-fructopyranose- (2 → 1) -and has molecular weights of 2000-2500 Da and 1000-1500 Da respectively. The preparation method of the codonopsis pilosula fructan compound comprises the following steps: (1) extracting total polysaccharide of codonopsis pilosula; (2) precipitating the Codonopsis pilosula total polysaccharide extract with ethanol; (3) and (4) separating codonopsis pilosula polysaccharide. The application of the radix Codonopsis fructan compound in medicine, health product, functional food and feed additive is provided.)

1. A codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus is characterized in that: comprises furan type fructan A and pyran type fructan B;

the structural general formula of the furan-type fructan A is as follows:

，

the structural general formula of the pyran fructan B is as follows:

。

2. the codonopsis pilosula fructan complex with pediococcus pentosaceus growth promoting effect according to claim 1, wherein: the molecular weight of the furan-type fructan A is 2000-2500 Da; the molecular weight of the pyran-type fructan B is 1000-1500 Da.

3. The codonopsis pilosula fructan complex with pediococcus pentosaceus growth promoting effect according to claim 1 or 2, wherein: the mass ratio of the furan type fructan A to the pyran type fructan B is 10: 1-1: 10.

4. A method for preparing the codonopsis pilosula fructan complex with pediococcus pentosaceus growth promoting effect according to any one of claims 1 to 3, which is characterized in that: the method comprises the following steps:

(1) extraction of Codonopsis pilosula polysaccharide

Soaking the codonopsis pilosula in a material-liquid ratio of 1: 10-1: 20, and then heating and refluxing for extraction;

(2) alcohol precipitation of dangshen polysaccharide

Concentrating the codonopsis pilosula polysaccharide extracting solution under reduced pressure to 1/4-1/3 volume, then precipitating by using 60-90% ethanol, then centrifuging for 15-30 minutes at 1000-1500 rpm, removing supernatant, washing precipitates for 3 times by using diethyl ether, ethyl acetate, acetone and absolute ethyl alcohol in sequence, and freeze-drying to obtain a codonopsis pilosula polysaccharide sample;

(3) and separation of Codonopsis pilosula polysaccharide

Dissolving radix Codonopsis polysaccharide with water, passing through 50000 molecular weight ultrafiltration membrane, preparing filtrate with Sephadex G-100 column chromatography, analyzing eluate chromatographic characteristics with HPGPC, and collecting target eluates of 33.550 min and 35.614min to obtain radix Codonopsis fructan complex.

5. Use of a codonopsis fructan complex as claimed in any one of claims 1 to 3 for the growth of pediococcus pentosaceus.

6. Use of the Codonopsis Pilosulae fructan complex of any one of claims 1 to 3 in the preparation of functional health food and feed additive.

Technical Field

The invention relates to the technical field of preparation methods of traditional Chinese medicine polysaccharide components, in particular to a codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus, a preparation method and application thereof in medicines, health products, functional foods and feed additives.

Background

Radix Codonopsis (Radix Codonopsis) is dried root of Radix Codonopsis, Radix Codonopsis Lanceolatae and Radix Codonopsis Sichuan of Codonopsis of Campanulaceae. Radix codonopsitis has the effects of tonifying middle-jiao and Qi, strengthening spleen and benefiting lung, and is clinically used for treating spleen-lung weakness, short breath, palpitation, poor appetite, loose stool, deficiency asthma, cough and other symptoms. The codonopsis pilosula mainly contains components such as polysaccharide, triterpene, alkyne glycoside, alkaloid, phenylpropanoid and the like, wherein the polysaccharide content is highest, and the codonopsis pilosula is an important pharmacological active component. Pediococcus pentosaceus (Pediococcus pentosaceus) Is an important probiotic lactic acid bacteria, can produce beneficial metabolites, and further has the effects of enhancing the immunity of the organism and preventing and treating diseases. At present, few reports are available about the growth of pediococcus pentosaceus of the codonopsis pilosula fructan complex.

Disclosure of Invention

The invention mainly separates the total polysaccharide of the codonopsis pilosula, and then screens the growth of pediococcus pentosaceus to obtain the codonopsis pilosula fructan compound with the growth of the pediococcus pentosaceus.

The invention is realized by adopting the following technical scheme:

a radix Codonopsis fructan complex with effect in promoting growth of Pediococcus pentosaceus comprises furan type fructan A and pyran type fructan B.

The structural general formula of the furan-type fructan A is as follows:

，

the structural general formula of the pyran fructan B is as follows:

。

the radix Codonopsis fructan compound mainly comprises furan type fructan A and pyran type fructan B. The furanose type fructan A is a fructan formed by connecting beta-D-fructofuranose group- (2 → 1) -and the pyranose type fructan B is a fructan formed by connecting beta-D-fructopyranose group- (2 → 1) -. The molecular weight of the furan-type fructan A is 2000-2500 Da; the molecular weight of the pyran-type fructan B is 1000-1500 Da. In the codonopsis pilosula fructan compound, the mass ratio of furan type fructan A to pyran type fructan B is 10: 1-1: 10.

The preparation method of the codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus comprises the following steps:

(1) extraction of Codonopsis pilosula polysaccharide

Soaking the codonopsis pilosula in a material-liquid ratio of 1: 10-1: 20, and then heating and refluxing for extraction;

(2) alcohol precipitation of dangshen polysaccharide

Concentrating the codonopsis pilosula polysaccharide extracting solution under reduced pressure to 1/4-1/3 volume, then precipitating by using 60-90% ethanol, then centrifuging for 15-30 minutes at 1000-1500 rpm, removing supernatant, washing precipitates for 3 times by using diethyl ether, ethyl acetate, acetone and absolute ethyl alcohol in sequence, and freeze-drying to obtain a codonopsis pilosula polysaccharide sample;

(3) and separation of Codonopsis pilosula polysaccharide

Dissolving radix Codonopsis polysaccharide with water, passing through 50000 molecular weight ultrafiltration membrane, preparing filtrate with Sephadex G-100 column chromatography, analyzing eluate chromatographic characteristics with HPGPC, and collecting target eluates of 33.550 min and 35.614min to obtain radix Codonopsis fructan complex.

The structural identification of the codonopsis pilosula fructan compound is as follows:

the molecular weight of the codonopsis pilosula fructan compound obtained by the invention is measured by adopting a high-efficiency gel permeation chromatography method:

chromatographic conditions are as follows: an Agilent 1260 HPLC system-RID detector, wherein the analytical column is a TSKgel G4000PWXL chromatographic column (7.8 mm multiplied by 30cm, 10 mu m), the mobile phase is ultrapure water, the flow rate is 0.3mL/min, the temperature of a differential detector is 35 ℃, the temperature of the column is 35 ℃, and the sample injection amount is 20 mu L.

Polysaccharide composition and structure analysis are carried out on the codonopsis pilosula fructan compound obtained by the invention: to be provided with¹H-NMR、¹³C-NMR and 2D-NMR analyses.

The above analysis proves that: the radix Codonopsis fructan compound mainly comprises furan type fructan and pyran type fructan. The fructofuranose is a fructan formed by connecting beta-D-fructofuranose- (2 → 1) -and the fructopyranose is a fructan formed by connecting beta-D-fructopyranose- (2 → 1) -and has molecular weights of 2000-2500 Da and 1000-1500 Da respectively.

The codonopsis pilosula fructan compound obtained by the invention has obvious promotion effect on growth of pediococcus pentosaceus in-vitro fecal bacteria culture, and the experiment comprises the following specific steps:

(1) culturing fecal bacteria in vitro: sterilizing the codonopsis pilosula fructan compound with 1% of MRS agar at a ratio of 2% under high pressure to prepare a flat plate, diluting excrement (taken from a young healthy female) with 0.1g/mL of PBS, filtering with gauze, spreading the filtrate on the flat plate for anaerobic culture, after bacteria grow out, inoculating the bacterial colony on the flat plate again, inoculating the bacterial colony to a lactic acid bacteria culture medium for expanded culture after three times of inoculation culture;

(2) and culturing dominant bacteria: inoculating into liquid with radix Codonopsis compound as sole carbon source, culturing, and detecting its growth;

(3) 16SrRNA sequence analysis: and (3) carrying out 16SrRNA sequence analysis on the bacteria cultured in the step (2) to identify the strain type.

The codonopsis pilosula fructan compound obtained by the invention can obviously promote the growth of pediococcus pentosaceus and has application in medicines, health products, functional foods and feed additives.

Drawings

FIG. 1 shows a gel permeation chromatogram of a Codonopsis fructan complex.

FIG. 2A shows HPLC chromatogram of acid hydrolysate of Codonopsis pilosula fructan complex.

FIG. 2B shows a standard D-fructose HPLC profile of Codonopsis pilosula fructan complex.

FIG. 2C shows a standard D-glucose HPLC profile of Codonopsis fructan complex.

FIG. 3A shows a Codonopsis fructan complex¹H-NMR spectrum.

FIG. 3B shows a Codonopsis fructan complex¹³C-NMR spectrum.

FIG. 3C shows the DEPT135 spectrum of Codonopsis pilosula fructan complex.

FIG. 4 shows the H-H COSY spectrum of Codonopsis pilosula fructan complex.

FIG. 5 shows the HSQC spectrum of the Codonopsis fructan complex.

FIG. 6 shows HMBC mapping of Codonopsis fructan complex.

FIG. 7 shows a graph of the growth of Pediococcus pentosaceus in a complex of Codonopsis fructan.

Detailed Description

The following provides a detailed description of specific embodiments of the present invention.

A preparation method of a codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus comprises the following steps:

(1) codonopsis pilosula polysaccharide extraction

Soaking the codonopsis pilosula for 30 minutes according to the material-liquid ratio of 1: 10-1: 20, heating and refluxing for extraction for 3 times, wherein each time lasts for 1.5 hours, and combining three extracting solutions.

(2) Preparation of Codonopsis pilosula polysaccharide

And (3) carrying out reduced pressure concentration on the codonopsis pilosula polysaccharide extract until the volume is 1/4-1/3, precipitating with 60-80% ethanol, centrifuging for 15 minutes at 1000 rpm, removing supernatant, sequentially washing the precipitate with diethyl ether, ethyl acetate, acetone and absolute ethyl alcohol for 3 times, and carrying out freeze drying to obtain a codonopsis pilosula polysaccharide sample.

(3) And separation of Codonopsis pilosula polysaccharide

Dissolving radix Codonopsis polysaccharide with water, passing through 50000 molecular weight ultrafiltration membrane, preparing filtrate with Sephadex G-100 column chromatography, analyzing chromatographic characteristics of eluate with HPGPC, and collecting target eluate to obtain radix Codonopsis fructan complex.

HPGPC analysis of Codonopsis pilosula polysaccharide

The chromatographic condition is Agilent 1260 HPLC system-RID detector, the analytical column is TSKgel G4000PWXL chromatographic column (7.8 mm multiplied by 30cm, 10 mu m), the mobile phase is ultrapure water, the flow rate is 0.3mL/min, the temperature of the differential detector is 30 ℃, the column temperature is 30 ℃, and the sample injection amount is 20 mu L, thus obtaining the HPGPC chart. The spectra exhibited the following characteristics: a distinct chromatographic peak (furan type fructan) at 33.55min and a chromatographic peak (pyran type fructan) at 35.614min, wherein the molecular weights of the corresponding polysaccharides are 2000-2500 Da and 1000-1500 Da respectively.

The results in FIG. 1 show that the codonopsis pilosula fructan compound mainly comprises two main chromatographic peaks of 33.550 minutes and 35.614 minutes, and the molecular weight of polysaccharide represented by the two main chromatographic peaks of 33.550 minutes and 35.614 minutes is 2000-2500 Da and 1000-1500 Da respectively according to the HPGPC molecular weight and time (minute) linear curve.

The results shown in FIGS. 2A, 2B and 2C show that the acidic hydrolysate of Codonopsis pilosula fructan complex is mainly D-fructose and D-glucose, which indicates that the monosaccharides constituting the polysaccharides of Codonopsis pilosula fructan complex are D-fructose and D-glucose.

II, NMR analysis of codonopsis pilosula polysaccharide

A sample of Codonopsis pilosula polysaccharide (50 mg) was dissolved in 1mL of deuterated water (D)₂O) in 400MHz NMR spectrometer¹H-NMR、¹³C-NMR、¹³C-DEPT135 and 2D-NMR analysis were performed to obtain NMR spectra of the fructan complex.

The results in FIGS. 3A, 3B and 3C show that the sugar signals of the Codonopsis fructan complex are mainly fructose signals and weak glucose signals, which are consistent with the acid hydrolysis results. According to the intensity of the carbon signal, polysaccharides represented by two main chromatographic peaks of 33.550 minutes and 35.614 minutes are beta-D-fructofuranosyl- (2 → 1) -linked fructan and beta-D-fructopyranosyl- (2 → 1) -linked fructan respectively.

The results in FIG. 4 clearly identify the hydrogen signals at positions 3, 4 and 5 of beta-D-fructofuranosyl and the hydrogen signals at positions 3, 4 and 5 of beta-D-fructopyranosyl.

FIG. 5 results clearly ascribe hydrogen and carbon signals at positions 1, 3, 4, 5 and 6 of β -D-fructopyranosyl and hydrogen and carbon signals at positions 1, 3, 4, 5 and 6 of β -D-fructopyranosyl.

The results in FIG. 6 clearly confirmed the- (2 → 1) -linkage pattern of the two fructosyl groups.

Table 1 codonopsis pilosula fructan complex H and C nuclear magnetic data attribution.

And thirdly, the obtained codonopsis pilosula fructan compound has the effect of promoting the growth of pediococcus pentosaceus in-vitro fecal bacteria culture.

(1) Culturing fecal bacteria in vitro: sterilizing the codonopsis pilosula fructan compound with 1% of MRS agar at a ratio of 2% under high pressure to prepare a flat plate, diluting excrement (taken from a young healthy female) with 0.1g/mL of PBS, filtering with gauze, spreading the filtrate on the flat plate for anaerobic culture, after bacteria grow out, inoculating the bacterial colony on the flat plate again, inoculating the bacterial colony to a lactic acid bacteria culture medium for expanded culture after three times of inoculation culture;

(2) and culturing dominant bacteria: inoculating into liquid with radix Codonopsis compound as sole carbon source, culturing, and detecting its growth;

(3) 16SrRNA sequence analysis: and (3) carrying out 16SrRNA sequence analysis on the bacteria cultured in the step (2) to identify the strain type.

According to the sequencing result of pediococcus pentosaceus 16SrRNA which promotes growth by taking the codonopsis pilosula fructan compound as a unique carbon source, the pediococcus pentosaceus is confirmed to be pediococcus pentosaceus by comparing with a database.

The results in FIG. 7 show that the Codonopsis fructan complex can significantly promote the growth of Pediococcus pentosaceus within 48 hours.

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the detailed description is made with reference to the embodiments of the present invention, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which shall be covered by the claims.

<110> university of Shanxi medical science

<120> codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus, preparation method and application thereof

<160>1

<210>1

<211>1479

<212>RNA

<213> Pediococcus pentosaceus (Pediococcus pentosaceus)

<220>

<223> probiotic lactic acid bacteria

<400>1

GGCGTGCGGG TGCTATACAT GCAAGTCGAA CGAACTTCCG TTAATTGATT ATGACGTACT 60

TGTACTGATT GAGATTTTAA CACGAAGTGA GTGGCGAACG GGTGAGTAAC ACGTGGGTAA 120

CCTGCCCAGA AGTAGGGGAT AACACCTGGA AACAGATGCT AATACCGTAT AACAGAGAAA 180

ACCGCATGGT TTTCTTTTAA AAGATGGCTC TGCTATCACT TCTGGATGGA CCCGCGGCGT 240

ATTAGCTAGT TGGTGAGGTA AAGGCTCACC AAGGCAGTGA TACGTAGCCG ACCTGAGAGG 300

GTAATCGGCC ACATTGGGAC TGAGACACGG CCCAGACTCC TACGGGAGGC AGCAGTAGGG 360

AATCTTCCAC AATGGACGCA AGTCTGATGG AGCAACGCCG CGTGAGTGAA GAAGGGTTTC 420

GGCTCGTAAA GCTCTGTTGT TAAAGAAGAA CGTGGGTAAG AGTAACTGTT TACCCAGTGA 480

CGGTATTTAA CCAGAAAGCC ACGGCTAACT ACGTGCCAGC AGCCGCGGTA ATACGTAGGT 540

GGCAAGCGTT ATCCGGATTT ATTGGGCGTA AAGCGAGCGC AGGCGGTCTT TTAAGTCTAA 600

TGTGAAAGCC TTCGGCTCAA CCGAAGAAGT GCATTGGAAA CTGGGAGACT TGAGTGCAGA 660

AGAGGACAGT GGAACTCCAT GTGTAGCGGT GAAATGCGTA GATATATGGA AGAACACCAG 720

TGGCGAAGGC GGCTGTCTGG TCTGCAACTG ACGCTGAGGC TCGAAAGCAT GGGTAGCGAA 780

CAGGATTAGA TACCCTGGTA GTCCATGCCG TAAACGATGA TTACTAAGTG TTGGAGGGTT 840

TCCGCCCTTC AGTGCTGCAG CTAACGCATT AAGTAATCCG CCTGGGGAGT ACGACCGCAA 900

GGTTGAAACT CAAAAGAATT GACGGGGGCC CGCACAAGCG GTGGAGCATG TGGTTTAATT 960

CGAAGCTACG CGAAGAACCT TACCAGGTCT TGACATCTTC TGACAGTCTA AGAGATTAGA 1020

GGTTCCCTTC GGGGACAGAA TGACAGGTGG TGCATGGTTG TCGTCAGCTC GTGTCGTGAG 1080

ATGTTGGGTT AAGTCCCGCA ACGAGCGCAA CCCTTATTAC TAGTTGCCAG CATTAAGTTG 1140

GGCACTCTAG TGAGACTGCC GGTGACAAAC CGGAGGAAGG TGGGGACGAC GTCAAATCAT 1200

CATGCCCCTT ATGACCTGGG CTACACACGT GCTACAATGG ATGGTACAAC GAGTCGCGAG 1260

ACCGCGAGGT TAAGCTAATC TCTTAAAACC ATTCTCAGTT CGGACTGTAG GCTGCAACTC 1320

GCCTACACGA AGTCGGAATC GCTAGTAATC GCGGATCAGC ATGCCGCGGT GAATACGTTC 1380

CCGGGCCTTG TACACACCGC CCGTCACACC ATGAGAGTTT GTAACACCCA AAGCCGGTGG 1440

GGTAACCTTT TAGGAGCTAG CCGTCTAAGT GACAGATTG 1479