WO2023010820A1 - 植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法 - Google Patents

植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法 Download PDF

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WO2023010820A1
WO2023010820A1 PCT/CN2022/074451 CN2022074451W WO2023010820A1 WO 2023010820 A1 WO2023010820 A1 WO 2023010820A1 CN 2022074451 W CN2022074451 W CN 2022074451W WO 2023010820 A1 WO2023010820 A1 WO 2023010820A1
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lactic acid
indole
fermentation supernatant
eluent
mobile phase
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French (fr)
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顾青
周青青
郦萍
顾容铖
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浙江工商大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
    • B01D15/1871—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/265—Adsorption chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • the invention belongs to the field of food biotechnology, and in particular relates to a method for extracting indole-3-lactic acid from the fermentation supernatant of plant lactic acid bacteria.
  • Indole-3-lactic acid is a metabolite of tryptophan with an indole ring, its molecular weight is 205Da, its molecular formula is C 11 H 11 NO 3 , and it has the formula I Structure.
  • Lactic acid bacteria such as Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium breve, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus reuteri, etc. can metabolize indole-3-lactic acid.
  • Indole-3-lactic acid the only tryptophan metabolite produced by Bifidobacteria, was found in relatively high levels in Bifidobacterium strains isolated from the intestinal tract of human infants compared with other strains, It is 22.17 ⁇ 33.12 ⁇ g/mL.
  • Lactobacillus plantarum UM55, Lactobacillus plantarum dy-1, and Lactobacillus plantarum F51 can metabolize indole-3-lactic acid by Lactobacillus plantarum strains, and the content is 4.30-30.70 ⁇ g/mL.
  • Indole-3-lactic acid has been reported to have antioxidant activity, participate in the induction of immune regulation, inhibit endogenous protein binding, and serve as a conserved biomarker for the diagnosis of alcoholic liver disease, gastric cancer, diabetes, inflammatory bowel disease, and some other metabolic Functional effects such as effective disease. Therefore, indole-3-lactic acid will be widely used in medicine, food, health care and other fields.
  • the separation of indole-3-lactic acid from the fermentation supernatant of lactic acid bacteria mainly adopts ultrafiltration and centrifugation.
  • the size of molecules intercepted by ultrafiltration centrifugation has a certain range, and substances with similar molecular weights are all centrifuged, and the difference in mechanical properties of filters can easily lead to differences in the content and purity of fractionated indole-3-lactic acid. Therefore, the screening of lactic acid bacteria with high indole-3-lactic acid production and the optimization of separation and purification methods are of great significance for the extraction of indole-3-lactic acid from lactic acid bacteria.
  • 2008100625229's invention "Lactobacillus plantarum ZJ316, the produced antimicrobial peptide and its preparation and application” provides a strain of Lactobacillus plantarum ZJ316 isolated from infant feces, the preservation number CCTCC NO: M 208077, and the obtained antibacterial peptide Has a broad-spectrum antibacterial effect.
  • the invention of 201911341978.3 provides a direct-injected lactic acid bacteria starter and a preparation method, wherein the direct-injected lactic acid bacteria starter is prepared using Lactobacillus plantarum ZJ316 among lactic acid bacteria, wherein the plant The deposit number of Lactobacillus is CCTCC No: M 208077.
  • the technical problem to be solved by the present invention is to provide a method for extracting indole-3-lactic acid from the fermentation supernatant of plant lactic acid bacteria, and to measure the antibacterial activity of the indole-3-lactic acid.
  • the present invention provides a method for purifying indole-3-lactic acid from the fermentation supernatant of plant lactic acid bacteria, utilizing the fermentation supernatant of Lactobacillus plantarum ZJ316 with the preservation number CCTCC NO:M 208077, comprising the following steps :
  • step 2) the concentrated solution obtained in step 1) is separated by Sephadex G25 to obtain the G25-2 component;
  • step 3 the G25-2 component obtained in step 2) is purified by reverse-phase high-performance liquid chromatography (RP-HPLC):
  • Mobile phase A is an aqueous trifluoroacetic acid solution with a volume concentration of 0.05%
  • the mobile phase B is a trifluoroacetic acetonitrile solution with a volume concentration of 0.05%;
  • the eluent is composed of mobile phase A and mobile phase B, the flow rate is 3 ⁇ 0.1mL/min, and the elution procedure is as follows:
  • the eluate is concentrated to 9-11% of the original volume to obtain a concentrate.
  • the step 1) is:
  • 5L Lactobacillus plantarum ZJ316 supernatant is adsorbed by 500g macroporous resin XAD-16 (flow rate is 1mL/min); For elution, the elution flow rate is 1 mL/min, and the eluate corresponding to 50% methanol with a pH of 7 is collected.
  • step 2) includes:
  • the concentration of the step 2) is as follows: the eluate is concentrated to near dryness, redissolved to 1 mL with ultrapure water, Get G25-2 component.
  • the step 3 is:
  • step 2 Take 500 ⁇ L of the G25-2 fraction (stored at 4°C) obtained in step 2), dilute it to 5mL with ultrapure water, and load it on Waters SunFire C18 Prep (5 ⁇ m 10 ⁇ 100mm) at a column temperature of 25°C to obtain H4 components;
  • the H4 component obtained in the step 3) is concentrated to nearly dryness to obtain indole-3-lactic acid.
  • the preparation method of fermentation supernatant is:
  • the Lactobacillus plantarum ZJ316 with the preservation number CCTCC NO: M 208077 was inoculated in the MRS medium for fermentation (v/v) according to the inoculum amount of 3% (volume %) bacteria solution, and the rotating speed was 180rpm, and it was cultivated at 37°C for 24h; the fermented liquid was centrifuged (8000rpm , 25min, 4°C) to obtain the supernatant (pH value is about 3.79).
  • the above-mentioned fermentation can utilize small-scale bacteria-type fermenters to ferment, that is, Lactobacillus plantarum ZJ316 is inoculated in 5L MRS to ferment by 3% bacterium liquid.
  • RP-HPLC Phase high performance liquid chromatography
  • RP-HPLC model and condition are Waters SunFire C18 (Prep 5 ⁇ m 10 ⁇ 100mm), mobile phase 0.05% trifluoroacetic acid/water (A) (v/v) and 0.05 % Trifluoroacetic acid/acetonitrile (B) (v/v).
  • the content of indole-3-lactic acid in the fermentation supernatant of Lactobacillus plantarum ZJ316 is determined by analytical HPLC. 3-Lactic acid standard sample (Sigma company) was analyzed, and the linear relationship of "peak area-concentration" was established to obtain a regression equation. Using this method, the indole-3-lactic acid content in the fermentation supernatant of Lactobacillus plantarum ZJ316 was determined to be 43.14 ⁇ g/mL, which is one of the reported lactic acid bacteria with the highest indole-3-lactic acid production.
  • the indole-3-lactic acid obtained in the invention has antibacterial properties and can effectively inhibit the growth of Salmonella, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, Pseudomonas aeruginosa and the like.
  • the present invention has the following technical advantages:
  • the present invention uses HPLC to determine the content of indole-3-lactic acid in the fermentation supernatant of Lactobacillus plantarum ZJ316, which is 43.14 ⁇ g/mL, which is one of the highest-yield Lactobacillus plantarum reported so far.
  • Fig. 1 is the regression equation figure of indole-3-lactic acid standard product.
  • Fig. 2 is the separation, purification and identification of indole-3-lactic acid in the fermentation supernatant of Lactobacillus plantarum ZJ316;
  • a is the separation of Sephadex G25; b is the separation and purification of reversed-phase high performance liquid chromatography (RP-HPLC); c is the liquid chromatogram of LC-MS; d is the mass spectrum of LC-MS.
  • RP-HPLC reversed-phase high performance liquid chromatography
  • Fig. 3 is the analysis of the purity of indole-3-lactic acid by analytical high performance liquid chromatography.
  • MRS liquid medium anhydrous glucose 20g, Tween-80 1mL, magnesium sulfate heptahydrate 0.2g, manganese sulfate 0.05g, tryptone 10g, yeast extract 5g, dipotassium hydrogen phosphate 2g, triammonium citrate 2g, beef Extract 10g, anhydrous sodium acetate 5g, dissolve with ultrapure water and dilute to 1L, adjust the pH of the medium to 6 with 37.5% concentrated hydrochloric acid.
  • MRS solid medium add 2% (mass concentration) bacterial culture medium agar to the above MRS liquid medium, and adjust the pH of the medium to 6 with 37.5% concentrated hydrochloric acid.
  • Embodiment 1 Determination of indole-3-lactic acid content in the fermentation supernatant of Lactobacillus plantarum ZJ316
  • step (2) Inoculate the culture solution obtained in step (1) into 150mL MRS liquid medium with 3% inoculum size (volume concentration) and continue static culture at 37°C for 24h;
  • step (3) Inoculate the culture solution obtained in step (2) with 3% inoculum size (volume concentration) in 5L MRS liquid medium for fermentation. Fermentation conditions: temperature 37°C, rotation speed 180rpm, time 24h.
  • the obtained fermentation broth was centrifuged: 8000rpm, 25min, 4°C, and the fermentation supernatant was stored at 4°C until use.
  • Mobile phase A 0.05% trifluoroacetic acid/water, that is, an aqueous solution of trifluoroacetic acid with a volume concentration of 0.05%;
  • Mobile phase B 0.05% trifluoroacetic acid/acetonitrile, that is, a solution of trifluoroacetic acid in acetonitrile with a volume concentration of 0.05%;
  • Step 1 Adsorption and elution of macroporous resin XAD-16
  • step (1) Concentrate the eluent (50% methanol eluent) obtained in step (1) to 200mL with a rotary evaporator to obtain a concentrated solution; rotary evaporation conditions: water bath temperature 37°C, pressure 30 ⁇ 5mbar, rotation speed 60rpm .
  • step (3) Use a rotary evaporator to concentrate the G25-2 (12 mL) obtained in step (2) to nearly dryness, and redissolve it to 1 mL with ultrapure water to obtain the G25-2 component, which is stored at 4°C until use; rotary evaporation conditions : The water bath temperature is 37°C, the pressure is 30 ⁇ 5mbar, and the rotation speed is 60rpm.
  • the RP-HPLC column model is Waters SunFire C18 Prep (5 ⁇ m 10 ⁇ 100mm), the column temperature is 25°C, the detector model is Waters 2998, the detector wavelength is 280nm, and the flow rate is 3mL/min.
  • the eluent consists of mobile phase A and mobile phase B:
  • Mobile phase A 0.05% trifluoroacetic acid/water, that is, an aqueous solution of trifluoroacetic acid with a volume concentration of 0.05%;
  • Mobile phase B 0.05% trifluoroacetic acid/acetonitrile, that is, a solution of trifluoroacetic acid in acetonitrile with a volume concentration of 0.05%;
  • the LC-MS instrument model is Agilent 1200-6210, the chromatographic column model is Waters SunFire C18 (5 ⁇ m 4.6 ⁇ 250mm), the column temperature is 30°C, the ion source is ESI, negative ion mode, the ion source gas temperature is 350°C, and the flow rate of the dry gas 9L/min, atomizing gas pressure 45psi, capillary voltage 3500V, fragmentation voltage 125V, mass scanning range 50-2000m/z, injection volume 10 ⁇ L.
  • HPLC eluent consists of mobile phase A and mobile phase B:
  • Mobile phase A 0.1% formic acid/water, that is, an aqueous solution of formic acid with a volume concentration of 0.1%;
  • Mobile phase B 0.1% formic acid/acetonitrile, that is, a solution of formic acid in acetonitrile with a volume concentration of 0.1%;
  • FIG. 2c is the liquid chromatogram of the H4 sample
  • Figure 2d is the mass spectrum of the H4 sample.
  • the molecular weight of the H4 sample was determined to be 205.
  • Example 3 Purity determination of isolated and purified indole-3-lactic acid
  • Table 4 shows the starting and ending time (min), peak area ( ⁇ V ⁇ s) and peak area ratio (%) of the main component peak (ie, indole-3-lactic acid) and two miscellaneous peaks.
  • the main component peak (i.e., indole-3-lactic acid) area is A
  • the sum of the main component peak and other miscellaneous peak peak areas is ⁇ A
  • the indole-3-lactic acid purity (%) is obtained.
  • the antibacterial activity of the indole-3-lactic acid purified in Step 3 of Example 2 was measured by the Oxford cup method. Add 1% (volume concentration) indicator bacteria, 150 ⁇ L concentration of 10 mg/mL indole-3-lactic acid (dissolved in 1% acetonitrile aqueous solution).
  • indicator bacteria see Table 5
  • Gram-positive bacteria Merococcus luteus, Staphylococcus aureus, Staphylococcus carnosus
  • Mimic Staphylococcus etc.
  • Gram-negative bacteria Escherichia coli, Salmonella paratyphi A/B, Salmonella enteritidis, Salmonella choleraesuis, Salmonella typhimurium, etc.
  • Comparative example 1 change the Lactobacillus plantarum ZJ316 in Example 1 into other existing Lactobacillus plantarum strains, specifically as described in Table 6 below, and the rest refer to Example 1 to detect indole in the Lactobacillus plantarum fermentation supernatant -3-Lactic acid content ( ⁇ g/mL).
  • the comparison between the obtained experimental results and the present invention is as described in Table 6.
  • the Lactobacillus plantarum ZJ316 fermentation supernatant that embodiment 1 gained is carried out ultrafiltration centrifugation, and the process parameter of ultrafiltration centrifugation is specifically as follows: Lactobacillus plantarum ZJ316 supernatant is crossed 0.22 ⁇ m filter membrane, centrifuged with 3kDa ultrafiltration The tubes (Millipore Amicon Ultra) were centrifuged (4000g, 4°C, 30min) and the filtrate was collected. The purity of the filtrate obtained under ultrafiltration and centrifugation was tested according to the method described in Example 3 above, and the purity of the obtained indole-3-lactic acid was about 40%.
  • Comparative Example 4-2 Change "30% methanol" in Step 1 of Example 2 to "35% methanol", and the rest are the same as Example 2. The purity of the obtained indole-3-lactic acid was about 94%.
  • Comparative example 8 change the elution procedure of the reversed-phase high-performance liquid chromatography (RP-HPLC) in step 3 of embodiment 2 to the following:
  • mobile phase B 10% 10-30min, mobile phase B 10-50%; 30-35min, mobile phase B 50-95%; 35-38min, mobile phase B 95-10%; 38-40min , mobile phase B 10%; the flow rate is 3mL/min.
  • mobile phase B 5% 10-30min, mobile phase B 5-40%; 30-35min, mobile phase B 40-95%; 35-38min, mobile phase B 95-5%; 38-40min , mobile phase B 5%; the flow rate is 3mL/min.
  • mobile phase B 10% 10-30min, mobile phase B 10-40%; 30-35min, mobile phase B 40-95%; 35-38min, mobile phase B 95-10%; 38-40min , mobile phase B 10%; the flow rate is 2mL/min.
  • mobile phase B 10% 10-30min, mobile phase B 10-40%; 30-35min, mobile phase B 40-95%; 35-38min, mobile phase B 95-10%; 38-40min , mobile phase B 10%; the flow rate is 4mL/min.
  • Example 2 The rest are equal to Example 2.
  • the purity of the obtained indole-3-lactic acid corresponds to about 92%, 96%, 96%, 90%, 82%.

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Abstract

本发明公开了一种从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法，利用保藏编号CCTCC NO:M 208077的植物乳杆菌ZJ316的发酵上清液，包括以下步骤：将发酵上清先用大孔树脂XAD-16吸附，然后洗脱，收集pH为7的50％甲醇对应的洗脱液，将洗脱液浓缩，得浓缩液；将浓缩液经葡聚糖凝胶G25分离，获得G25-2组分；将G25-2组分经反相高效液相色谱纯化，将收集的洗脱液进行浓缩，得吲哚-3-乳酸。本发明所得的吲哚-3-乳酸，纯度为99.00％，具有广谱抗菌特性。

Description

植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法 技术领域
本发明属于食品生物技术领域，具体涉及一种植物乳酸菌发酵上清中提取吲哚-3-乳酸的方法。
背景技术
吲哚-3-乳酸(Indole-3-lactic acid，ILA)，是一种带有吲哚环的色氨酸代谢产物，分子量为205Da，分子式为C 11H 11NO 3，具有式I所示的结构。乳酸菌如长双歧杆菌、婴儿双歧杆菌、短双歧杆菌、唾液乳杆菌、植物乳杆菌、罗伊氏乳杆菌等可代谢产生吲哚-3-乳酸。与其他菌株相比，吲哚-3-乳酸是双歧杆菌产生的唯一色氨酸代谢物，从人类婴儿肠道分离的双歧杆菌菌株中发现的吲哚-3-乳酸含量相对较高，为22.17～33.12μg/mL。而植物乳杆菌菌株代谢产生吲哚-3-乳酸的菌株仅有植物乳杆菌UM55、植物乳杆菌dy-1、植物乳杆菌F51等，含量为4.30～30.70μg/mL。
Figure PCTCN2022074451-appb-000001
据报道，吲哚-3-乳酸具有抗氧化活性，参与诱导免疫调节，抑制内源性蛋白结合，作为酒精性肝病、胃癌诊断的保守生物标志物，对糖尿病、炎症性肠病和其他一些代谢疾病有效等功能作用。因此，吲哚-3-乳酸在医药、食品、保健等领域将有广泛应用。
目前，从乳酸菌发酵上清液中分离吲哚-3-乳酸，主要采用超滤离心法。超滤离心截留的分子大小具有一定的范围，相近分子量的物质均被离心截留，且过滤器机械性能的不同，易导致分馏的吲哚-3-乳酸含量及纯度存在差异。因此，筛选高产吲哚-3-乳酸的乳酸菌，并优化分离纯化的方法对乳酸菌源吲哚-3-乳酸的提取意义十分重大。
2008100625229的发明《Lactobacillus plantarum ZJ316、产生的抗菌肽及其制备与应用》提供了一株从婴儿粪便中分离到的植物乳杆菌——Lactobacillus plantarum ZJ316，保藏编号CCTCC NO：M 208077，所得的抗菌肽具有广谱的抑菌作用。
201911341978.3的发明《直投式乳酸菌发酵剂和制备方法》提供了一直投式乳酸菌发酵 剂和制备方法，其中所述直投式乳酸菌发酵剂，应用乳酸菌中的植物乳杆菌ZJ316制备，其中所述植物乳杆菌的保藏编号CCTCC No：M 208077。
发明内容
本发明要解决的技术问题是提供一种从植物乳酸菌发酵上清中提取吲哚-3-乳酸的方法，并测定该吲哚-3-乳酸的抗菌活性。
为解决上述技术问题，本发明提供一种从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法，利用保藏编号CCTCC NO:M 208077的植物乳杆菌ZJ316的发酵上清液，包括以下步骤：
1)、将发酵上清先用大孔树脂XAD-16吸附，然后依次用超纯水、30％甲醇、pH为7的50％甲醇进行洗脱，洗脱流速均为1±0.1mL/min，收集pH为7的50％甲醇对应的洗脱液；
将所述洗脱液进行浓缩，得浓缩液；
2)、将步骤1)所得的浓缩液经葡聚糖凝胶G25分离，获得G25-2组分；
3)、将步骤2)所得的G25-2组分经反相高效液相色谱(RP-HPLC)纯化：
流动相A为体积浓度为0.05％的三氟乙酸水溶液；
流动相B为体积浓度为0.05％的三氟乙酸乙腈溶液；
洗脱剂由流动相A、流动相B组成，流速为3±0.1mL/min，洗脱程序如下：
RP-HPLC洗脱程序
Figure PCTCN2022074451-appb-000002
即：0～10min，流动相B 10％；10～30min，流动相B 10～40％；30～35min，流动相B 40～95％；35～38min，流动相B 95～10％；38～40min，流动相B 10％；
收集出峰时间(起止时间)为23.9273～24.5716min的洗脱液，获得H4组分；将收集的洗脱液进行旋转蒸发仪浓缩，得吲哚-3-乳酸。
作为本发明的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法的改进：所述步骤1)中，将洗脱液进行浓缩至原体积的9～11％，得浓缩液。
作为本发明的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法的进一步改进：所述步骤1)为：
5L植物乳杆菌ZJ316上清液经500g大孔树脂XAD-16吸附(流速为1mL/min)；然后依次用2L的超纯水、2L的30％甲醇、2L的pH为7的50％甲醇进行洗脱，洗脱流速均为1mL/min，收集pH为7的50％甲醇对应的洗脱液。
作为本发明的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法的进一步改进，步骤2)包括：
取步骤1)所得的浓缩液2mL，过0.22μm滤膜，上样至Sephadex G25葡聚糖凝胶柱(柱高80cm，柱直径1.6cm)；
以超纯水为洗脱液，流速1±0.1mL/min，每隔3min接收1管洗脱液，收集t39、t40、t41、t42这4管洗脱液(为280nm波长下第2个色谱峰中吸光度值最高的4管)；
将洗脱液浓缩，得G25-2组分。
作为本发明的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法的进一步改进，所述步骤2)的浓缩为：将洗脱液浓缩近干，用超纯水重溶至1mL，得G25-2组分。
作为本发明的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法的进一步改进：所述步骤3)为：
取步骤2)所得的G25-2组分(储存于4℃)500μL，用超纯水稀释至5mL后，上样至Waters SunFire C18 Prep(5μm 10×100mm)，柱温为25℃，获得H4组分；
所述步骤3)所得的H4组分浓缩近干，得吲哚-3-乳酸。
作为本发明的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法的进一步改进：发酵上清的制备方法为：
将保藏编号CCTCC NO:M 208077的植物乳杆菌ZJ316按3％(体积％)菌液接种量接种于MRS培养基中发酵(v/v)，转速180rpm，37℃培养24h；发酵液离心(8000rpm，25min，4℃)，获得上清(pH值约为3.79)。
上述发酵可利用小型细菌型发酵罐发酵，即，将植物乳杆菌ZJ316按3％菌液接种于5L MRS中发酵。
在本发明中，5L发酵上清液选用500g大孔树脂XAD-16吸附，获得约2L 50％甲醇洗脱液；再经葡聚糖凝胶G25分离，获得G25-2组分；后经反相高效液相色谱(RP-HPLC)制备纯化，RP-HPLC型号及条件为Waters SunFire C18(Prep 5μm 10×100mm)，流动相0.05％三氟乙酸/水(A)(v/v)和0.05％三氟乙酸/乙腈(B)(v/v)。
在本发明中，植物乳杆菌ZJ316发酵上清液中吲哚-3-乳酸的含量是利用分析型HPLC，色谱柱型号为Waters SunFire C18(5μm 4.6×250mm)对一系列浓度梯度的吲哚-3-乳酸标品(Sigma公司)进行分析，建立“峰面积—浓度”的线性关系，得回归方程。利用该方法测定植物乳杆菌ZJ316发酵上清液中吲哚-3-乳酸含量为43.14μg/mL，是目前已有报道的乳酸菌中吲哚-3-乳酸产量最高的植物乳杆菌之一。
本发明所得的吲哚-3-乳酸具有抗菌特性，可有效抑制沙门氏菌、大肠杆菌、藤黄微球菌、金黄色葡萄球菌、铜绿假单胞菌等的生长。
与现有技术相比，本发明具有如下技术优势：
1、本发明利用HPLC测定植物乳杆菌ZJ316发酵上清液中吲哚-3-乳酸的含量为43.14μg/mL，为目前报道的产量最高的植物乳杆菌之一。
2、应用“大孔树脂XAD-16—葡聚糖凝胶G25—反相高效液相色谱(RP-HPLC)”三步法从植物乳杆菌ZJ316发酵上清液中获得吲哚-3-乳酸，纯度为99.00％，且具有广谱抗菌特性。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1是吲哚-3-乳酸标准品回归方程图。
图2是植物乳杆菌ZJ316发酵上清液中吲哚-3-乳酸的分离纯化及鉴定；
图2中：a为葡聚糖凝胶G25分离；b为反相高效液相色谱(RP-HPLC)分离纯化；c为LC-MS的液相色谱图；d为LC-MS的质谱图。
图3是分析型高效液相色谱对吲哚-3-乳酸纯度的分析。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
MRS液体培养基：无水葡萄糖20g，吐温-80 1mL，七水硫酸镁0.2g，硫酸锰0.05g，胰蛋白胨10g，酵母提取物5g，磷酸氢二钾2g，柠檬酸三铵2g，牛肉浸膏10g，无水乙酸钠5g，用超纯水溶解并定容至1L，用37.5％浓盐酸调节培养基pH＝6。
MRS固体培养基：在上述MRS液体培养基中加入2％(质量浓度)的细菌培养基琼脂，用37.5％浓盐酸调节培养基pH＝6。
实施例1：植物乳杆菌ZJ316发酵上清液中吲哚-3-乳酸含量测定
(1)将植物乳杆菌ZJ316菌种划线于MRS固体培养基，37℃培养36h，挑取单菌落于10mL MRS液体培养基中37℃静置培养24h；
(2)将步骤(1)所得培养液以3％接种量(体积浓度)接种于150mL MRS液体培养基中37℃继续静置培养24h；
(3)将步骤(2)所得培养液以3％接种量(体积浓度)接种于5L MRS液体培养基中发酵。发酵条件：温度37℃，转速180rpm，时间24h。
将所得的发酵液离心：8000rpm、25min、4℃，发酵上清液储存于4℃待用。
(4)紫外检测条件：检测器型号Waters 2498、检测器波长：280nm，柱温：25℃，进样量：30μL。对一系列浓度梯度(1.00、2.50、5.00、10.00、25.00、50.00、100.00μg/mL)的吲哚-3-乳酸标准品(购买于Sigma公司)测定。建立出峰的峰面积与浓度的标准曲线，并计算得回归方程y＝6687.8x+1905.4(R 2＝0.9994)，如图1所示。
(5)、分析型HPLC测定步骤(3)所得的发酵上清中吲哚-3-乳酸含量，色谱柱型号为Waters SunFire C18(5μm 4.6×250mm)，取30μL步骤(3)所得的发酵上清液(储存于4℃)注入色谱柱内；洗脱剂由流动相A和流动相B组成：
流动相A：0.05％三氟乙酸/水，即，为体积浓度为0.05％的三氟乙酸水溶液；
流动相B：0.05％三氟乙酸/乙腈，即，为体积浓度为0.05％的三氟乙酸乙腈溶液；
洗脱程序如下表1所述。
表1、HPLC洗脱程序
Figure PCTCN2022074451-appb-000003
280nm紫外检测波长下，将与吲哚-3-乳酸标准品保留时间对应的色谱峰进行峰面积积分，所得峰面积为290395，即y＝290395，代入图1回归方程y＝6687.8x+1905.4(R 2＝0.9994)计算，得植物乳杆菌ZJ316发酵上清液中吲哚-3-乳酸的含量为43.14μg/mL。
实施例2：吲哚-3-乳酸的分离纯化及鉴定
步骤一：大孔树脂XAD-16吸附洗脱
(1)5L植物乳杆菌ZJ316发酵上清液(实施例1步骤(3)所得)经500g大孔树脂XAD-16吸附，流速为1mL/min；然后依次用2L的超纯水、30％甲醇、50％甲醇(37.5％浓 盐酸调节pH＝7)，流速为1mL/min，收集pH＝7的50％甲醇对应的洗脱液(约2L)；
(2)将步骤(1)所得的洗脱液(50％甲醇洗脱液)用旋转蒸发仪浓缩至200mL，得浓缩液；旋转蒸发条件：水浴温度37℃，压力30±5mbar，旋转速度60rpm。
步骤二：葡聚糖凝胶柱Sephadex G25分离
(1)采用Sephadex G25葡聚糖凝胶柱对步骤一所得的浓缩液进行柱分离(柱高80cm，柱直径1.6cm)。
取步骤一所得的浓缩液2mL，过0.22μm滤膜，以超纯水为洗脱液，流速1mL/min，用自动接样器每隔3min接收1管洗脱液，即，每管G25洗脱液的体积为3mL；
(2)用紫外分光光度计检测每管洗脱液在280nm波长下的吸光度值，记录并绘制“吸光度值—管数”，如图2a所示。收集管数“t39、t40、t41、t42”对应的样品，总体积为12mL，记为“G25-2”；
(3)用旋转蒸发仪将步骤(2)所得的G25-2(12mL)浓缩近干，用超纯水重溶至1mL，得G25-2组分，储存于4℃待用；旋转蒸发条件：水浴温度37℃，压力30±5mbar，旋转速度60rpm。
步骤三：反相高效液相色谱(RP-HPLC)纯化
取步骤二所得的G25-2组分(储存于4℃)500μL，用超纯水稀释至5mL。RP-HPLC色谱柱型号为Waters SunFire C18 Prep(5μm 10×100mm)，柱温为25℃，检测器型号Waters 2998，测器波长为280nm，流速为3mL/min。
洗脱剂由流动相A和流动相B组成：
流动相A：0.05％三氟乙酸/水，即，为体积浓度为0.05％的三氟乙酸水溶液；
流动相B：0.05％三氟乙酸/乙腈，即，为体积浓度为0.05％的三氟乙酸乙腈溶液；
洗脱程序如下表2所述。
RP-HPLC色谱图如图2b所示，收集其中出峰起止时间为23.9273～24.5716min出峰样品，所得记为“H4”。
表2、RP-HPLC洗脱程序
Figure PCTCN2022074451-appb-000004
Figure PCTCN2022074451-appb-000005
步骤四：LC-MS分子量测定
将步骤三所得的H4样品(约1.93mL)，用旋转蒸发仪浓缩近干，用超纯水重溶至50μL，旋转蒸发条件：水浴温度37℃，压力30±5mbar，旋转速度60rpm，将浓缩所得物进行分子量鉴定。
LC-MS仪器型号为安捷伦1200-6210，色谱柱型号为Waters SunFire C18(5μm 4.6×250mm)，柱温为30℃，离子源为ESI，负离子模式，离子源气体温度为350℃，干燥气体流量为9L/min，雾化气压力为45psi，毛细管电压为3500V，碎裂电压为125V，质量数扫描范围为50-2000m/z，进样量为10μL。
HPLC洗脱剂由流动相A和流动相B组成：
流动相A：0.1％甲酸/水，即，为体积浓度为0.1％的甲酸水溶液；
流动相B：0.1％甲酸/乙腈，即，为体积浓度为0.1％的甲酸乙腈溶液；
洗脱程序如下表3所述。
流动相：0.1％甲酸水溶液(A)和乙腈(B)，
浓缩的H4样品的LC-MS图谱如图2c、2d所示，图2c为H4样品的液相色谱图，图2d为H4样品的质谱图，确定H4样品的分子量为205。
因此，证明H4组分浓缩后所得物确实为吲哚-3-乳酸。
表3、HPLC洗脱程序
Figure PCTCN2022074451-appb-000006
实施例3：分离纯化的吲哚-3-乳酸的纯度测定
取实施例2步骤三的浓缩样品“H4”，按照实施例1步骤(5)中所述的分析型HPLC检测条件和洗脱程序，得到吲哚-3-乳酸的色谱图，如图3所示。主成分峰(即，吲哚-3-乳酸)及2个杂峰的出峰起止时间(min)、峰面积(μV·s)、峰面积比例(％)如表4所示。根据峰面积归一化法，主成分峰(即，吲哚-3-乳酸)面积为A，主成分峰与其余杂峰峰面积总和为 ∑A，得吲哚-3-乳酸纯度(％)计算公式：
吲哚-3-乳酸纯度(％)＝A/∑A×100％。
表4、HPLC色谱峰的峰面积
Figure PCTCN2022074451-appb-000007
因此，通过实施例2步骤一～三分离纯化得到：
吲哚-3-乳酸纯度(％)＝A/(A+A1+A2)×100％
＝691433/(691433+4028+2933)×100％
＝99.00％。
实施例4：吲哚-3-乳酸的抗菌活性
采用牛津杯法对实施例2步骤三纯化得到的吲哚-3-乳酸(即，浓缩H4，纯度99.00％)进行抗菌活性的测定。添加1％(体积浓度)的指示菌，150μL浓度10mg/mL吲哚-3-乳酸(1％乙腈水溶液溶解)。结果显示该植物乳杆菌ZJ316产生的吲哚-3-乳酸对指示菌的生长均有抑制作用(见表5)，包括革兰氏阳性菌(藤黄微球菌、金黄色葡萄球菌、肉葡萄球菌、模仿葡萄球菌等)，革兰氏阴性菌(大肠杆菌、甲/乙型副伤寒沙门氏菌、肠炎沙门氏菌、猪霍乱沙门氏菌、鼠伤寒沙门氏菌等)。
表5、植物乳杆菌ZJ316产生的吲哚-3-乳酸的抑菌谱
Figure PCTCN2022074451-appb-000008
Figure PCTCN2022074451-appb-000009
对比例1、将实施例1中的植物乳杆菌ZJ316，改成现有的其余植物乳杆菌菌株，具体如下表6所述，其余参照实施例1，检测植物乳杆菌发酵上清液中吲哚-3-乳酸的含量(μg/mL)。所得实验结果与本发明的对比如表6所述。
表6
Figure PCTCN2022074451-appb-000010
对比例2、将实施例1所得的植物乳杆菌ZJ316发酵上清液进行超滤离心，超滤离心的工艺参数具体如下：植物乳杆菌ZJ316上清液过0.22μm滤膜，用3kDa超滤离心管(Millipore Amicon Ultra)离心(4000g、4℃、30min)，收集滤液。将超滤离心条件下的所得滤液，按照上述实施例3中所述方法进行纯度的检测，所得吲哚-3-乳酸的纯度约为40％。
对比例3、将实施例2步骤一中的“大孔树脂XAD-16”改成“大孔树脂XAD-2”，其余等同于实施例2。所得吲哚-3-乳酸的纯度对应约为95％。
对比例4-1、将实施例2步骤一中的“30％甲醇”改成“25％甲醇”，其余等同于实施例2。所得吲哚-3-乳酸的纯度约为83％。
对比例4-2、将实施例2步骤一中的“30％甲醇”改成“35％甲醇”，其余等同于实施例2。所得吲哚-3-乳酸的纯度约为94％。
对比例4-3、取消实施例2步骤一中的“30％甲醇”的使用，即，改成“用2L的超纯水、50％甲醇(pH＝7)”，其余等同于实施例2。所得吲哚-3-乳酸的纯度约为35％。
对比例4-4、将实施例2步骤一中的“50％甲醇(pH＝7)”改成“50％甲醇”，即，取消对50％甲醇的pH调节，其余等同于实施例2。所得吲哚-3-乳酸的纯度约为75％。
对比例5、将实施例2步骤二中的“葡聚糖凝胶柱Sephadex G25”改成“葡聚糖凝胶柱Sephadex G50柱”或“葡聚糖凝胶柱Sephadex G15柱”，其余等同于实施例2。改用“葡聚糖凝胶柱Sephadex G50柱”，无法得到与图2a类似的色谱图，只含有1个样品峰，即无法得到组分G25-2；改用“葡聚糖凝胶柱Sephadex G15柱”，可得到与图2a类似的色谱图，含有2个样品峰，即，可得到组分G25-2，其余等同于实施例2。所得吲哚-3-乳酸的纯度对应约为78％。
对比例6、将实施例2步骤二中的葡聚糖凝胶柱Sephadex G25对应的流速“1mL/min”改成“0.5mL/min”或“1.5mL/min”，其余等同于实施例2。所得吲哚-3-乳酸的纯度对应约为98％、70％。
对比例7、将实施例2步骤三中的反相高效液相色谱(RP-HPLC)所用的色谱柱“Waters SunFire C18 Prep(5μm 10×100mm)”改成“YMC-Pack Pro C18(5μm 20×150mm)”，其余等同于实施例2。则无法得到与图2b类似的色谱图，即，无法得到单峰组分H4。
对比例8、将实施例2步骤三中的反相高效液相色谱(RP-HPLC)洗脱程序改为如下：
0～10min，流动相B 5％；10～30min，流动相B 5～50％；30～35min，流动相B 50～95％；35～38min，流动相B 95～5％；38～40min，流动相B 5％；流量为3mL/min。
或0～10min，流动相B 10％；10～30min，流动相B 10～50％；30～35min，流动相B 50～95％；35～38min，流动相B 95～10％；38～40min，流动相B 10％；流量为3mL/min。
或0～10min，流动相B 5％；10～30min，流动相B 5～40％；30～35min，流动相B 40～95％；35～38min，流动相B 95～5％；38～40min，流动相B 5％；流量为3mL/min。
或0～10min，流动相B 10％；10～30min，流动相B 10～40％；30～35min，流动相B 40～95％；35～38min，流动相B 95～10％；38～40min，流动相B 10％；流量为2mL/min。
或0～10min，流动相B 10％；10～30min，流动相B 10～40％；30～35min，流动相B 40～95％；35～38min，流动相B 95～10％；38～40min，流动相B 10％；流量为4mL/min。
其余等同于实施例2。所得吲哚-3-乳酸的纯度对应约为92％、96％、96％、90％、82％。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。

Claims (7)

  1. 从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法，利用保藏编号CCTCC NO:M 208077的植物乳杆菌ZJ316的发酵上清，其特征在于包括以下步骤：
    1)、将发酵上清先用大孔树脂XAD-16吸附，然后依次用超纯水、30％甲醇、pH为7的50％甲醇进行洗脱，洗脱流速均为1±0.1mL/min，收集pH为7的50％甲醇对应的洗脱液；
    将所述洗脱液进行浓缩，得浓缩液；
    2)、将步骤1)所得的浓缩液经葡聚糖凝胶G25分离，获得G25-2组分；
    3)、将步骤2)所得的G25-2组分经反相高效液相色谱纯化：
    流动相A为体积浓度为0.05％的三氟乙酸水溶液；
    流动相B为体积浓度为0.05％的三氟乙酸乙腈溶液；
    洗脱剂由流动相A、流动相B组成，流速为3±0.1mL/min，洗脱程序如下：
    RP-HPLC洗脱程序
    Figure PCTCN2022074451-appb-100001
    收集出峰时间为23.9273～24.5716min的洗脱液，作为H4组分；经浓缩，得吲哚-3-乳酸。
  2. 根据权利要求1所述的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法，其特征在于：所述步骤1)中，将洗脱液进行浓缩至原体积的9～11％，得浓缩液。
  3. 根据权利要求1或2所述的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法，其特征在于所述步骤1)为：
    5L植物乳杆菌ZJ316上清液经500g大孔树脂XAD-16吸附；然后依次用2L的超纯水、2L的30％甲醇、2L的pH为7的50％甲醇进行洗脱，洗脱流速均为1mL/min，收集pH为7的50％甲醇对应的洗脱液。
  4. 根据权利要求1所述的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法，其特征在于所述步骤2)包括：
    取步骤1)所得的浓缩液2mL，过0.22μm滤膜，上样至Sephadex G25葡聚糖凝胶柱；
    以超纯水为洗脱液，流速1±0.1mL/min，每隔3min接收1管洗脱液，收集t39、t40、t41、t42这4管洗脱液；
    将洗脱液浓缩，得G25-2组分。
  5. 根据权利要求4所述的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法，其特征在于：所述步骤2)的浓缩为：将洗脱液浓缩近干，用超纯水重溶至1mL，得G25-2组分。
  6. 根据权利要求4或5所述的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法，其特征在于：所述步骤3)为：
    取步骤2)所得的G25-2组分500μL，用超纯水稀释至5mL后，上样至Waters SunFire C18 Prep，柱温为25℃，获得H4组分；
    所述步骤3)所得的H4组分浓缩近干，得吲哚-3-乳酸。
  7. 根据权利要求1～6任一所述的从植物乳酸菌发酵上清中纯化吲哚-3-乳酸的方法，其特征在于发酵上清的制备方法为：
    将保藏编号CCTCC NO:M 208077的植物乳杆菌ZJ316按3％菌液接种量接种于MRS培养基中发酵，转速180rpm，37℃培养24h；发酵液离心，获得上清。
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