CN111358798A - New application of oligosaccharide ferulic acid ester - Google Patents
New application of oligosaccharide ferulic acid ester Download PDFInfo
- Publication number
- CN111358798A CN111358798A CN202010174590.5A CN202010174590A CN111358798A CN 111358798 A CN111358798 A CN 111358798A CN 202010174590 A CN202010174590 A CN 202010174590A CN 111358798 A CN111358798 A CN 111358798A
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- CN
- China
- Prior art keywords
- ferulic acid
- acid ester
- oligosaccharide
- clostridium difficile
- lactobacillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Images
Classifications
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
The application belongs to the technical field of microorganisms, and particularly relates to a new application of oligosaccharide ferulic acid ester. In a first aspect, the present application provides the use of a mixed system of the oligosaccharides ferulic acid ester and lactobacillus for inhibiting the growth of clostridium difficile. In a second aspect, the present application provides a composition for inhibiting the growth of clostridium difficile comprising the oligosaccharides ferulic acid ester and lactobacillus. The application discovers the new application of oligosaccharide ferulic acid ester, enriches the types of medicaments for inhibiting the growth of clostridium difficile, and thus provides a new medicament for treating clostridium difficile infection.
Description
Technical Field
The application belongs to the technical field of microorganisms, and particularly relates to a new application of oligosaccharide ferulic acid ester.
Background
The Clostridium is a gram-positive obligate spore-forming anaerobic bacterium, comprising more than 100 species. Most of them can hydrolyze carbohydrate and protein in food, are greatly influenced by host diet, are microorganisms closely related to food metabolism and absorption, and are closely related to various diseases of the host. Difficile is a representative species of the genus clostridium, and is a normal flora in the human intestinal tract. When the intestinal flora homeostasis is destroyed, clostridium difficile can multiply in a large quantity and produce enterotoxin and cytotoxin, thereby destroying the intestinal mucosa barrier and causing fatal diseases such as diarrhea, pseudomembranous enteritis and the like. Clinically, about 10% to 20% antibiotic-associated diarrhea, 75% antibiotic-associated enteritis, and nearly 100% pseudomembranous enteritis are associated with this bacterium.
Clostridium Difficile Infection (CDI) has attracted worldwide attention. In some asian countries, CDI rates have been on the rise with the heavy use of broad-spectrum antibiotics over the last 20 years. Studies have shown that the mortality rate caused by CDI is 2% to 7%. Not only does CDI lead to prolonged patient hospitalization, it also causes the transmission of c. Metronidazole and vancomycin are currently the first choice drugs for treating CDI. However, researches show that the early recurrence rate of CDI patients after receiving treatment of vancomycin and metronidazole is as high as 15-35%, and the patients with recurrence are difficult to treat and have high treatment cost. Clostridium difficile strains with reduced sensitivity to metronidazole and vancomycin are found, and the drug resistance rate of Clostridium difficile to metronidazole is gradually increased, which brings difficulty to clinical treatment of CDI. Research shows that vancomycin treatment can reduce the proportion of normal obligate anaerobe in the whole intestinal flora and increase the proportion of facultative anaerobe and microaerophilic flora. Therefore, it is of great significance to study functional components that can inhibit the growth of clostridium difficile.
Disclosure of Invention
In view of the above, the present application finds a new application of oligosaccharide ferulic acid ester, and enriches the drug classes for inhibiting the growth of clostridium difficile.
In a first aspect, the present application provides the use of a mixed system of the oligosaccharides ferulic acid ester and lactobacillus for inhibiting the growth of clostridium difficile.
The oligosaccharide ferulic acid ester is a water-soluble compound formed by esterifying ferulic acid and oligosaccharide, contains structural characteristics of both ferulic acid and hydrophilic oligosaccharide groups, has dual activities of ferulic acid and oligosaccharide, and is an active substance with physiological functions of ferulic acid and oligosaccharide.
Preferably, the ratio of lactobacillus to clostridium difficile is 1: 1.
Preferably, the concentration of the oligosaccharide ferulic acid ester is 5 mg/mL-20 mg/mL.
Preferably, the lactobacillus is selected from lactobacillus plantarum.
Preferably, the preparation method of the oligosaccharide ferulic acid ester comprises the following steps:
the cellulose raw material is subjected to enzymolysis or acidolysis to obtain oligosaccharide ferulic acid ester.
Specifically, the cellulosic material is selected from one or more of bagasse, corn bran, wheat bran, and rice bran.
Wherein the oligosaccharide ferulic acid ester extracted from corn bran has been confirmed by structure:
(5-O-FA--L-Ara)-1,3-Xyl-1,4-D-Xyl)；
O--D-Xyl-1,2-(O-5-O-FA-L-Ara)-O--D-Xyl-1,3--D-Xyl-1,2-O-(5-O-FA-L-Ara)；
O-L-Gal-1,4-O-D-Xyl-1.2-(5-O-FA-L-Ara)-O-4-O-FA--D-Xyl-1,6-D-Gluc；
-D-Xyl-1,3--L-Gal-1,2--D-Xyl-1,2–5-O-FA-L-Ara；
-D-Gal-1, 3-L-Gal-1, 2-D-Xyl-1, 2-5-O-FA-L-Ara. (wherein FA represents ferulic acid, Xyl represents a xylose residue, Ara represents an arabinose residue, and Gal represents a galactose residue).
The preparation method of the oligosaccharide ferulic acid ester comprises the steps of drying and crushing corn bran, sieving the crushed corn bran with a 40-mesh sieve, removing residual starch by using high-temperature resistant α -amylase, removing protein by using protease, boiling to inactivate the enzyme, filtering to leave insoluble residues, drying, cooking for 30min at 121 ℃ for extracting the oligosaccharide ferulic acid ester by using a feed-liquid ratio of 1:10 and an oxalic acid concentration of 0.6% (w/v), filtering to obtain an acidolysis solution, filtering the acidolysis solution by using an ultrafiltration membrane with a molecular weight of 5ku and a nanofiltration membrane with a molecular weight of 150u to remove macromolecular impurities such as macromolecular impurities and oxalic acid, purifying an extracting solution subjected to ultrafiltration and nanofiltration treatment by using resin, concentrating, and freeze-drying to obtain a powdery extract.
The acid hydrolysis extract after ultrafiltration and nanofiltration also contains a large amount of monosaccharide, oligosaccharide and free ferulic acid which are not esterified with ferulic acid. The oligosaccharide ferulic acid ester is further purified by resin to remove the impurities. In the embodiment, common D301 and HP-20 resins are selected to purify oligosaccharide ferulic acid ester, ethanol is adopted to elute oligosaccharide ferulic acid ester adsorbed by the D301 and HP-20 resins, ethanol eluent is subjected to vacuum concentration, and then spray drying is carried out, so that high-purity solid oligosaccharide ferulic acid ester is obtained.
Preferably, in the above preparation method, the volume percentage of ethanol is 70%, and the application finds that the elution rate is increased along with the increase of the ethanol concentration, the elution rate reaches the highest when the ethanol concentration is 70%, and the elution rate of the oligosaccharide ferulic acid ester begins to decrease when the ethanol concentration continues to increase.
Preferably, in the preparation method, the resin is HP-20 resin, and the application finds that HP-20 has stronger adsorption performance on ferulic acid and oligosaccharide ferulic acid ester than D301, and is easier to elute when ethanol is used for elution.
The indexes of the corn bran oligosaccharide ferulic acid ester prepared by the method are respectively as follows: the total sugar content is 734.33mg/g, the reducing sugar content is 178.94mg/g, the total ferulic acid content is 93.58mg/g, the free ferulic acid content is 5.79mg/g, and the average polymerization degree of oligosaccharide is 4.1. It can be seen that only 6.19% of the total ferulic acid in the oligosaccharide ferulic acid ester prepared in this example is free ferulic acid, and 93.81% of ferulic acid is combined with oligosaccharide.
The application provides a preparation method of a second oligosaccharide ferulic acid ester, which specifically comprises the following steps: mixing the pretreated cellulosic raw material with 1-2% acid liquor, mixing under a heating condition, and filtering to obtain filtrate and filter residue; removing the soluble macromolecules which are not acidolyzed from the filtrate by ultrafiltration to obtain a permeate; the mass volume ratio of the pretreated cellulosic raw material to the acid liquor is 0.05-0.1 kg/L.
The pretreatment method specifically comprises the steps of crushing, starch removing and protein removing, wherein the crushing is to inactivate enzyme and dry the cellulosic raw material at 105 ℃ for 4 hours and crush the cellulosic raw material to 80-120 meshes, the starch removing is to uniformly mix the crushed cellulosic raw material and water according to the mass-to-volume ratio of 0.1kg/L to obtain a mixed solution, the mixed solution is gelatinized at 90 ℃ for 30 minutes, then high-temperature resistant α -amylase is added, the addition amount of the high-temperature resistant α -amylase is 0.2-0.3% of the mass of the mixed solution, enzymolysis is carried out at 90 ℃ for 2-3 hours, the protein removing is to reduce the temperature of the starch-removed cellulosic raw material to 65 ℃, protease is added, the addition amount of the protease is 1% of the mass of the starch-removed cellulosic raw material, the reaction is carried out for 30 minutes, then the filtering is carried out, and filter residues are washed by water for 3-5 times to obtain the pretreated cellulosic raw material.
Specifically, the acid in the acid solution is selected from oxalic acid, hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid.
Specifically, the heating temperature is 60-100 ℃, and the stirring time is 2-3 hours; the cut-off molecular weight of the ultrafiltration is 3000-10000.
Preferably, the method further comprises the steps of subjecting the cellulosic raw material to enzymolysis or acidolysis, adsorbing the product by ion exchange resin, and eluting to obtain the oligosaccharide ferulic acid ester.
Specifically, the ion exchange resin adsorption treatment specifically includes: after the permeation liquid obtained by enzymolysis or acidolysis of the cellulosic raw material is absorbed by resin, the resin is washed by water to remove oligosaccharide; then eluting with ethanol water solution with the volume percentage concentration of 50-60 percent to obtain ethanol eluent; concentrating the ethanol eluate to obtain a concentrated solution; drying the concentrated solution to obtain oligosaccharide ferulic acid ester; the resin is a D301 macroporous weakly basic anion exchange resin.
Specifically, the solid-liquid volume ratio of the resin to the permeate is (3-4) to 1.
Specifically, the resin is washed with water until the volume of the washing liquid reaches 8 times of the volume of the resin; the volume of the ethanol eluent is 5 times that of the resin; the volume of the concentrated solution is 1/20-1/40 of the volume of the ethanol eluent.
Specifically, the concentration is vacuum concentration, and the concentration temperature is 40-60 ℃; the drying is spray drying, and the drying temperature is 80-100 ℃.
It should be noted that trans-ferulic acid in the cellulose of the present application is mainly bonded to polysaccharide and lignin by ester bonds, and weak glycoside bonds can be broken by acid treatment while most of ferulic acid ester bonds are retained, thereby forming oligosaccharide ferulic acid esters with different sugar chain lengths. In the acid treatment process, after the sugar chain segment without ferulic acid is acidolyzed, oligosaccharide is formed; in addition, a small amount of ferulic acid ester bond is also subjected to acidolysis to form free ferulic acid. The oligosaccharide ferulic acid ester and free ferulic acid can be retained on the resin by adsorption of D301 macroporous weak base anion exchange resin, while oligosaccharide without ferulic acid is removed by washing, and the oligosaccharide ferulic acid ester with weak adsorption capability is eluted by 50-60% ethanol solution according to the strong adsorption capability of the D301 resin to the free ferulic acid, thereby obtaining high-purity oligosaccharide ferulic acid ester solution. The solution is concentrated in vacuum to remove most of ethanol, and finally spray-dried to obtain a high-purity oligosaccharide ferulic acid ester solid product.
Specifically, the oligosaccharide ferulic acid ester provided by the application is a product containing 2-12 monosaccharides and a hydroxyl group of part of arabinose esterified by ferulic acid, and the specific structural formula is as follows:
the oligosaccharide ferulic acid ester is a water-soluble compound (structure formula 1) formed by esterifying carbohydrate hydroxyl and ferulic acid carboxyl at different positions in oligosaccharide, and can be obtained by enzymolysis or acidolysis of ferulic acid polysaccharide (formed by esterifying ferulic acid with arabinose, galactose or galactose residue in pectin) in plant cell wall. Compared with ferulic acid polysaccharide, the oligosaccharide ferulic acid ester is easier to ferment in colon, and has dual functions of oligosaccharide and ferulic acid.
In a second aspect, the present application provides a composition for inhibiting the growth of clostridium difficile comprising the oligosaccharides ferulic acid ester and lactobacillus.
In particular, the compositions of the present application are useful for inhibiting the growth of clostridium difficile.
In particular, the application of the composition in the preparation of products for inhibiting the growth of clostridium difficile.
Preferably, the ratio of lactobacillus to clostridium difficile is 1: 1.
More preferably, the concentration of the oligosaccharide ferulic acid ester is 5 mg/mL-20 mg/mL.
Preferably, the lactobacillus is selected from lactobacillus plantarum.
Preferably, the preparation method of the oligosaccharide ferulic acid ester comprises the following steps:
the cellulose raw material is subjected to enzymolysis or acidolysis to obtain oligosaccharide ferulic acid ester.
Experiments prove that the single oligosaccharide ferulic acid ester does not inhibit the action of clostridium difficile. However, the present application found that the mixed system of oligosaccharide ferulic acid ester and lactobacillus plantarum has the effect of inhibiting clostridium difficile.
The lactobacillus plantarum and the clostridium difficile are mixed and cultured, and the influence of oligosaccharide ferulic acid ester on the form and the number ratio of the two bacteria in a mixed culture system is researched by means of a microscope. The results show that the oligosaccharide ferulic acid ester has no obvious direct inhibition effect on clostridium difficile. However, in the mixed culture system, the oligosaccharide ferulic acid ester promotes the growth of lactobacillus plantarum, but inhibits the growth of clostridium difficile.
In conclusion, the oligosaccharide ferulic acid ester can be used as an additive in the food industry. After being ingested, the oligosaccharide ferulic acid ester can promote the growth of lactobacillus and inhibit the growth of clostridium difficile in intestinal tracts, which is beneficial to exerting the normal physiological function of the intestinal tracts and maintaining the health of organisms.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a graph showing the results of the action of oligosaccharide ferulic acid ester on Lactobacillus plantarum provided in the examples of the present application;
FIG. 2 is a graph showing the results of the present application on different concentrations of oligosaccharide ferulic acid ester for treating total bacteria in a mixed system of Lactobacillus plantarum and Clostridium difficile;
FIG. 3 is a graph showing the results of the ratio of oligosaccharide ferulic acid ester to Lactobacillus plantarum in a mixed system of Lactobacillus plantarum and Clostridium difficile provided in the examples of the present application;
FIG. 4 is a graph showing the results of oligosaccharide ferulic acid ester on the ratio of Clostridium difficile in a mixed system of Lactobacillus plantarum and Clostridium difficile provided in the examples of the present application;
FIG. 5 shows the form of Clostridium difficile, Lactobacillus plantarum, a mixed strain thereof, and a mixed strain thereof under an oil lens (10 × 100) treated with an FOs solution for 48 hours according to the present embodiment;
figure 6 is a graph of the effect of the present application on different concentrations of oligosaccharide ferulic acid ester alone on clostridium difficile.
Detailed Description
The application provides a method for preparing the same, which is used for overcoming the technical defects in the prior art.
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Wherein, clostridium difficile ATCC 700057 and lactobacillus plantarum ATCC 8014 are purchased from Guangdong province microorganism strain collection center; vancomycin hydrochloride (biotech grade) was purchased from Shanghai Merlin Biotech limited;
the culture medium for culturing clostridium difficile is brain heart infusion agar medium (BHI). BHI is a common non-selective medium formulated to which L-Cys is added as a reducing agent from time to time.
Minimum Inhibitory Concentration (MIC): the lowest drug concentration which can inhibit the growth of pathogenic bacteria in a culture medium after culturing the bacteria in vitro for a certain time is an index for measuring the antibacterial activity of the antibacterial drug;
optical Density (OD): the optical density of the light absorbed by the test object is a term used in the detection method, and the detection unit is expressed by OD value, 1OD is log10(1/trans), wherein trans is the light transmittance T value of the detection object. The energy difference between the front and the back when the light passes through the object to be detected is the energy absorbed by the object to be detected, and the concentration of the same object to be detected and the absorbed energy are in quantitative relation under specific wavelength.
OD600: called turbidity, at 600nm there is a linear relationship between the concentration of regularly shaped microorganisms and the absorbance, such as bacteria, yeast, etc. It is a standard method for tracking the growth of microorganisms in liquid culture. The culture solution without the addition of the bacterial solution is used as a blank solution, and then the cultured bacterial-containing culture solution is quantitatively determined.
Example 1
The application provides a test for preparing oligosaccharide ferulic acid ester by using corn bran, and the specific preparation method comprises the following steps:
referring to the effects of Huangru qing (Huangru qing, Oeshi. oxalic acid cooking treatment and surfactant on the preparation of oligosaccharide ferulic acid ester by enzyme method, Chinese food science, 2012,05,71-75) or a method of patent No. CN101628922B, the oligosaccharide ferulic acid ester is extracted by hydrolyzing corn bran at high temperature with oxalic acid or hydrochloric acid.
The method mainly comprises the steps of drying and crushing corn bran, sieving the corn bran with a 40-mesh sieve, removing residual starch by using high-temperature resistant α -amylase, removing protein by using protease, boiling to inactivate enzyme, filtering to leave insoluble residues, drying, cooking for 30min at 121 ℃ according to a feed-liquid ratio of 1:10 and an oxalic acid concentration of 0.6% (w/v) to extract oligosaccharide ferulic acid ester, filtering to obtain acidolysis solution, filtering the acidolysis solution by using an ultrafiltration membrane with a molecular weight of 5ku and a nanofiltration membrane with a molecular weight of 150u to remove macromolecular impurities, oxalic acid and other small molecular impurities, purifying the extracting solution subjected to ultrafiltration and nanofiltration treatment by using resin, concentrating, and freeze-drying to obtain a powdery extract.
The acid hydrolysis extract after ultrafiltration and nanofiltration also contains a large amount of monosaccharide, oligosaccharide and free ferulic acid which are not esterified with ferulic acid. The oligosaccharide ferulic acid ester is further purified by resin to remove the impurities. In this embodiment, common D301 and HP-20 resins are selected to purify oligosaccharide ferulic acid ester, then ethanol is used to elute oligosaccharide ferulic acid ester adsorbed by D301 and HP-20 resins, ethanol eluent is vacuum-concentrated, and then spray-dried to obtain high-purity solid oligosaccharide ferulic acid ester (FOs for short), and the solid oligosaccharide ferulic acid ester is dissolved by deionized water to obtain a FOs liquid medicine (which is a mother solution of FOs liquid medicine) with high concentration.
In this example, HP-20 was found to have a stronger adsorption of ferulic acid and oligosaccharide ferulic acid ester than D301 and to elute more easily when eluted with ethanol, so that HP-20 was subsequently used as a resin for oligosaccharide ferulic acid ester purification. In this example, it is found that the elution rate increases with the increase of the ethanol concentration, the elution rate reaches the highest when the ethanol concentration is 70%, and the elution rate of the oligosaccharide ferulic acid ester starts to decrease when the ethanol concentration continues to increase. Therefore, 70% ethanol was finally determined to be used as eluent.
The free ferulic acid and the total ferulic acid of the oligosaccharide ferulic acid ester prepared in the example are measured by the following method:
measuring free ferulic acid by directly measuring ferulic acid content in bran acidolysis solution by HPLC (Zhaoyang method) (Zhaoyang, Oushaiyi, Linqiling, Linhaiwei, oligosaccharide ferulic acid ester content rapid measurement method. food science 2010,18, 329-plus 332), wherein the liquid phase conditions are Agilent Eclipse C18 chromatographic column (4.6mm × 250mm,5 μm), the mobile phase comprises volume fraction of 1% acetic acid solution-methanol (72:28, V/V), the detection wavelength is 313nm, the flow rate is 1mL/min, the column temperature is 40 ℃, the sample introduction amount is 10 μ L, the retention time is used for qualitative determination, the area normalization method is used for calculating the relative content, the standard curve of the ferulic acid standard is prepared by respectively preparing 25, 50, 75, 100 and 125 μ g/mL ferulic acid standard ethanol solutions, and the linear equation of y and the mass concentration x is y-27134, R51160 x-271342＝0.9999。
Determination of total ferulic acid: 10mL of bran acidolysis solution is taken to be put in a 100mL conical flask, 10mL of NaOH with the concentration of 2mol/L is added, the mixture is placed in a shaking table for light-shielding oscillation reaction for 60min at the temperature of 35 ℃, then the excessive NaOH is neutralized to be neutral by hydrochloric acid with the concentration of 2mol/L, and then the total ferulic acid content is determined by the method for determining free ferulic acid.
The indexes of the oligosaccharide ferulic acid ester powder prepared by purifying the resin HP-20 are respectively as follows: the total sugar content is 734.33mg/g, the reducing sugar content is 178.94mg/g, the total ferulic acid content is 93.58mg/g, the free ferulic acid content is 5.79mg/g, and the average polymerization degree of oligosaccharide is 4.1. It can be seen that only 6.19% of the total ferulic acid in the oligosaccharide ferulic acid ester prepared in this example is free ferulic acid, and 93.81% of ferulic acid is combined with oligosaccharide.
Example 2
The embodiment of the application provides a preparation test of clostridium difficile bacterial liquid, which comprises the following specific steps:
5.22g of BHI solid medium and 4.31g of Brookfield agar were weighed and dissolved in 100mL of distilled water, respectively, to prepare a solid medium. 3.7g of BHI brain-heart extract broth and 0.05g of L-Cys were weighed and dissolved in 100mL of distilled water to prepare a liquid medium. The prepared liquid culture medium, solid culture medium, subpackaged flat plates and 10mL centrifuge tubes are wrapped by double-layer newspaper, and then the wrapped flat plates and centrifuge tubes are placed in a high-pressure steam sterilization pot at the temperature of 121 ℃ for sterilization for 20 min. Transferring the sterilized culture medium to a clean bench, pouring the solid culture medium into a flat plate after the solid culture medium is cooled to about 50 ℃, and subpackaging the liquid culture medium into sterilized 10mL centrifuge tubes. And finally, transferring the flat plate and the centrifuge tube to an anaerobic workstation for pre-reduction.
According to the clinical laboratory standards Committee (CLSI), the frozen strain must be subcultured at least twice in medium, the turbidity of the inoculum in the dilution test is 0.5 McLeod, which corresponds to a suspension containing (1-4) × 107Clostridium difficile ATCC 700057 per mL. The preparation of the inoculum during dilution adopts a growth method, selects growth bacterial colonies which are incubated on a Buchner agar plate for 48 hours and have the diameter of 1mm, gently picks 1-3 bacterial colonies which are well separated and have similar shapes, inoculates the bacterial colonies in brain-heart infusion broth, and incubates the bacterial colonies for 10 hours at 37 ℃ to achieve sufficient bacterial liquid turbidity. The OD of the bacterial suspension at this time was measured6001.003, OD after 100-fold dilution600Is 0.017 (OD)6000.01≈1×106CFU/mL), closer to the prescribed inoculum turbidity standard, made 1 × 106C, CFU/mL of clostridium difficile bacterial liquid.
Example 3
The embodiment of the application provides a test for culturing oligosaccharide ferulic acid ester and lactobacillus plantarum after mixing, which comprises the following specific steps:
sterilized 96-well plates, BHI broth were placed in an anaerobic workstation for pre-reduction for 24h prior to use. And taking out 2mL of each of the frozen drug diluents, and putting the drug diluents into an anaerobic workstation for later use. Three columns are arranged, three parallel groups of n1, n2 and n3 are arranged in each column, the first column is a blank group, and only 200 mu l of BHI broth is contained in the hole without lactobacillus plantarum liquid; the second column is a positive control, and the wells contained 100. mu.l of BHI broth and 100. mu.l of Lactobacillus plantarum bacterial suspension (OD of Lactobacillus plantarum bacterial suspension)600Is 0.010 ≈ 1 × 106CFU/mL), without FOs solution. The third row contains the Lactobacillus plantarum solution and the FOs solution obtained in example 1And adding 200 mu l of FOs liquid medicine into three parallel holes in the third row respectively, sucking 100 mu l of the FOs liquid medicine into the next hole respectively, adding 100 mu l of culture medium filtered by a microporous membrane to double-dilute the medicine, sucking 100 mu l of the diluted liquid medicine into the next hole, repeating the operation, and discarding 100 mu l of the last hole. After dilution, 100. mu.l of Lactobacillus plantarum suspension (OD of Lactobacillus plantarum solution) was added to each well of the third column600Is 0.010 ≈ 1 × 106CFU/mL), thus forming three replicates of determining the MIC value of FOs. Final FOs (mg/mL) drug concentrations per well were: 20; 10; 5; 2.5; 1.25. incubating the 96-well plate in an anaerobic constant temperature box at 37 ℃ for 48h, taking out the 96-well plate, and measuring OD (optical density) of each well by using an enzyme-labeling instrument600。
The data were analyzed using SPSS 24.0 software. Multiple comparisons of different samples were performed using the Duncan's multiple range test (p < 0.05) to determine significant differences between means. The results are shown in FIG. 1, and FIG. 1 is a graph showing the effect of oligosaccharide ferulic acid ester on Lactobacillus plantarum provided in the examples of the present application.
The results showed that when the concentration of FOs in the FOs solution was 1.25mg/mL, the OD of the Lactobacillus plantarum suspension increased with the increase in the concentration of FOs600Is obviously improved, which shows that FOs has obvious promotion effect on lactobacillus plantarum.
Example 4
The embodiment of the application provides a test for mixed culture of oligosaccharide ferulic acid ester, lactobacillus plantarum and clostridium difficile bacterial liquid, which comprises the following specific steps:
taking out the clostridium difficile and lactobacillus plantarum conservation tube from a refrigerator at the temperature of minus 80 ℃, transferring the tube to an anaerobic workstation by using ice blocks, respectively using sterile inoculating loops to take a small amount of conservation bacteria liquid to be scribed on a Brinell plate, rotating the plate by 90 ℃ after scribing an 1/4 area, using a new inoculating loop, continuing scribing in a Z shape, and repeating the operation on the plate for four times to ensure the separation and purification of single bacterial colony. After subculture, transferring 1-3 colonies growing for 48h to BHI broth, growing for 10h until OD of two bacterial suspensions600When the values are relatively similar (Clostridium difficile OD)600Is 0.017; lactobacillus plantarum OD600Is 0.010; wherein OD of Clostridium difficile bacterial liquid600Is 0.017 ≈ 1 × 106CFU/mL, OD of Lactobacillus plantarum solution600Is 0.010 ≈ 1 × 106CFU/mL), the number of bacteria in the bacterial suspension was about the same. Then mixing the two bacterial suspensions in a ratio of 1:1 to prepare a mixed bacterial liquid, and incubating the mixed bacterial liquid in a 96-well plate containing the FOs liquid medicine with gradient dilution, wherein the final FOs (mg/mL) medicine concentration of each well is respectively as follows: 20; 10; 5; 2.5; OD was performed with microplate reader after 1.25, 48h600Measurement and microscopic observation.
The microscopic observation comprises the steps of firstly dropping a small drop of physiological saline in the center of a glass slide, selecting single bacterial colonies (lactobacillus plantarum and clostridium difficile) with good shapes from a flat plate by using an inoculating loop, uniformly mixing the single bacterial colonies with the water drops on the glass slide, coating the single bacterial colonies into a thin layer, quickly moving the single bacterial colonies to and fro 3-4 times outside an alcohol lamp flame after smearing to dry and fix the single bacterial colonies, ensuring that the temperature of the glass slide is not more than 60 ℃, preferably ensuring that the back of the glass slide is not scalded when touching the skin of the back of the hand, placing the glass slide for cooling, dyeing the glass slide by using a gram kit, observing the normal shapes of the clostridium difficile and the lactobacillus plantarum by using an oil lens (10 ×), sucking 100 mu l of mixed bacterial suspension processed by FOs and 20mg/mL of FOs liquid medicine liquid for 48 hours into a centrifuge tube, sucking the culture medium in the bacterial suspension after centrifugal precipitation, adding 100 mu l of physiological saline to uniformly mix the mixed bacterial suspension, transferring the mixed bacterial suspension onto a cover glass slide after drying and fixing, and observing the morphological change of the two bacterial cells after the medicine.
The results are shown in fig. 2-5, fig. 2 is a graph of the results of the application of treating total bacteria in a mixed system of Lactobacillus plantarum and clostridium difficile with different concentrations of oligosaccharide ferulic acid ester, fig. 3 is a graph of the results of the application of oligosaccharide ferulic acid ester on the ratio of Lactobacillus plantarum in a mixed system of Lactobacillus plantarum and clostridium difficile, fig. 4 is a graph of the results of the application of oligosaccharide ferulic acid ester on the ratio of clostridium difficile in a mixed system of Lactobacillus plantarum and clostridium difficile, fig. 5 is a graph of the mixture of clostridium difficile, Lactobacillus plantarum and the mixture thereof under an oil lens (10 ×) provided by the application of example after the mixture of clostridium difficile, Lactobacillus difficile and the mixture thereof is treated with FOs liquid medicine for 48h, wherein a in fig. 4 is the form of clostridium difficile ATCC 057 under the oil lens (10 × 100), B is the form of Lactobacillus plantarum ATCC 8014 under the oil lens (10 ×) and C7007 under the mixture of Lactobacillus plantarum and Lactobacillus difficile ATCC 7007 under the mixture of Lactobacillus plantarum (10) and Lactobacillus difficile ATCC 70048 under the culture liquid medicine (10) under the mixture of Lactobacillus plantarum and Lactobacillus difficile 3, C70048, C10 is the mixture of Lactobacillus plantarum and Lactobacillus plantarum strain ATCC 8014 under the strain 12 under the strain ATCC 8014 under the strain (10,5948 under the strain of Lactobacillus difficile 3) cultured with Lactobacillus strain (10,48 under the strain of Lactobacillus difficile strain under the strain 3 under the strain of Lactobacillus strain.
The results show that the ratio of the number of the lactobacillus plantarum particles is greater than that of the clostridium difficile particles no matter the concentration of the added FOs liquid medicine in the mixed culture system, which indicates that the growth of the lactobacillus plantarum in the mixed culture system is dominant. In addition, with the increase of the concentration of the FOs liquid medicine, the ratio of the clostridium difficile is slightly increased and then gradually decreased, and the ratio of the lactobacillus plantarum is just opposite, so that the trend of decreasing firstly and then increasing is shown, which indicates that the addition of the FOs in the mixed culture system is beneficial to the growth of the lactobacillus plantarum and is not beneficial to the growth of the clostridium difficile. The results in the single-bacterium culture system show that the FOs have promotion effects on the growth of both bacteria, which indicates that in the mixed culture system, the FOs can promote the lactobacillus plantarum to produce more bacteriostatic substances, such as lactic acid, lactobacillus and the like, so that the growth of clostridium difficile is inhibited.
The result shows that, in normal conditions, clostridium difficile is a bacillus elongatus, the size is (1.3-1.6) mu m × (3.6-6.4) mu m, the clostridium difficile can move or cannot move, the motile strain is periwinkle, spores are oval, are positioned at the second extreme of thalli, are not capsular and are gram-positive, but tend to be gram-negative after 48 hours of culture, lactobacillus plantarum is a eubacterium stropharis, is (0.9-1.2) mu m × (3.0-8.0) mu m, and is single, paired or short-chain.
As shown in FIG. 5, the gram-stained bacteria were red, indicating that they turned gram-negative, but remained rod-shaped and did not produce spores. The lactobacillus plantarum is purple after gram staining and is a positive bacterium and exists in a single or chain form. The forms of the single bacteria of the clostridium difficile and the lactobacillus plantarum after being added with the FOs liquid medicine for incubation for 48 hours are not obviously changed, and after being added with the FOs liquid medicine for mixed culture for 48 hours, the middle or one end of part of the bacteria generate bulges, and some bacteria generate bifurcations, which indicates that the forms of the mixed bacteria after being treated with the FOs liquid medicine for 48 hours are changed. Comparing C and F in fig. 5, it can be seen that: the treatment of FOs resulted in a decrease in the number of clostridium difficile and an increase in the number of lactobacillus plantarum in the mixed bacteria culture system, and in general, an increase in the total number of bacteria in the mixed system of lactobacillus plantarum and clostridium difficile, which is consistent with the results of fig. 2 to 4.
Comparative example 1
The application provides a test of co-culture of oligosaccharide ferulic acid ester and clostridium difficile, which comprises the following specific steps:
sterilized 96-well plates, BHI broth were placed in an anaerobic workstation for pre-reduction for 24h prior to use. And taking out 2mL of each of the frozen drug diluents, and putting the drug diluents into an anaerobic workstation for later use. Three columns are arranged, three parallel groups of n1, n2 and n3 are arranged in each column, the first column is a blank group, and only 200 mu l of BHI broth is contained in the hole without clostridium difficile liquid; the second column was a positive control, and the wells contained 100. mu.l of BHI broth and 100. mu.l of Clostridium difficile (OD of Clostridium difficile broth)600Is 0.017 ≈ 1 × 106CFU/mL), without FOs solution. The third row contains the Lactobacillus plantarum bacterial liquid and the FOs liquid prepared in example 1, 200. mu.l of the FOs liquid is added into each of the three parallel wells of the third row, 100. mu.l of the FOs liquid is sucked into the next well, 100. mu.l of the culture medium filtered by the microporous membrane is added to double-dilute the FOs liquid, 100. mu.l of the diluted bacterial liquid is sucked into the next well, the above operations are repeated, and 100. mu.l of the last well is discarded. After dilution, 100. mu.l of Clostridium difficile suspension (OD of Clostridium difficile liquid) was added to each well of the third column600Is 0.017 ≈ 1 × 106CFU/mL), thus forming three replicates of determining the MIC value of FOs. Final FOs (mg/mL) drug concentrations per well were: 20; 10; 5; 2.5; 1.25. incubating the 96-well plate in an anaerobic constant temperature box at 37 ℃ for 48h, taking out the 96-well plate, and measuring OD (optical density) of each well by using an enzyme-labeling instrument600。
The data were analyzed using SPSS 24.0 software. Multiple comparisons of different samples were performed using the Duncan's multiple range test (p < 0.05) to determine significant differences between means. The results are shown in fig. 6, and fig. 6 is a graph of the effect of oligosaccharide ferulic acid ester provided by the comparative example of the application on clostridium difficile alone.
The results show that when the concentration of FOs is 1.25mg/mL, the turbidity of the clostridium difficile suspension has a slight trend of decreasing, but no significant difference exists; when the concentration reaches 5mg/mL, the FOs have a remarkable promoting effect on clostridium difficile.
As described above, the present examples and comparative examples show that FOs alone does not inhibit the action of clostridium difficile, but the present inventors found that a mixed system of FOs and lactobacillus plantarum has the effect of inhibiting clostridium difficile. The oligosaccharide ferulic acid ester has the function of promoting the propagation of lactobacillus plantarum and clostridium difficile; the lactobacillus plantarum and the clostridium difficile are mixed and cultured, and the influence of oligosaccharide ferulic acid ester on the form and the quantity ratio of the two bacteria in a mixed culture system is researched by means of a microscope. The results show that the oligosaccharide ferulic acid ester has no obvious direct inhibition effect on clostridium difficile. However, in the mixed culture system, the oligosaccharide ferulic acid ester promotes the growth of lactobacillus plantarum, but inhibits the growth of clostridium difficile.
It is seen that FOs can find application in the food industry as an additive. After the FOs are ingested, the growth of lactobacillus can be promoted and the growth of clostridium difficile can be inhibited in the intestinal tract, which is beneficial to exerting the normal physiological function of the intestinal tract and maintaining the health of the organism.
The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.
Claims (10)
1. The application of the mixed system of oligosaccharide ferulic acid ester and lactobacillus in inhibiting the growth of clostridium difficile.
2. The use according to claim 1, wherein the ratio of lactobacillus to clostridium difficile is 1: 1.
3. The use according to claim 2, wherein the oligosaccharide ferulic acid ester is at a concentration of 5mg/mL to 20 mg/mL.
4. Use according to claim 2, wherein the lactobacillus is selected from lactobacillus plantarum.
5. The use according to claim 2, wherein the oligosaccharide ferulic acid ester is prepared by a process comprising the steps of:
the cellulose raw material is subjected to enzymolysis or acidolysis to obtain oligosaccharide ferulic acid ester.
6. The use of claim 5, wherein the step of subjecting the cellulosic material to enzymatic or acid hydrolysis further comprises subjecting the product to ion exchange resin adsorption and elution to obtain the oligosaccharide ferulic acid ester.
7. A composition for inhibiting the growth of Clostridium difficile comprises the oligosaccharides ferulic acid ester and Lactobacillus.
8. A composition for inhibiting the growth of Clostridium difficile according to claim 7, wherein the ratio of the Lactobacillus to the Clostridium difficile is 1: 1; the concentration of the oligosaccharide ferulic acid ester is 5 mg/mL-20 mg/mL.
9. Clostridium difficile growth inhibiting composition according to claim 7, wherein the Lactobacillus is selected from Lactobacillus plantarum.
10. A Clostridium difficile growth inhibiting composition according to claim 7, wherein the oligosaccharide ferulic acid ester is prepared by a process comprising the steps of:
the cellulose raw material is subjected to enzymolysis or acidolysis to obtain oligosaccharide ferulic acid ester.
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