Preparation method and application of specific molecular weight mannose polysaccharide composite inhibitor

Abstract

The invention belongs to the technical field of bacteriostatic agents, and discloses a preparation method and application of a specific molecular weight mannose polysaccharide composite inhibitor, and particularly relates to the following steps: preparing specific molecular weight mannose polysaccharide with the weight-average molecular weight of 6.1-7.7 kDa from saccharomycetes, adding the specific molecular weight mannose polysaccharide into a fermentation culture medium inoculated with lactobacillus plantarum, and fermenting to obtain fermentation liquor; and collecting the supernatant of the fermentation liquor to obtain the specific molecular weight mannose polysaccharide composite inhibitor. The preparation method is green, environment-friendly and low in cost, and the specific molecular weight mannose polysaccharide composite inhibitor is added into the culture medium, so that the growth of escherichia coli and staphylococcus aureus can be effectively inhibited. The invention is suitable for feed additives.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial agents, and specifically relates to a preparation method and application of a mannosan composite inhibitor with a specific molecular weight. Background Art

[0002] Prebiotics can promote probiotics to produce more beneficial metabolites, such as organic acids and short-chain fatty acids, to create a good internal environment for probiotics and maintain the balance of intestinal flora. In addition, probiotics can metabolize specific prebiotics to produce beneficial secondary metabolites. Among the many prebiotics, polysaccharides with mannose as the main monosaccharide unit are called mannosaccharides, which are widely found in plants, fungi and some microorganisms.

[0003] Due to the different number of monosaccharides or connection methods that make up mannosan, the physicochemical properties and functions of different mannosans will vary to a certain extent, limiting the stable beneficial effects of mannosans in specific directions. Among them, if the molecular weight of mannosans is high, it has poor water solubility and high solution viscosity, which is not conducive to the absorption and utilization of mannosans in the body. It will also reduce the biological activity of mannosans, limiting its application in specific directions such as immunity, anti-oxidation or anti-inflammatory.

[0004] At present, most of the mannoses with smaller molecular weight are obtained by enzymatic degradation of polysaccharides using enzymes. For example, konjac glucomannan is enzymatically hydrolyzed by the extracellular enzymes of Bacillus licheniformis, Bacillus subtilis, and alkaliphilic Bacillus to obtain glucomannan oligosaccharides. If the degradation is insufficient, the distribution of the degree of polymerization of the product is too wide, which is not conducive to the application of mannoses with smaller molecular weight, and excessive degradation increases the cost. Overall, the production efficiency of the enzymatic method is low, and the industrial production cost is too high. In addition, there are also public technical reports on the method of obtaining mannoses by chemical degradation, such as U.S. Patent 4965354, which uses high temperature and hydrochloric acid degradation to obtain mannoses with smaller molecular weight. However, the industrial pollution problem caused by chemical degradation cannot be ignored.

[0005] Therefore, how to produce mannan polysaccharides of specific molecular weight in a greener and more efficient way, and how to make mannan polysaccharides stably exert their prebiotic effects in a specific direction have become key issues to be solved. Summary of the invention

[0006] In order to solve the above deficiencies in the prior art, the present invention aims to provide a method for preparing a mannosan compound inhibitor with a specific molecular weight. The inventor unexpectedly discovered that mannosan with a weight average molecular weight of 6.1 to 7.7 kDa but not other molecular weights can effectively inhibit the growth of Escherichia coli and Staphylococcus aureus. By adjusting the concentration of ethanol in the alcohol precipitation process, mannosan with a specific molecular weight can be obtained, and the compound inhibitor can be obtained by fermentation culture, thereby achieving the purpose of green and efficient production and stable beneficial effects.

[0007] Another object of the present invention is to provide an application of the above-mentioned specific molecular weight mannosan compound inhibitor, which is suitable for use as a feed additive and added to animal feed to achieve the purpose of promoting animal intestinal health.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing a specific molecular weight mannopolysaccharide composite inhibitor, comprising taking yeast and subjecting it to autolysis and high temperature extraction to obtain crude mannopolysaccharide, and then subjecting it to protein removal, decolorization, alcohol precipitation and chromatography purification to obtain a specific molecular weight mannopolysaccharide with a weight average molecular weight of 6.1 kDa to 7.7 kDa;

[0010] The specific molecular weight mannoses are added to a fermentation medium inoculated with plant lactobacillus for fermentation to obtain a fermentation liquid; the supernatant of the fermentation liquid is collected as the specific molecular weight mannoses composite inhibitor.

[0011] As a limitation of the present invention, the addition amount of the specific molecular weight mannan polysaccharide is 5g-10g per liter of fermentation medium inoculated with Lactobacillus plantarum; the inoculation amount of Lactobacillus plantarum in the fermentation medium is 2%-5%, and the Lactobacillus plantarum is activated to an OD value of 0.3-0.8 before inoculation.

[0012] As a further limitation of the present invention, after decolorization, spray drying is performed to obtain mannosan powder, and alcohol precipitation is performed by dissolving the mannosan powder in water and then adding anhydrous ethanol until the volume fraction of the anhydrous ethanol is 70% to 90%.

[0013] As another limitation of the present invention, the temperature of high temperature extraction is 110° C. to 125° C.; and the protein is removed by using trichloroacetic acid accounting for 4% to 6% of the weight of the crude mannopolysaccharide.

[0014] Among them, the protein removal methods using Sevage method and trichloroacetic acid method were compared, and it was unexpectedly found that the protein removal effect of trichloroacetic acid method exceeded expectations.

[0015] As a further limitation of the present invention, decolorization is performed by activated carbon. The deproteinized mannopolysaccharide is decolorized by hydrogen peroxide or activated carbon, wherein the activated carbon decolorization effect is better.

[0016] As a further limitation of the present invention, the fermentation medium is MRS medium.

[0017] As a further limitation of the present invention, the raw materials constituting the MRS medium include: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 5 g / L diammonium hydrogen citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 0.5 g / L L-cysteine, and 20 g / L lactose.

[0018] As a further limitation of the present invention, the autolysis time of the yeast is 40 hours to 50 hours.

[0019] As a further limitation of the present invention, the fermentation temperature is 35° C. to 38° C., and the fermentation time is 18 to 28 hours.

[0020] The present invention also provides the use of the above-mentioned specific molecular weight mannan polysaccharide composite inhibitor, which is added to the culture medium at a volume ratio of 20% to 35% to inhibit the growth of Escherichia coli and Staphylococcus aureus.

[0021] Due to the adoption of the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0022] The present invention first discovered that a specific molecular weight mannosan compound inhibitor prepared by fermentation with a weight average molecular weight of 6.1 to 7.7 kDa can have a stabilizing effect in the antibacterial direction, can effectively inhibit the growth of Escherichia coli and Staphylococcus aureus, and can be added to animal feed as an additive to help the intestinal health of animals. When preparing the specific molecular weight mannosan, the molecular weight of the mannosan is adjusted by changing the volume fraction of anhydrous ethanol in the alcohol precipitation process, and the method is simple to operate.

[0023] The preparation method of the specific molecular weight mannosan compound inhibitor of the present invention is based on Lactobacillus plantarum fermentation and adopts a biological fermentation method. Compared with low molecular weight mannosan that needs to be further enzymatically hydrolyzed by mannosan, the method is simple and efficient to operate, is green and environmentally friendly, has low application toxicity, and significantly improves economic benefits.

[0024] The invention is suitable for industrial production of mannosan compound inhibitor with specific molecular weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The growth results of Escherichia coli in Examples 1-3 and Comparative Examples 1-3 of the present invention are shown in FIG.

[0026] Figure 2 The growth results of Staphylococcus aureus in Examples 1-3 and Comparative Examples 1-3 of the present invention are shown in FIG.

[0027] Figure 3The results of the Staphylococcus aureus inhibition zone test of Examples 1-3 and Comparative Examples 1-3 of the present invention are shown;

[0028] Figure 4 This is a graph showing the results of the Escherichia coli inhibition zone experiment of Examples 1-3 of the present invention and Comparative Examples 1-3. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below through the accompanying drawings and specific embodiments. It should be understood that the described embodiments are only used to explain the present invention, but not to limit the present invention.

[0030] Example 1 Preparation method of specific molecular weight mannosan compound inhibitor A

[0031] The specific molecular weight mannosan compound inhibitor A is prepared from mannosan with a weight average molecular weight of 7.7 kDa by fermentation and other processes. The specific preparation method is as follows:

[0032] (I) Preparation of mannopolysaccharide with specific molecular weight

[0033] (1) taking commercially available yeast powder and adding deionized water to form a bacterial suspension with a mass fraction of 30%, placing it in a 37°C incubator and standing it for 40 hours to allow it to autolyze; after the autolysis is completed, the precipitate is collected by centrifugation, washed with deionized water three times, and then deionized water is added to form a bacterial suspension, which is subjected to high-temperature extraction at 121°C for 8 hours; after the high-temperature extraction is completed, the supernatant is collected by centrifugation to obtain crude mannopolysaccharide, i.e., mannopolysaccharide crude extract;

[0034] (2) Protein removal: add trichloroacetic acid (6% by weight of crude mannan polysaccharide) to the crude extract of mannan polysaccharide and stir at 60°C for 40 min to remove protein;

[0035] (3) Decolorization: Add 1% activated carbon to the crude extract of mannan polysaccharide and stir at 40°C for 50 min for decolorization;

[0036] (4) spray drying: spray drying the treated mannosan solution to obtain mannosan powder;

[0037] (5) Alcohol precipitation: Add deionized water to the mannosan powder to obtain a mannosan aqueous solution with a mass volume ratio of 20% (i.e., mannosan solid mass / deionized water volume = 20%), add anhydrous ethanol to the solution so that the volume fraction of ethanol in the solution is 90% (i.e., ethanol volume / (ethanol volume + mannosan aqueous solution volume) = 90%), and let stand overnight at 4°C;

[0038] (6) Chromatographic purification: The precipitate was collected by centrifugation, added with deionized water and stirred evenly, and added to a gel column filled with Sephadex G-150 for chromatography purification to obtain mannosan with a weight average molecular weight of 7.7 kDa.

[0039] (II) Fermentation to prepare specific molecular weight mannopolysaccharide complex inhibitor

[0040] Prepare fermentation medium: prepare fermentation medium according to 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 5 g / L diammonium hydrogen citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 0.5 g / L L-cysteine ​​and 20 g / L lactose, adjust pH to 6.8, sterilize and set aside.

[0041] Fermentation preparation: Lactobacillus plantarum is inoculated into MRS medium at a volume ratio of 2%, cultured at 37°C for 24 hours, and after three consecutive passages, the OD value of Lactobacillus plantarum is adjusted to 0.5 to complete activation. Mannosan with a weight average molecular weight of 7.7 kDa is added to the fermentation medium at 10 g / L, Lactobacillus plantarum is inoculated into the fermentation medium at a volume ratio of 2%, fermented and cultured at 37°C for 24 hours, the fermented liquid is centrifuged at 10000 r / min for 10 minutes, and the supernatant of the fermented liquid is collected, which is the specific molecular weight mannosan compound inhibitor A.

[0042] Example 2 Preparation method of specific molecular weight mannopolysaccharide composite inhibitor B

[0043] The specific molecular weight mannosan compound inhibitor B is prepared by fermentation and other processes from mannosan with a weight average molecular weight of 6.5 kDa. The preparation method is basically the same as that of Example 1, except that in step (1), the yeast autolysis time is 45 hours; in step (2), mannosan with a weight average molecular weight of 6.5 kDa is added to the fermentation medium at a rate of 7.5 g / L, and Lactobacillus plantarum is inoculated into the fermentation medium at an inoculation rate of 3% by volume.

[0044] Example 3 Preparation method of specific molecular weight mannosan compound inhibitor C

[0045] The specific molecular weight mannosan compound inhibitor B is prepared by fermentation and other processes from mannosan with a weight average molecular weight of 6.1 kDa. The preparation method is basically the same as that of Example 1, except that in step (1), the yeast autolysis time is 50 hours; in step (2), mannosan with a weight average molecular weight of 6.1 kDa is added to the fermentation medium at 7.5 g / L, and Lactobacillus plantarum is inoculated into the fermentation medium at an inoculation rate of 3% by volume.

[0046] Comparative Example 1 Preparation method of inhibitor 1

[0047] Inhibitor 1 is an inhibitor without mannosaccharide based on Example 1. Its preparation method is to inoculate Lactobacillus plantarum at a volume ratio of 2% into MRS medium, culture at 37°C for 24 hours, and continuously pass down for 3 times. Inoculate Lactobacillus plantarum at a volume ratio of 2% into the same fermentation medium as Example 1, ferment and culture at 37°C for 24 hours, take the fermentation liquid and centrifuge it at 10000r / min for 10 minutes, and collect the supernatant of the fermentation liquid, which is inhibitor 1.

[0048] Comparative Example 2 Preparation method of inhibitor 2

[0049] Inhibitor 2 is based on Example 1, but without step (ii), and only contains mannopolysaccharide with a weight average molecular weight of 7.7 kDa as the inhibitor.

[0050] Comparative Example 3 Preparation method of inhibitor 3

[0051] Inhibitor 3 is a composite inhibitor with the weight average molecular weight of mannosan adjusted on the basis of Example 1. Its preparation method is basically the same as that of Example 1, except that in step (5), anhydrous ethanol is added to make the volume fraction in the solution 60%, and the weight average molecular weight of the obtained mannosan is 96 kD. The remaining steps are the same as those of Example 1.

[0052] Antibacterial effect detection experiment:

[0053] The inhibitors prepared in Examples 1-3 and Comparative Examples 1-3 were analyzed for their effects on the growth of Escherichia coli and Staphylococcus aureus by absorbance detection and inhibition zone test. The details are as follows:

[0054] (1.1) Effects of composite inhibitors A–C and inhibitors 1–3 on the growth of Escherichia coli

[0055] The Escherichia coli was inoculated into LB medium at a volume ratio of 2%, and cultured at 37°C and 180 rpm for 24 hours, and subcultured three times to obtain an Escherichia coli culture liquid;

[0056] The E. coli liquid was added to each group of inoculated liquid in Table 1 at a volume ratio of 5%, and the volume of each group of inoculated liquid was the same; the liquid was cultured at 37°C and 180r / min for 48h, and the growth of E. coli was measured at an absorbance of 600nm. The results are as follows: Figure 1 .

[0057] Table 1 Experimental group settings for antibacterial effect detection

[0058]

[0059]

[0060] (1.2) Effects of compound inhibitors A-C and inhibitors 1-3 on the growth of Staphylococcus aureus

[0061] According to the method of (1.1), Escherichia coli was replaced by Staphylococcus aureus. Other experimental methods were the same. The effects of compound inhibitors A-C and inhibitors 1-3 on the growth of Staphylococcus aureus were tested. The results are as follows Figure 2 .

[0062] Depend on Figure 1 and Figure 2 It can be seen that the composite inhibitors A to C and inhibitors 1 to 3 all have an inhibitory effect on Escherichia coli and Staphylococcus aureus. The composite inhibitors A, B, and C fermented with specific molecular weight mannan polysaccharide in step (ii) have the best inhibitory effect on the two pathogens, and the antibacterial effect is higher than that of inhibitors 1 to 3. According to calculation, the antibacterial rates of composite inhibitor A on Escherichia coli and Staphylococcus aureus can reach 62% and 47.2%, respectively.

[0063] (1.3) Antibacterial effects of compound inhibitors A-C and inhibitors 1-3 on Escherichia coli

[0064] Take 0.1mL 10 5 CFU / mL Escherichia coli and Staphylococcus aureus were evenly spread on the agar medium plate, and after solidification, a sterilized Oxford cup was placed on the plate. 0.1mL of composite inhibitors A-C and inhibitors 1-3 were placed in the Oxford cup respectively, and cultured at 37℃ for 24h. The size of the inhibition zone was observed and measured.

[0065] Among them, the results of the plate inhibition zone experiment coated with Staphylococcus aureus are as follows Figure 3 As shown, the results of the E. coli inhibition zone test are as follows Figure 4 As shown in the figure, the average antibacterial circle diameter of composite antibacterial agents A to C against Escherichia coli is 21.8 mm, and the average antibacterial diameter against Staphylococcus aureus is 23.1 mm; therefore, the antibacterial rate of plant lactobacillus fermentation supernatant fermented with mannan polysaccharide having a weight average molecular weight of 6.1 to 7.7 kDa against both bacteria is significantly higher than the antibacterial effect of mannan polysaccharide alone or plant lactobacillus fermentation broth alone, indicating that the small molecule mannan polysaccharide with a specific molecular weight of the present invention is more easily utilized by plant lactobacillus to produce more metabolites with antibacterial function to inhibit the growth of pathogenic bacteria.

[0066] Example 4 Preparation method of specific molecular weight mannosan compound inhibitor

[0067] The preparation method of this embodiment is basically the same as that of Example 1, except that the autolysis time of yeast is 43 hours; alcohol precipitation is performed by dissolving mannan polysaccharide powder in water and then adding anhydrous ethanol to a volume fraction of anhydrous ethanol of 70%; the addition amount of the mannan polysaccharide of specific molecular weight is 5 g per liter of fermentation medium inoculated with Lactobacillus plantarum; the inoculation amount of Lactobacillus plantarum in the fermentation medium is 2%, and the Lactobacillus plantarum is activated to an OD value of 0.5 before inoculation; the fermentation temperature is 35° C., and the fermentation time is 23 hours.

[0068] Example 5 Preparation method of specific molecular weight mannosan compound inhibitor

[0069] The preparation method of this embodiment is basically the same as that of embodiment 1, except that the autolysis time of yeast is 46 hours; alcohol precipitation is performed by dissolving mannan polysaccharide powder in water and then adding anhydrous ethanol to a volume fraction of anhydrous ethanol of 80%; the addition amount of the mannan polysaccharide of specific molecular weight is 8 g per liter of fermentation medium inoculated with Lactobacillus plantarum; the inoculation amount of Lactobacillus plantarum in the fermentation medium is 5%, and the Lactobacillus plantarum is activated to an OD value of 0.3 before inoculation; the fermentation temperature is 37.8° C., and the fermentation time is 28 hours.

[0070] Example 6 Preparation method of specific molecular weight mannosan compound inhibitor

[0071] The preparation method of this embodiment is basically the same as that of embodiment 1, except that the autolysis time of yeast is 40h to 50h; alcohol precipitation is performed by dissolving mannan polysaccharide powder in water and then adding anhydrous ethanol to a volume fraction of anhydrous ethanol of 70%; the addition amount of the mannan polysaccharide of specific molecular weight is 10g per liter of fermentation medium inoculated with Lactobacillus plantarum; the inoculation amount of Lactobacillus plantarum in the fermentation medium is 4.3%, and the Lactobacillus plantarum is activated to an OD value of 0.8 before inoculation; the fermentation temperature is 38°C, and the fermentation time is 18h.

[0072] In other embodiments, the specific molecular weight mannan complex inhibitor is a mannan with a weight average molecular weight of 7kDa, 7.6kDa, 6.8kDa or 7.5kDa; it is obtained by high temperature extraction at a temperature of 110°C, 115°C, 118°C or 125°C; and the protein is removed by using trichloroacetic acid at a weight percentage of 4%, 4.5%, 5.5% or 5.6% of the crude mannan weight.

[0073] Example 7 Application of specific molecular weight mannopolysaccharide composite inhibitor

[0074] The specific molecular weight mannan polysaccharide composite inhibitors prepared according to the preparation methods of Examples 1 to 6 were added to the culture medium at a volume ratio of 20% to 35%. The specific group design is shown in Table 2. The antibacterial effect test results showed that the specific molecular weight mannan polysaccharide composite inhibitors A to C could inhibit the growth of Escherichia coli and Staphylococcus aureus.

[0075] Table 2 Experimental group settings for application of specific molecular weight mannopolysaccharide complex inhibitors

[0076]

[0077] The specific molecular weight mannosan compound inhibitors prepared by the preparation methods of Examples 1 to 6 were respectively used as additives and added to animal feed. According to the intestinal flora abundance test, consuming the animal feed added with the specific molecular weight mannosan compound inhibitors can inhibit the growth of Escherichia coli and Staphylococcus aureus, thereby promoting the intestinal health of animals.

[0078] It has been verified that the above embodiments of the present invention all achieve similar application effects of inhibiting the growth of Escherichia coli and Staphylococcus aureus, which are similar to the antibacterial effect detection experimental results of the specific molecular weight mannan polysaccharide composite inhibitor A.

[0079] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a specific molecular weight mannosan compound inhibitor, characterized in that: The yeast was autolyzed and extracted at high temperature to obtain crude mannoses, which were then deproteinized, decolorized, precipitated with alcohol and purified by chromatography to obtain mannoses with a specific molecular weight of 6.1 kDa to 7.7 kDa. The specific molecular weight mannoses are added to a fermentation medium inoculated with plant lactobacillus for fermentation to obtain a fermentation liquid; the supernatant of the fermentation liquid is collected as the specific molecular weight mannoses composite inhibitor.

2. The method for preparing the specific molecular weight mannosan compound inhibitor according to claim 1, characterized in that: The addition amount of the specific molecular weight mannan polysaccharide is 5g-10g per liter of fermentation medium inoculated with Lactobacillus plantarum; the inoculation amount of Lactobacillus plantarum in the fermentation medium is 2%-5%, and the Lactobacillus plantarum is activated to an OD value of 0.3-0.8 before inoculation.

3. The method for preparing the specific molecular weight mannosan compound inhibitor according to claim 2, characterized in that: After decolorization, spray drying is performed to obtain mannopolysaccharide powder; Alcohol precipitation is to dissolve mannan polysaccharide powder in water, and then add anhydrous ethanol until the volume fraction of anhydrous ethanol is 70%~90%.

4. The method for preparing the specific molecular weight mannosan compound inhibitor according to claim 3, characterized in that: The temperature of high temperature extraction is 110℃~125℃; The protein was removed by using trichloroacetic acid accounting for 4% to 6% of the weight of the crude mannopolysaccharide.

5. The method for preparing the specific molecular weight mannosan compound inhibitor according to claim 4, characterized in that: Decolorization is carried out using activated carbon.

6. The method for preparing the specific molecular weight mannosan compound inhibitor according to claim 5, characterized in that: The fermentation medium is MRS medium.

7. The method for preparing the specific molecular weight mannosan compound inhibitor according to claim 6, characterized in that: The raw materials constituting the MRS culture medium include: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 5 g / L diammonium hydrogen citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 0.5 g / L L-cysteine, and 20 g / L lactose.

8. The method for preparing the specific molecular weight mannosan compound inhibitor according to claim 7, characterized in that: The autolysis time of the yeast is 40h~50h.

9. The method for preparing a specific molecular weight mannosan compound inhibitor according to any one of claims 1 to 8, characterized in that: The fermentation temperature is 35℃~38℃, and the fermentation time is 18~28h.

10. Use of a specific molecular weight mannosan compound inhibitor prepared by the preparation method according to any one of claims 1 to 9, characterized in that: It is added to the culture medium at a volume ratio of 20%~35% to inhibit the growth of Escherichia coli and Staphylococcus aureus.

Images