阅读说明：本技术 一种壳寡糖衍生物、制备方法及在预防和/或治疗猪腹泻中的应用 (Chitosan oligosaccharide derivative, preparation method and application of chitosan oligosaccharide derivative in preventing and/or treating porcine diarrhea ) 是由 杜昱光 任立世 闫雅璐 焦思明 程功 孙明 于 2020-08-25 设计创作，主要内容包括：本发明属于糖工程应用技术领域,公开了一种壳寡糖衍生物、制备方法及在预防和/或治疗猪腹泻中的应用。所述制备方法包括以下步骤：1)将乳糖和壳寡糖以一定的质量比混合,加入乳糖酶,在加热条件下进行混合搅拌反应得到反应液；2)使反应液中的乳糖酶失活,再将反应液冻干,得到产物。本发明通过壳寡糖和乳糖合成出一种壳寡糖衍生物,其具有与传统壳寡糖不同的结构及性能,能够在抑制产肠毒素大肠杆菌K88粘附肠上皮细胞中发挥优异的作用,从而可以作为预防和/或治疗猪腹泻的饲料添加剂/口服液使用,有效降低由产肠毒素大肠杆菌K88导致的猪腹泻,不仅有效降低成本,且不易产生耐药性。(The invention belongs to the technical field of sugar engineering application, and discloses a chitosan oligosaccharide derivative, a preparation method and application thereof in preventing and/or treating porcine diarrhea. The preparation method comprises the following steps: 1) mixing lactose and chitosan oligosaccharide in a certain mass ratio, adding lactase, and carrying out mixing and stirring reaction under a heating condition to obtain a reaction solution; 2) inactivating lactase in the reaction solution, and freeze-drying the reaction solution to obtain the product. The chitosan oligosaccharide derivative is synthesized by chitosan oligosaccharide and lactose, has different structure and performance from the traditional chitosan oligosaccharide, can play an excellent role in inhibiting enterotoxigenic escherichia coli K88 from adhering to intestinal epithelial cells, can be used as a feed additive/oral liquid for preventing and/or treating pig diarrhea, effectively reduces pig diarrhea caused by enterotoxigenic escherichia coli K88, effectively reduces the cost, and is not easy to generate drug resistance.)

1. A preparation method of a chitosan oligosaccharide derivative is characterized by comprising the following steps: the method comprises the following steps:

1) mixing lactose and chitosan oligosaccharide in a certain mass ratio, adding lactase, and carrying out mixing and stirring reaction under the heating condition to obtain a reaction solution;

2) inactivating lactase in the reaction solution, and freeze-drying the reaction solution to obtain the product.

2. The method for producing a chitosan oligosaccharide derivative according to claim 1, wherein: the mass ratio of the lactose to the chitosan oligosaccharide is (1-3): 5.

3. the method for producing a chitosan oligosaccharide derivative according to claim 1 or 2, wherein: in the step 1), the heating temperature is 30-50 ℃, and the stirring reaction time is not less than 24 h.

4. A process for preparing a chitosan oligosaccharide derivative according to claim 3, wherein: the lactase in the step 2) is inactivated by the following mode: and heating the reaction solution to above 80 ℃ and maintaining the temperature for 20-30 minutes.

5. A chitosan oligosaccharide derivative, which is characterized in that: the chitosan oligosaccharide derivative of any one of claims 1 to 4.

6. The chitosan oligosaccharide derivative according to claim 5, wherein: in the detection chromatogram of HPLC of the chitosan oligosaccharide derivative, the peak-off time of the strongest characteristic peak is 8.988min, and the peak-off time of the secondary intensity characteristic peak is 13.389 min.

7. Use of the chitosan oligosaccharide derivative of claim 5 for inhibiting adhesion of enterotoxigenic Escherichia coli to intestinal cells.

8. Use of a chitosan oligosaccharide derivative according to claim 7, for inhibiting adhesion of enterotoxigenic e.coli, wherein: the enterotoxigenic escherichia coli comprises escherichia coli K88, and/or the enterocytes comprise porcine intestinal epithelial cells.

9. A feed additive/oral liquid for preventing and/or treating porcine diarrhea is characterized in that: comprising the chitosan oligosaccharide derivative of claim 5 or 6.

10. The feed additive/oral liquid for preventing and/or treating porcine diarrhea according to claim 9, wherein: the concentration of the chitosan oligosaccharide derivative is 5-50 mg/mL when the chitosan oligosaccharide derivative is used as an oral liquid.

Technical Field

The invention belongs to the technical field of sugar engineering application, and discloses a chitosan oligosaccharide derivative, a preparation method and application thereof in preventing and/or treating porcine diarrhea.

Background

According to the data of the national statistical bureau, the pork yield of China accounts for 56 percent of the total meat yield (the data comes from the national statistical bureau). With the enlargement of pig raising scale in China, the death number of piglets is continuously increased and further increased, about 1.4 hundred million piglets die each year, and the death rate fluctuates from 7% to 37%. Death of piglets causes huge economic losses for the pig industry every year. The death of piglets is caused by many factors, such as piglet's own function, feed and feed, stress and the like. The diarrhea of piglets is the main cause of death of piglets, and about 4000 to ten thousand piglets die from the diarrhea.

At present, aiming at piglet diarrhea, the common method is to add antibiotics into the feed for piglets and lactating sows, reduce the risk of infection of the piglets with bacteria and viruses, improve the anti-infection capacity of the piglets, treat diarrhea diseases of the piglets and the like. The method can play a role in effective prevention and treatment to a certain extent, but has the defects of low complete cure rate, high repeated morbidity, immunosuppression of piglets, long treatment time and the like. Especially, with the generation of bacterial drug resistance, the effect of antibiotics is greatly reduced, and the prevention and treatment of multifactorial diseases such as diarrhea are far from satisfied.

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of diarrhea in piglets. Among them, ETEC K88, K99 and 987P are the main pathogenic bacteria, and about 56% to 70% of piglet diarrhea is related to ETEC infection. The existing methods for treating diarrhea are mainly fluid replacement or antibiotic treatment. The disablement of antibiotics has forced the swine industry to find other prophylactic and therapeutic approaches.

Enterotoxigenic escherichia coli is pathogenic in two steps, firstly, ETEC is adhered to piglet intestinal epidermal cells by flagella, so that the ETEC is fixedly planted in a host; ETEC after colonization will release enterotoxins (ST and LT) allowing water and electrolytes to enter the intestine through Cl "ion channels, causing diarrhea.

Adhesion is a crucial link for pathogenic bacteria such as ETEC and the like, and the pathogenic bacteria are adhered to host cells through adhesion proteins on the surfaces of the bacteria, so that the cleaning mechanism of the host can be resisted, the colonization is facilitated, nutrient substances can be conveniently obtained, and mass propagation is facilitated; in addition, the release of toxins or other pathogenic agents can be promoted. The adhesion proteins have a Carbohydrate recognition domain (glycoprotein) that recognizes glycoproteins on the host cell to establish adhesion, thus inhibiting or preventing initial adhesion of ETEC to host epithelial cells, often referred to as anti-adhesion therapy, is becoming an alternative to antibiotics and a strategy for combating bacterial infections.

Chitosan oligosaccharide is called chitosan oligosaccharide and oligomeric chitosan, is an oligosaccharide product with the polymerization degree of 2-20, is obtained by degrading chitosan through a special biological enzyme technology (reports of using chemical degradation and microwave degradation technologies), has the molecular weight of less than or equal to 3200Da, and is a low-molecular-weight product with good water solubility, large functional effect and high biological activity. It has several unique functions of high solubility, complete water solubility, easy absorption and utilization by organism, etc. and its action is 14 times that of chitosan.

Based on the excellent characteristics of the chitosan oligosaccharide, the chitosan oligosaccharide has been used as an additive for preventing and treating piglet diarrhea in the prior art, and through search, related applications have been disclosed, for example, an application with the Chinese patent application number of 201910856823.7 and the publication date of 2019, 12 and 13 discloses a feed additive for preventing and treating piglet diarrhea, which comprises the following raw materials in parts by weight: 6-12 parts of bighead atractylodes rhizome extract, 6-12 parts of coptis root extract, 4-8 parts of codonopsis pilosula extract, 1-3 parts of liquorice extract, 0.6-1.3 parts of chitosan, 1-3 parts of linseed oil acid and 2-4 parts of modified zinc oxide; wherein, the modified zinc oxide is mainly prepared from zinc oxide, sodium stearate and gluconic acid. The feed additive mainly depends on the traditional zinc oxide to reduce the diarrhea rate of piglets, and the chitosan and the linseed oleic acid are added to mainly improve the palatability when the piglets are fed with the zinc oxide and enhance the immunity of the piglets, so that the chitosan oligosaccharide has excellent properties, and can not play a role in reducing the diarrhea rate of the piglets by itself, and the preparation of a large amount of substances causes overhigh cost.

Based on the defects of the prior art, a new method and a product which are low in cost, convenient to use and capable of effectively reducing the diarrhea rate of piglets are needed to be invented.

Disclosure of Invention

1. Problems to be solved

Aiming at the problem that in the method for reducing the diarrhea rate of piglets in the prior art, the adopted antibiotics are easy to generate drug resistance; the invention synthesizes a novel chitosan oligosaccharide derivative through chitosan oligosaccharide and lactose, which has different structure and performance from the traditional chitosan oligosaccharide and can play an excellent role in inhibiting enterotoxigenic escherichia coli K88 from adhering to intestinal epithelial cells, thereby being used as a feed additive/oral liquid, effectively reducing pig diarrhea caused by enterotoxigenic escherichia coli K88, not only effectively reducing the cost, but also being difficult to generate drug resistance.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the invention provides a preparation method of a chitosan oligosaccharide derivative, which comprises the following steps:

1) mixing lactose and chitosan oligosaccharide in a certain mass ratio, adding lactase, and carrying out mixing and stirring reaction under the heating condition;

2) inactivating lactase in the reaction solution, and then freeze-drying the reaction solution to obtain the product.

As a further improvement of the invention, the mass ratio of the lactose to the chitosan oligosaccharide is (1-3): 5.

as a further improvement of the present invention, the lactase is inactivated in step 2) by: heating the reaction solution to above 80 ℃ and maintaining the temperature for 20-30 minutes to inactivate the enzyme.

As a further improvement of the invention, in the step 1), the heating temperature is 30-50 ℃, and the stirring reaction time is not less than 24 hours.

As a further improvement, the invention provides a chitosan oligosaccharide derivative, and in a detection map by HPLC, the peak-off time of the strongest characteristic peak is 8.988min, and the peak-off time of the secondary intensity characteristic peak is 13.389 min.

As a further improvement of the invention, the size exclusion chromatography assay contains two peaks, wherein the first peak has a relative average molecular mass of 659.72Da and the second peak has a relative average molecular mass of 212.81 Da.

As a further improvement, the invention provides an application method of the chitosan oligosaccharide derivative in inhibiting adhesion of enterotoxigenic Escherichia coli and enterocytes.

As a further development of the invention, the enterotoxigenic Escherichia coli comprises Escherichia coli K88, and/or the intestinal cells comprise porcine intestinal epithelial cells.

As a further improvement, the invention provides a feed additive/oral liquid for preventing and/or treating porcine diarrhea, wherein the feed additive/oral liquid contains the chitosan oligosaccharide derivative.

As a further improvement of the invention, when the chitosan oligosaccharide derivative is used as a feed additive, the chitosan oligosaccharide derivative can be added into feed for use.

When the chitosan oligosaccharide derivative is used as an oral liquid, the chitosan oligosaccharide derivative is prepared into a chitosan oligosaccharide derivative solution with the concentration of 5-50 mg/mL.

As a further improvement of the invention, the effective use concentration is preferably 5-10 mg/mL.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) the chitosan oligosaccharide derivative can effectively inhibit the adhesion of enterotoxigenic escherichia coli K88 and porcine small intestine epithelial cells, so that the problem of porcine diarrhea is fundamentally solved, compared with the prior art that antibiotics are added for preventing and treating porcine diarrhea, the chitosan oligosaccharide derivative is not easy to generate drug resistance, compared with a special diarrhea-preventing additive such as zinc oxide and the like, the chitosan oligosaccharide derivative has better palatability, can exert excellent performance without adding a large amount of compounds, and has excellent effect.

(2) The chitosan oligosaccharide derivative disclosed by the invention shows a stable antibacterial effect in a concentration range of 5-50 mg/mL, the antibacterial rate of 10mg/mL can reach 64.37%, the antibacterial adhesion rate of lactose of 10mg/mL is only 6.90%, the antibacterial adhesion rate of unmodified chitosan oligosaccharide is only 7.47%, and a coating experiment also shows that the chitosan oligosaccharide derivative disclosed by the invention shows a more remarkable inhibition phenomenon of enterotoxigenic escherichia coli K88 adhesion than the traditional chitosan oligosaccharide. Therefore, the chitosan oligosaccharide derivative prepared by compounding lactose and chitosan oligosaccharide generates a structure different from the traditional chitosan oligosaccharide, thereby generating an unexpected adhesion inhibition effect on enterotoxigenic Escherichia coli K88 and porcine small intestine epithelial cells, and obtaining the confirmation information of the related structure of the product from the results of HPLC analysis, LC-MS analysis and molecular exclusion chromatography analysis performed on the product.

(3) The oligosaccharide derivative is prepared from the traditional chitosan oligosaccharide and lactose by a simple method, can exert excellent bacteriostatic adhesion performance by adding the product, and can effectively reduce the cost compared with the prior art which needs to add various additives. Meanwhile, based on good water solubility of the chitosan oligosaccharide derivative, the chitosan oligosaccharide derivative can be directly prepared into a solution or added into feed, and the use mode is flexible; the oligosaccharide derivative has low cost and excellent effect, and is beneficial to popularization and use.

Drawings

FIG. 1 is an HPLC chromatogram of a conventional chitosan oligosaccharide;

FIG. 2 is an HPLC detection chromatogram of chitosan oligosaccharide derivative RX COS-K88 prepared by the present invention;

FIG. 3 is a representation of LC-MS detection of chitosan oligosaccharide derivative RX COS-K88 prepared by the present invention;

FIG. 4 shows the results of size exclusion chromatography detection of chitosan oligosaccharide derivative RX COS-K88 prepared by the present invention;

FIG. 5 is a polyethylene glycol molecular exclusion chromatography detection profile of different molecular masses;

FIG. 6 is a bar graph comparing the bacteriostatic adhesion rates of various oligosaccharides from example 2;

FIG. 7 shows the colony growth of oligosaccharide-plated bacteria in example 2.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, and it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.

It should be noted that the terms "upper", "lower", "left", "right" and "middle" used in the present specification are for the sake of clarity, and are not intended to limit the scope of the present invention, and changes or adjustments of the relative relationship thereof may be made without substantial technical changes.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.