WO2017016022A1 - 一种壳寡糖-o-曲酸-曼尼希碱衍生物抗菌剂及其制备方法 - Google Patents

一种壳寡糖-o-曲酸-曼尼希碱衍生物抗菌剂及其制备方法 Download PDF

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WO2017016022A1
WO2017016022A1 PCT/CN2015/088398 CN2015088398W WO2017016022A1 WO 2017016022 A1 WO2017016022 A1 WO 2017016022A1 CN 2015088398 W CN2015088398 W CN 2015088398W WO 2017016022 A1 WO2017016022 A1 WO 2017016022A1
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mannich base
kojic acid
chitosan oligosaccharide
acid
antibacterial agent
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夏文水
刘晓丽
姜启兴
许艳顺
于沛沛
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A23B2/779—Sugars; Derivatives thereof

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  • the invention belongs to the technical field of food additives, and in particular relates to a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and a preparation method thereof.
  • Chitooligosaccharide is a low molecular weight basic aminooligosaccharide obtained by cleavage of chitosan backbone by physical, chemical or enzymatic degradation. It is biodegradable, biocompatible, biologically non-toxic and chemically reactive. It inhibits a range of microorganisms including bacteria and fungi and is considered an ideal material for the development of new natural food preservatives.
  • chitosan oligosaccharide is a natural macromolecular product, when used as a food antiseptic antibacterial agent, it has disadvantages such as low antibacterial activity compared with conventional common chemical preservatives, so the application in the food industry is not very current. universal. Therefore, the structure of chitosan oligosaccharide can be chemically modified. Due to the chemically reactive amino group and hydroxyl group in the molecular chain, these sites are ideal sites for chemical modification of chitooligosaccharides, which can be further improved. Its antibacterial activity, which is also an effective method for research at home and abroad.
  • an object of the present invention is to provide a chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent and a preparation method thereof, and an active antibacterial group in kojic acid- ⁇ -pyran
  • the keto group and N-methylpiperazine are introduced into the chitosan oligosaccharide molecular chain to synergize with the chitooligosaccharide molecule to enhance the antibacterial activity of chitooligosaccharide, and obtain a well-soluble and synergistic chitosan derivative.
  • the chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent proposed by the present invention has the chemical structural formula shown below, wherein n is 6-20 and the degree of substitution is 1.21-1.78.
  • the invention also provides a preparation method of the chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent, comprising the following steps:
  • the method further comprises the step of purifying after the step (2), dialysis the dried product, adding an organic solvent to precipitate, suction filtration, and freeze-drying to finally obtain purified chitooligosaccharide-O-kojic acid. - Mannich base derivative.
  • the ratio by mass of the chitooligosaccharide Schiff base to the chlorodeteric acid-Mannich base is 2:(1-5).
  • the chitosan oligosaccharide has a molecular weight of 1000 to 5000 Da and a degree of deacetylation of 90 to 95%.
  • the organic solvent for dissolving the chloroderivative-Mannich base is dimethyl sulfoxide or dimethylformamide, and the amount of the chloroderivative-Mannich base is 2 -4 times;
  • the organic solvent for dissolving chitooligosaccharide Schiff base is dimethylformamide and pyridine, the amount of dimethylformamide is 2-4 times that of chitooligosaccharide Schiff base, and the amount of pyridine is chitosan oligosaccharide 2-6 times the Schiff base.
  • the reaction temperature is -35 ° C to 40 ° C, and the reaction time is 2 to 6 h.
  • the reaction temperature is room temperature
  • the reaction time is 12-24 h.
  • the preparation method of the chlorodeteric acid-Mannich base comprises the following steps:
  • the ratio by mass of the N-methylpiperazine and the chloroderivative acid is 1: (2-3), and the ratio of the volume of the methanol to the formalin solution is (10-20):1. .
  • the present invention has at least the following advantages: the present invention is based on the idea of substructure linkage, and the active group - ⁇ -pyranone and N-methyl piperazine in kojic acid are linked to chitooligosaccharide molecules. In the chain, the three have synergistic antibacterial activity, and developed a new type of food antiseptic and bacteriostatic agent.
  • Figure 1 is an infrared spectrum of chitosan oligosaccharide
  • Example 2 is an infrared spectrum diagram of a chitosan oligosaccharide derivative prepared in Example 1 of the present invention
  • Figure 3 is a 1 H-NMR chart of the chitosan oligosaccharide derivative prepared in Example 1 of the present invention.
  • Figure 4 is a 13 C-NMR chart of the chitosan oligosaccharide derivative prepared in Example 1 of the present invention.
  • Figure 5 is an infrared spectrum diagram of a chitosan oligosaccharide derivative prepared in Example 2 of the present invention.
  • Figure 6 is an infrared spectrum of a chitosan oligosaccharide derivative prepared in Example 3 of the present invention.
  • the preparation method of chitosan oligosaccharide-O-kojic acid-Mannich base derivative having good water solubility and antibacterial activity is as follows: using N-methylpiperazine and chloroderivative acid as raw materials, firstly obtained by Mannich reaction Chlorothetrex-Mannich base is then alkylated with an amino-protected chitooligosaccharide Schiff base, and then subjected to amino deprotection and purification to obtain a chitosan oligosaccharide-O-koroic acid which retains the active amino group.
  • Mannich base derivatives the synthetic route is as follows:
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • the preparation method of the chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent comprises the following steps:
  • N-methylpiperazine and chlorodeteric acid were dissolved in 100 mL of methanol and 10 mL of formalin at a mass ratio of 1:2, and rapidly stirred at 25 ° C to form a brown precipitate, collected, filtered, and dried. The mixture was washed with methanol for 5 times, recrystallized from anhydrous methanol, and dried to give chlorotrimetic acid-Mannich base.
  • the precipitate was subjected to Soxhlet extraction with ethanol and acetone to remove dimethylformamide and pyridine. After lyophilization, 1 g of chitooligosaccharide Schiff base-O-kojic acid-Mannich base derivative was obtained.
  • the yield of the chitosan oligosaccharide derivative was 62.1%, which was characterized by infrared spectroscopy and nuclear magnetic resonance.
  • Fig. 1 is an infrared spectrum of chitosan oligosaccharide, wherein 3422.36 cm -1 is the stretching vibration absorption peak of OH and NH, 2923.83 cm -1 is the absorption peak of CH stretching vibration, and 1628.76 cm -1 is the bending vibration absorption of NH 2 .
  • the peaks, 1155.97 cm -1 and 1071.52 cm -1 are absorption peaks of CO stretching vibration, and 893.24 cm -1 is a ring stretching vibration absorption peak.
  • Figure 3 is a 1 H-NMR chart of the chitosan oligosaccharide derivative in the present example, the peak at which the chemical shift occurs at 1.968 ppm corresponds to the proton peak of -CH 3 on the acetamido residue; at 2.47-2.796 ppm The peak corresponds to the proton peak of H 2 on the glucosamine residue and the acetamido residue; the peak at 3.089-3.837 ppm corresponds to the corresponding proton peak at H3, H4, H5, H6 on glucosamine and acetylglucosamine The peak at 4.7 ppm is the solvent peak; two new chemical shifts appear at 4.26 and 6.82 ppm, which can be assigned to the -CH2 (H-7') and H-3, respectively, on the 5-hydroxypyrone skeleton of kojic acid.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the preparation method of the chitosan oligosaccharide-O-kojic acid-Mannich base derivative antibacterial agent comprises the following steps:
  • the reaction mixture was magnetically stirred in a water bath at -5 ° C for 4 hours, poured into excess acetone to precipitate the product, and the precipitate was filtered, and the precipitate was subjected to Soxhlet extraction with ethanol and acetone to remove dimethylformamide and Pyridine, and finally 1.8 g of the product after vacuum freeze-drying was obtained.
  • the yield of the chitooligosaccharide derivative was 65.2%.
  • IR spectra for samples of FIG. 5, wherein, wherein ⁇ - glycosidic bonds pyran corresponding to an absorption peak at 892.24cm -1, 1518.32cm -1 derivative for C C stretching vibration absorption peaks, 1226.23cm -1 is the COC stretching vibration absorption peak of the derivative, and 971.91 cm -1 is the absorption peak of the covalently bonded bond of chloric acid-Mannich base and chitooligosaccharide, and the existence of 1226.23 cm -1 and 971.91 cm -1 Prove the formation of the target product.
  • N-methylpiperazine and chlorodeteric acid were dissolved in 200 mL of methanol and 10 mL of formalin at a mass ratio of 1:3, and rapidly stirred at 25 ° C to form a brown precipitate, which was collected, filtered, and dried. The mixture was washed with methanol for 5 times, recrystallized from anhydrous methanol, and dried to give chlorotrimetic acid-Mannich base.
  • reaction mixture was magnetically stirred in a 40 ° C water bath for 6 hours, poured into excess acetone to precipitate the product, and the precipitate was filtered, and the precipitate was subjected to Soxhlet extraction with ethanol and acetone to remove dimethylformamide and pyridine, respectively. Finally, 3 g of the product after vacuum freeze-drying was obtained.
  • the samples in the first embodiment, the second embodiment and the third embodiment are respectively named as derivative 1, derivative 2, and derivative 3, and chitosan oligosaccharide, kojic acid, derivative 1, derivative 2 and
  • the derivative 3 was dissolved and formulated into a solution having a concentration gradient of 0.01, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 mg/mL, and filtered through a 0.22 ⁇ m microporous membrane. ,spare.
  • N1 the total number of colonies in the initial culture solution
  • N2 Total number of colonies in the culture medium containing the antibacterial agent.
  • the IC 50 value is obtained.
  • the antibacterial agent prepared by the present invention has good antibacterial activity against Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Salmonella typhimurium, Shigella dysenteriae and Escherichia coli. Moreover, the antibacterial property is much better than that of chitosan oligosaccharide and kojic acid, and has potential application value in food preservation.
  • the principle of the present invention is as follows: there are three chemically modified active sites in the molecular structure of chitosan oligosaccharide. If an alkylation reaction occurs, the amino acid at the C-2 position first occurs according to the magnitude of the activity at three positions. The upper one is followed by the primary hydroxyl group at the C-6 position, and the second is the secondary hydroxyl group at the C-3 position. Since the C-2 amino group in the chitosan oligosaccharide molecule is an active group which acts as an antibacterial agent in an acidic solution, the active amino group at the C-2 position is first protected with benzaldehyde, and an alkane is formed.
  • the beneficial effects of the present invention are as follows: (1) O-alkylation reaction of a hydroxy group at the C-6 position of a chitooligosaccharide with a chloroderivative-Mannich base, and an active antibacterial group- ⁇ in kojic acid -pyridyl
  • the ketone group and N-methylpiperazine are introduced into the chitooligosaccharide molecular chain to synergize with the chitooligosaccharide molecule, which significantly enhances its antibacterial activity.
  • These new derivatives are low in toxicity, have good water solubility, can be dissolved in various inorganic and organic solvents, avoid the use of organic solvents, are more environmentally friendly, and expand their application fields. It has a wide range of applications in the fields of medicine, food, cosmetics and agriculture.

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Abstract

一种壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂及其制备方法，该方法包括以下步骤：（1）将N-甲基哌嗪和氯代曲酸混合并反应，得到产物氯代曲酸-曼尼希碱；（2）将壳寡糖希夫碱与氯代曲酸-曼尼希碱混合并反应，经过后处理得到壳寡糖希夫碱-O-曲酸-曼尼希碱衍生物，经过氨基去保护反应和纯化得到壳寡糖-O-曲酸-曼尼希碱衍生物。

Description

一种壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂及其制备方法
本申请要求了申请日为2015年07月24日，申请号为2015104438518，发明名称为“一种壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于食品添加剂技术领域，尤其涉及一种壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂及其制备方法。
背景技术
近年来，食品安全问题已成为全球公众优先考虑的世界性焦点问题之一。食品在生产加工、运输、贮藏和销售等过程中都可能会遭受到一些有害微生物的污染，这些有害微生物能够污染食品，使食品腐败变质，不仅会造成食品资源的严重浪费，而且还会对误食者的身体造成严重的伤害，因此各类食品的防腐保鲜始终是一个亟待解决的重要问题。天然防腐抑菌剂如壳聚糖和曲酸，以其安全无毒、热稳定性好等特性受到人们的亲睐，通过化学改性开发出具有更强抑菌活性和更广抑菌谱的天然食品防腐剂已经成为人们研究的热点。
壳寡糖是壳聚糖主链经物理、化学或酶降解断裂后得到的低分子量碱性氨基寡糖。具有生物可降解性、生物相容性、生物无毒性和化学反应活性，对包括细菌、真菌在内的一系列微生物都有抑制作用，被视为开发新型天然食品防腐剂的理想材料。
然而，由于壳寡糖为天然大分子产物，当作为食品防腐抗菌剂使用时，与传统的常用化学防腐剂相比，仍有抗菌活性低等缺点，所以目前在食品工业中的应用并不是很普遍。因此，可以通过对壳寡糖的结构进行化学修饰，由于其分子链上具有化学反应活性的氨基和羟基，这些位点均是对壳寡糖进行化学改性的理想作用位点，可以进一步提高其抗菌活性，这也是目前国内外研究较多的有效方法。
有鉴于上述的缺陷，本设计人，积极加以研究创新，以期创设一种壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂及其制备方法，使其更具有产业上的利用价值。
发明内容
为解决上述技术问题，本发明的目的是提供一种壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂及其制备方法，将曲酸中的活性抗菌基团—γ-吡喃酮基和N-甲基哌嗪引入到壳寡糖分子链中，与壳寡糖分子产生协同作用，以增强壳寡糖的抗菌活性，得到溶解性好、相互增效的壳寡糖衍生物。
本发明提出的一种壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂，其化学结构式如下所示，其中，n为6-20，取代度为1.21-1.78。
Figure PCTCN2015088398-appb-000001
本发明还提出了壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法，包括以下步骤：
(1)将壳寡糖希夫碱与氯代曲酸-曼尼希碱用有机溶剂分别溶解后，混合并搅拌，反应结束后添加有机溶剂沉淀产物，过滤，沉淀产物用有机溶剂索氏抽提，冷冻干燥即得壳寡糖希夫碱-O-曲酸-曼尼希碱衍生物；
(2)向所述步骤(1)的壳寡糖希夫碱-O-曲酸-曼尼希碱衍生物中添加0.25mol/L的盐酸/乙醇(V/V＝1:4)混合液，混合并搅拌，反应结束后调节pH至中性，用有机溶剂洗涤，抽滤，冷冻干燥得到样品。
进一步的，所述方法还包括在步骤(2)之后的纯化步骤，将所述干燥好的产品透析，加入有机溶剂沉淀，抽滤，冷冻干燥最终得到经纯化的壳寡糖-O-曲酸-曼尼希碱衍生物。
进一步的，所述壳寡糖希夫碱与氯代曲酸-曼尼希碱的质量份比例为2∶(1-5)。
进一步的，所述壳寡糖的分子量为1000～5000Da，脱乙酰度为90-95％。
进一步的，所述步骤(1)中，溶解氯代曲酸-曼尼希碱的有机溶剂为二甲亚砜或二甲基甲酰胺，用量为氯代曲酸-曼尼希碱质量的2-4倍；溶解壳寡糖希夫碱的有机溶剂为二甲基甲酰胺和吡啶，二甲基甲酰胺的用量为壳寡糖希夫碱的2-4倍，吡啶的用量为壳寡糖希夫碱的2-6倍。
进一步的，所述步骤(1)中，反应温度为-35℃～40℃，反应时间为2～6h。
进一步的，所述步骤(2)中，反应温度为室温，反应时间为12-24h。
进一步的，所述步骤(1)中，所述氯代曲酸-曼尼希碱的制备方法包括以下步骤：
将N-甲基哌嗪和氯代曲酸溶解在甲醇和福尔马林溶液中，在室温下快速搅拌，生成褐色沉淀，收集、过滤，用无水甲醇洗涤若干次，用无水甲醇重结晶、干燥，即得到氯代曲酸-曼尼希碱。
进一步的，所述N-甲基哌嗪和氯代曲酸的质量份比例为1∶(2-3)，所述甲醇和福尔马林溶液的体积份比例为(10-20)∶1。
借由上述方案，本发明至少具有以下优点：本发明基于亚结构连接的思想，把曲酸中的活性基团—γ-吡喃酮基和N-甲基哌嗪接入到壳寡糖分子链中，使三者具有协同抗菌活性，开发出新型的食品防腐抑菌剂。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发 明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是壳寡糖的红外光谱图；
图2是本发明实施例一中制备的壳寡糖衍生物的红外光谱图；
图3是本发明实施例一中制备的壳寡糖衍生物的1H-NMR图；
图4是本发明实施例一中制备的壳寡糖衍生物的13C-NMR图；
图5是本发明实施例二中制备的壳寡糖衍生物的红外光谱图；
图6是本发明实施例三中制备的壳寡糖衍生物的红外光谱图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
具有良好水溶性和抗菌活性的壳寡糖-O-曲酸-曼尼希碱衍生物的制备方法如下：采用N-甲基哌嗪和氯代曲酸为原料，首先经曼尼希反应得到氯代曲酸-曼尼希碱，然后与经氨基保护的壳寡糖希夫碱发生烷基化反应，再经氨基脱保护反应和纯化得到保留活性氨基的壳寡糖-O-曲酸-曼尼希碱衍生物，其合成路线如下：
Figure PCTCN2015088398-appb-000002
实施例一：
壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法包括以下步骤：
1、氯代曲酸-曼尼希碱的制备
N-甲基哌嗪和氯代曲酸按质量比1:2溶解在100mL的甲醇和10mL的福尔马林溶液中，在25℃下快速搅拌，生成褐色沉淀，收集、过滤，用无水甲醇洗涤5次，用无水甲醇重结晶、干燥，即得到氯代曲酸-曼尼希碱。
2、壳寡糖-O-曲酸-曼尼希碱衍生物的制备
快速搅拌下，把0.5g氯代曲酸-曼尼希碱溶于2mL的二甲亚砜中，1g壳寡糖希夫碱(分子量为1000Da，脱乙酰度为90％)溶于2mL的二甲基甲酰胺和2mL的吡啶混合液中，将溶于二甲基甲酰胺和吡啶混合液中的壳寡糖希夫碱逐滴加入溶于二甲基甲酰胺的氯代曲酸-曼尼希碱中，反应混合物在-35℃水浴中磁力搅拌2小时后，倒入过量丙酮使产品沉淀，沉淀物过滤，沉淀物分别用乙醇、丙酮进行索氏提取以除去二甲基甲酰胺和吡啶，冷冻干燥得即得到1g壳寡糖希夫碱-O-曲酸-曼尼希碱衍生物。
在上述制得的壳寡糖希夫碱-O-曲酸-曼尼希碱衍生物中添加0.25mol/L的盐酸乙醇(V/V＝1:4)混合液20mL，室温搅拌12h后，用10％的Na2CO3调节pH至中性，用丙酮反复洗涤5次，抽滤，冷冻干燥。
将1克粗品悬于5mL超纯水中，放入透析袋中，透析24小时，浓缩，加入足量的丙酮沉淀，抽滤，冷冻干燥最终得到经纯化的褐色的壳寡糖-O-曲酸-曼尼希碱衍生物。
该壳寡糖衍生物的得率为62.1％，使用红外光谱和核磁共振对其进行表征。
图1为壳寡糖的红外光谱图，其中，3422.36cm-1为O-H与N-H的伸缩振动吸收峰，2923.83cm-1为C-H伸缩振动的吸收峰，1628.76cm-1为NH2的弯曲振动吸收峰，1155.97cm-1与1071.52cm-1为C-O伸缩振动的吸收峰，893.24cm-1为环伸缩振动吸收峰。
图2是本实施中壳寡糖衍生物的红外光图谱，其中，890.44cm-1处对应的β-吡喃型糖苷键的特征吸收峰，1519.37cm-1为该衍生物中C＝C伸缩振动吸收峰，1223.02cm-1为该衍生物中C-O-C伸缩振动吸收峰，970.39cm-1为氯代曲酸-曼尼希碱与壳寡糖共价结合键的吸收峰，1223.02cm-1和970.39cm-1的存在证明目标产物的形成。
图3是本实施例中壳寡糖衍生物的1H-NMR图，化学位移出现在1.968ppm处的峰对应的是乙酰氨基残基上的-CH3的质子峰；2.447-2.796ppm处的峰对应的是氨基葡萄糖残基和乙酰氨基残基上H2的质子峰；3.089-3.837ppm处的峰对应的是氨基葡萄糖和乙酰氨基葡萄糖上相应的H3,H4,H5,H6处的质子峰；4.7ppm处的峰为溶剂峰；4.26和6.82ppm处出现了两个新的化学位移，可分别归属为曲酸5-羟基吡喃酮骨架上-CH2(H-7’)和H-3’各质子的化学位移；而δ＝2.98ppm处的氨基葡萄糖残基上H2的化学位移仍然存在，此结果与红外光谱的结果一致，所以这些新的化学位移证明曲酸中5-羟基吡喃酮与壳寡糖的氨基发生了烷基化；2.88，3.07和3.12ppm处为N-甲基哌嗪环上的的化学位移；因此，通过以上化学位移的归属，可以证明，氯代曲酸-曼尼希碱与壳寡糖希夫碱发生了O-烷基化反应，壳寡糖-O-曲酸-曼尼希碱衍生物制备成功。
图4为本实施例中壳寡糖衍生物的13C-NMR图，δ＝22.74，56.33，60.46，71.03，75.11，76.95，98.63，174.18ppm分别归属为壳寡糖分子中-CH3，C2，C6，C3，C5，C4，C1和-C＝O的化学位移；δ＝45.83，47.08，56.38ppm，分别归属为衍生物中N-甲基哌嗪环上的-CH2和-CH3中的C。δ＝60.44，114.64，139.82，145.87，159.35和176.99ppm 处的化学位移归属为衍生物中的5-羟基吡喃酮基中C-7’，C-3’，C-6’，C-5’，C-2’和C-4’的化学位移，表明壳寡糖-O-曲酸-曼尼希碱衍生物制备成功。
实施例二：
壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法包括以下步骤：
1、氯代曲酸-曼尼希碱的制备
N-甲基哌嗪和氯代曲酸按质量比1:2.5溶解在150mL的甲醇和10mL的福尔马林溶液中，在25℃下快速搅拌，生成褐色沉淀，收集、过滤，用无水甲醇洗涤5次，用无水甲醇重结晶、干燥，即得到氯代曲酸-曼尼希碱。
2、壳寡糖-O-曲酸-曼尼希碱衍生物的制备
快速搅拌下，把1.5g氯代曲酸-曼尼希碱溶于3mL的二甲基甲酰胺中，1g壳寡糖希夫碱(分子量为3000Da，脱乙酰度为93％)溶于3mL的二甲基甲酰胺和4mL的吡啶混合液中，将溶于二甲基甲酰胺和吡啶混合液中的壳寡糖希夫碱逐滴加入溶于二甲基甲酰胺的氯代曲酸-曼尼希碱中，反应混合物在-5℃水浴中磁力搅拌4小时后，倒入过量丙酮使产品沉淀，沉淀物过滤，沉淀物分别用乙醇、丙酮进行索氏提取以除去二甲基甲酰胺和吡啶，最后得到经真空冷冻干燥后的产品1.8g。
在上述制得的壳寡糖希夫碱-O-曲酸-曼尼希碱衍生物中添加0.25mol/L的盐酸乙醇(V/V＝1:4)混合液36mL，室温搅拌12h后，用10％的Na2CO3调节pH至中性，用丙酮反复洗涤5次，抽滤，冷冻干燥。
将1克粗品悬于5mL超纯水中，放入透析袋中，透析24小时，浓缩，加入足量的丙酮沉淀，抽滤，冷冻干燥最终得到经纯化的褐色 的壳寡糖-O-曲酸-曼尼希碱衍生物。
该壳寡糖衍生物的得率为65.2％。图5为该样品的红外光谱图，其中，892.24cm-1处对应的β-吡喃型糖苷键的特征吸收峰，1518.32cm-1为该衍生物中C＝C伸缩振动吸收峰，1226.23cm-1为该衍生物中C-O-C伸缩振动吸收峰，971.91cm-1为氯代曲酸-曼尼希碱与壳寡糖共价结合键的吸收峰，1226.23cm-1和971.91cm-1的存在证明目标产物的形成。
实施例三：
壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法包括以下步骤：
1、氯代曲酸-曼尼希碱的制备
N-甲基哌嗪和氯代曲酸按质量比1:3溶解在200mL的甲醇和10mL的福尔马林溶液中，在25℃下快速搅拌，生成褐色沉淀，收集、过滤，用无水甲醇洗涤5次，用无水甲醇重结晶、干燥，即得到氯代曲酸-曼尼希碱。
2、壳寡糖-O-曲酸-曼尼希碱衍生物的制备
快速搅拌下，把2.5g氯代曲酸-曼尼希碱溶于4mL的二甲亚砜中，1g壳寡糖希夫碱(分子量为5000Da，脱乙酰度为95％)溶于4mL的二甲基甲酰胺和6mL的吡啶混合液中，将溶于二甲基甲酰胺和吡啶混合液中的壳寡糖希夫碱逐滴加入溶于二甲基甲酰胺的氯代曲酸-曼尼希碱中，反应混合物在40℃水浴中磁力搅拌6小时后，倒入过量丙酮使产品沉淀，沉淀物过滤，沉淀物分别用乙醇、丙酮进行索氏提取以除去二甲基甲酰胺和吡啶，最后得到经真空冷冻干燥后的产品3g。
在上述制得的壳寡糖希夫碱-O-曲酸-曼尼希碱衍生物中添加0.25mol/L的盐酸乙醇(V/V＝1:4)混合液60mL，室温搅拌12h后， 用10％的Na2CO3调节pH至中性，用丙酮反复洗涤5次，抽滤，冷冻干燥。
将1克粗品悬于5mL超纯水中，放入透析袋中，透析24小时，浓缩，加入足量的丙酮沉淀，抽滤，冷冻干燥最终得到经纯化的褐色的壳寡糖-O-曲酸-曼尼希碱衍生物。
该壳寡糖衍生物的得率为63.5％。图6为该样品的红外光谱图，其中，893.34cm-1处对应的β-吡喃型糖苷键的特征吸收峰，1514.18cm-1为该衍生物中C＝C伸缩振动吸收峰，1223.73cm-1为该衍生物中C-O-C伸缩振动吸收峰，970.39cm-1为氯代曲酸与壳寡糖共价结合键的吸收峰，1223.13cm-1和970.39cm-1的存在证明目标产物的形成。
抗菌性的检测
1、抗菌剂溶液的制备
将实施例一、实施例二、实施例三中的样品分别命名为衍生物1、衍生物2、衍生物3，用去离子水将壳寡糖、曲酸、衍生物1、衍生物2和衍生物3溶解，配成浓度梯度为0.01、0.05、0.1、0.5、1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0和9.0mg/mL的溶液，用0.22μm的微孔滤膜过滤，备用。
2、抗菌活性测定
将1mL的初始菌液与98mL灭菌后的营养肉汤培养混合，再向其中加入1mL的不同浓度梯度的上述抗菌剂，以1mL去离子水代替抗菌剂溶液作为空白，37℃下摇床培养。在培养过程中，8h后取出1mL培养液，倍比稀释后涂布平板法计算菌落数，每个样品重复3次。抗菌率(I)按照以下公式计算：
I(％)＝N1-N2/N1×100
其中，N1：初始培养液中的菌落总数；
N2：含有抗菌剂培养液中的菌落总数。
以抗菌剂的浓度为横坐标，抑制率为纵坐标作图，得到IC50值
表1抗菌剂性能的检测
Figure PCTCN2015088398-appb-000003
从表1中可以看出，本发明制得的抗菌剂对金黄色葡萄球菌、酿脓链球菌、枯草芽孢杆菌、鼠伤寒沙门氏菌、痢疾志贺氏菌和大肠杆菌都具有很好的抗菌活性，而且抗菌性能大大优于壳寡糖和曲酸，在食品防腐方面具有潜在的应用价值。
综上所述，本发明的原理如下：壳寡糖的分子结构中有三个化学改性活性位点，如果发生烷基化反应，按照三个位置活性的大小，首先发生在C-2位氨基上，其次是C-6位伯羟基上，最后是C-3位仲羟基上。由于在酸性溶液中，壳寡糖分子中的C-2氨基质子化之后，是起抗菌作用的活性基团，因此，首先将C-2位的活性氨基用苯甲醛进行保护，待发生了烷基化反应之后，再在酸醇溶液中脱保护，释放活性氨基。而C-3位的羟基由于位阻效应，不参与反应。因此C-6位的羟基与氯代曲酸-曼尼希碱发生O-烷基化反应。
本发明的有益效果如下：(1)通过壳寡糖的C-6位的羟基与氯代曲酸-曼尼希碱发生O-烷基化反应，将曲酸中的活性抗菌基团—γ-吡 喃酮基和N-甲基哌嗪引入到壳寡糖分子链中，与壳寡糖分子产生协同作用，显著增强其抗菌活性。(2)此类新的衍生物毒性低，且有很好的水溶性，可溶解于多种无机和有机溶剂中，避免了有机溶剂的使用，更有利于环境保护，而且扩大了其应用领域，在医药、食品、化妆品和农业等领域有广泛的应用价值。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (10)

  1. 一种壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂，其特征在于：其化学结构式如下所示，其中，n为6-20，取代度为1.21-1.78。
    Figure PCTCN2015088398-appb-100001
  2. 根据权利要求1所述的壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法，其特征在于：包括以下步骤：
    (1)将壳寡糖希夫碱与氯代曲酸-曼尼希碱用有机溶剂分别溶解后，混合并搅拌，反应结束后添加有机溶剂沉淀产物，过滤，沉淀产物用有机溶剂索氏抽提，冷冻干燥即得壳寡糖希夫碱-O-曲酸-曼尼希碱衍生物；
    (2)向所述步骤(1)的壳寡糖希夫碱-O-曲酸-曼尼希碱衍生物中添加0.25mol/L的盐酸/乙醇(V/V＝1:4)混合液，混合并搅拌，反应结束后调节pH至中性，用有机溶剂洗涤，抽滤，冷冻干燥得到样品。
  3. 根据权利要求2所述的壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法，其特征在于：所述方法还包括在步骤(2)之后的纯化步骤，将所述干燥好的产品透析，加入有机溶剂沉淀，抽滤，冷冻干燥最终得到经纯化的壳寡糖-O-曲酸-曼尼希碱衍生物。
  4. 根据权利要求2所述的壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法，其特征在于：所述壳寡糖希夫碱与氯代曲酸-曼尼希碱的质量份比例为2∶(1-5)。
  5. 根据权利要求4所述的壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法，其特征在于：所述壳寡糖的分子量为1000～5000Da，脱乙酰度为90-95％。
  6. 根据权利要求2所述的壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法，其特征在于：所述步骤(1)中，溶解氯代曲酸-曼尼希碱的有机溶剂为二甲亚砜或二甲基甲酰胺，用量为氯代曲酸-曼尼希碱质量的2-4倍；溶解壳寡糖希夫碱的有机溶剂为二甲基甲酰胺和吡啶，二甲基甲酰胺的用量为壳寡糖希夫碱的2-4倍，吡啶的用量为壳寡糖希夫碱的2-6倍。
  7. 根据权利要求2所述的壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法，其特征在于：所述步骤(1)中，反应温度为-35℃～40℃，反应时间为2～6h。
  8. 根据权利要求2所述的壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法，其特征在于：所述步骤(2)中，反应温度为室温，反应时间为12-24h。
  9. 根据权利要求2所述的壳寡糖-O-曲酸-曼尼希碱衍生物抗菌剂的制备方法，其特征在于：所述步骤(1)中，所述氯代曲酸-曼尼希碱的制备方法包括以下步骤：
    将N-甲基哌嗪和氯代曲酸溶解在甲醇和福尔马林溶液中，在室温下快速搅拌，生成褐色沉淀，收集、过滤，用无水甲醇洗涤若干次，用无水甲醇重结晶、干燥，即得到氯代曲酸-曼尼希碱。
  10. 根据权利要求9所述的壳寡糖-O-曲酸-曼尼希碱衍生物抗菌 剂的制备方法，其特征在于：所述N-甲基哌嗪和氯代曲酸的质量份比例为1：(2-3)，所述甲醇和福尔马林溶液的体积份比例为(10-20)：1。
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CN102321196A (zh) * 2011-10-11 2012-01-18 北京联合大学生物化学工程学院 O-水杨酸酯化低聚壳聚糖水杨醛席夫碱抑菌剂及其制备方法
CN103304683A (zh) * 2013-06-09 2013-09-18 江南大学 一种壳寡糖曲酸衍生物及其制备方法
CN104558244A (zh) * 2014-12-19 2015-04-29 华南理工大学 一种o-吡啶酸酯壳聚糖及其制备方法与应用

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