CN113632910B - 丁香酚-槐糖脂纳米乳液及其制备方法与应用 - Google Patents
丁香酚-槐糖脂纳米乳液及其制备方法与应用 Download PDFInfo
- Publication number
- CN113632910B CN113632910B CN202110900426.2A CN202110900426A CN113632910B CN 113632910 B CN113632910 B CN 113632910B CN 202110900426 A CN202110900426 A CN 202110900426A CN 113632910 B CN113632910 B CN 113632910B
- Authority
- CN
- China
- Prior art keywords
- eugenol
- sophorolipid
- nanoemulsion
- bacillus cereus
- staphylococcus aureus
- 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.)
- Active
Links
- 239000007908 nanoemulsion Substances 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- RRAFCDWBNXTKKO-UHFFFAOYSA-N eugenol Chemical compound COC1=CC(CC=C)=CC=C1O RRAFCDWBNXTKKO-UHFFFAOYSA-N 0.000 claims abstract description 79
- NPBVQXIMTZKSBA-UHFFFAOYSA-N Chavibetol Natural products COC1=CC=C(CC=C)C=C1O NPBVQXIMTZKSBA-UHFFFAOYSA-N 0.000 claims abstract description 39
- 239000005770 Eugenol Substances 0.000 claims abstract description 39
- UVMRYBDEERADNV-UHFFFAOYSA-N Pseudoeugenol Natural products COC1=CC(C(C)=C)=CC=C1O UVMRYBDEERADNV-UHFFFAOYSA-N 0.000 claims abstract description 39
- 229960002217 eugenol Drugs 0.000 claims abstract description 39
- ZTOKUMPYMPKCFX-CZNUEWPDSA-N (E)-17-[(2R,3R,4S,5S,6R)-6-(acetyloxymethyl)-3-[(2S,3R,4S,5S,6R)-6-(acetyloxymethyl)-3,4,5-trihydroxyoxan-2-yl]oxy-4,5-dihydroxyoxan-2-yl]oxyoctadec-9-enoic acid Chemical compound OC(=O)CCCCCCC/C=C/CCCCCCC(C)O[C@@H]1O[C@H](COC(C)=O)[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](COC(C)=O)O1 ZTOKUMPYMPKCFX-CZNUEWPDSA-N 0.000 claims abstract description 34
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 9
- 244000005700 microbiome Species 0.000 claims abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- 239000002245 particle Substances 0.000 claims description 18
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- 239000002904 solvent Substances 0.000 claims description 14
- 239000012153 distilled water Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 230000032683 aging Effects 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 238000000861 blow drying Methods 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims 2
- 241000193755 Bacillus cereus Species 0.000 abstract description 32
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 15
- 241000607768 Shigella Species 0.000 abstract description 14
- 238000000576 coating method Methods 0.000 abstract description 4
- 239000004064 cosurfactant Substances 0.000 abstract description 4
- 239000011248 coating agent Substances 0.000 abstract description 3
- 229920006280 packaging film Polymers 0.000 abstract description 3
- 239000012785 packaging film Substances 0.000 abstract description 3
- 239000003755 preservative agent Substances 0.000 abstract description 3
- 230000002195 synergetic effect Effects 0.000 abstract description 3
- 239000004615 ingredient Substances 0.000 abstract 1
- 230000002335 preservative effect Effects 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 17
- 238000007664 blowing Methods 0.000 description 11
- 239000000839 emulsion Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 5
- 108010053775 Nisin Proteins 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 239000004309 nisin Substances 0.000 description 5
- 235000010297 nisin Nutrition 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 244000078673 foodborn pathogen Species 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 230000001629 suppression Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 244000223014 Syzygium aromaticum Species 0.000 description 3
- 235000016639 Syzygium aromaticum Nutrition 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000341 volatile oil Substances 0.000 description 3
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 229930186217 Glycolipid Natural products 0.000 description 2
- 241000186779 Listeria monocytogenes Species 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- MLFHJEHSLIIPHL-UHFFFAOYSA-N isoamyl acetate Chemical compound CC(C)CCOC(C)=O MLFHJEHSLIIPHL-UHFFFAOYSA-N 0.000 description 2
- 239000004973 liquid crystal related substance Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 238000009631 Broth culture Methods 0.000 description 1
- 241001508786 Citeromyces matritensis Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003876 biosurfactant Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 231100000206 health hazard Toxicity 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229940117955 isoamyl acetate Drugs 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000017066 negative regulation of growth Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229910052762 osmium Inorganic materials 0.000 description 1
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- -1 semisolid Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3481—Organic compounds containing oxygen
- A23L3/349—Organic compounds containing oxygen with singly-bound oxygen
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3544—Organic compounds containing hetero rings
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种丁香酚‑槐糖脂纳米乳液及其制备方法与应用，其中，所述丁香酚‑槐糖脂纳米乳液由丁香酚、槐糖脂和水组成，对蜡样芽孢杆菌(Bacilluscereus)、志贺氏菌(Shigella Castellani)、金黄色葡萄球菌(Staphylococcus aureus)中的至少一种微生物起抑制作用。本发明丁香酚‑槐糖脂纳米乳液稳定性好，不仅可以使丁香酚增溶，而且槐糖脂与丁香酚起到协同抑菌效果，可以作为配料应用于防腐剂、抑菌涂膜、抗菌包装膜等领域中，且制备方法简单、不需要助表面活性剂，成本低，具有很大的市场前景。
Description
技术领域
本发明属于抗菌技术，特别是涉及一种丁香酚-槐糖脂纳米乳液及其制备方法与应用。
背景技术
食品安全一直是全球重要的公共卫生问题，其中食源性致病菌引起的健康危害备受关注。常见的食源性致病菌包括大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphyloccocus aureus)、蜡样芽胞杆菌(Bacillus cereus)、单增李斯特菌(Listeriamonocytogenes)、沙门氏菌(Samonella)等。使用抑菌剂是有效抑制和杀灭食品中病原微生物的有效方法。随着消费者对健康产品的追求，绿色、安全、高效的天然抑菌剂已成为抑菌剂领域研究和应用发展的必然趋势。
从丁香花蕾中提取出的丁香精油，呈淡黄色或无色的油状液体，具有特殊的馨香气味，是一种用途广泛的抗菌药物。丁香精油能够显著抑制细菌、真菌的生长繁殖。丁香酚是丁香精油的主要抗菌活性成分。但丁香酚水溶性差、易挥发，在含水体系应用效果不佳，又由于丁香酚为脂溶性成分，易与脂质等成分结合，影响其功效发挥。通过由表面活性剂、水、油相或者助表面活性剂组成的乳液体系可以达到增油溶性成分、稳定活性的作用。
槐糖脂是由微生物发酵转化获得的一类糖脂类表面活性剂，具有良好的乳化性、表/界面活性、生物降解性等，同时具有抑菌活性、诱导肿瘤细胞凋亡和变异等生理活性，在食品、化妆品、环保、医药、材料等领域有潜在的应用价值。尤其可作为乳化剂应用于乳液体系的构建。在无助表面活性剂的条件下，槐糖脂、丁香酚与水可以形成具有各向同性且热力学稳定的透明或半透明分散体系，其分散相质点为1～200nm。由于丁香酚和槐糖脂均具有抑菌活性，其丁香酚-槐糖脂纳米乳液不仅能增溶丁香酚，还具有抑菌协同性。
目前还没有以丁香酚-槐糖脂纳米乳液及其制备以及在抑制食源致病菌生长的应用报道。
发明内容
发明目的：针对上述现有技术，本发明提供了一种丁香酚-槐糖脂纳米乳液及其制备方法以及应用。
技术方案：本发明所述的一种丁香酚-槐糖脂纳米乳液，包括丁香酚、槐糖脂和蒸馏水，每1L水中含有的丁香酚和槐糖脂的总质量为0.5g～10g，其中，丁香酚、槐糖脂的质量比为1:9～9:1。
进一步的，所述纳米乳液粒径小于500nm，测定纳米乳液的浊度A600为 0.01～1.0，进一步也可以通过纳米粒度仪确定粒径大小。
本发明所述丁香酚-槐糖脂纳米乳液的制备方法，包括以下步骤：
(1)取配方量的丁香酚、槐糖脂，溶于有机溶剂，吹干溶剂并干燥；
(2)逐滴加入蒸馏水，涡旋混匀即得纳米乳液。
步骤(1)中，所述有机溶剂选自氯仿、丙酮、乙酸乙酯、甲醇、乙醇中的一种。
步骤(1)中，采用氮吹仪吹干，氮气流速1.2～2.5L/min，温度为20～45℃，真空干燥过夜，确保无溶剂残留。
步骤(2)中，按照50～100uL逐次滴入蒸馏水，每加完一次，混合液在 800～1500rpm条件下涡旋混匀10min，静置30min，控制加水次数，使混合液浊度A600为0.01～1.0，获得纳米乳液。
所述丁香酚-槐糖脂纳米乳液在抑制微生物生长中的应用也在本发明的保护范围内。
其中，所述微生物选自蜡样芽孢杆菌(Bacillus cereus)、志贺氏菌(ShigellaCastellani)、金黄色葡萄球菌(Staphylococcus aureus)。
所述应用包括以下步骤：丁香酚-槐糖脂纳米乳液加入培养基中，并接种蜡样芽孢杆菌(Bacillus cereus)、志贺氏菌(Shigella Castellani)、金黄色葡萄球菌(Staphylococcus aureus)中的至少一种微生物，起效时间1～2h，抑菌时效24～72h。
进一步的，所述丁香酚-槐糖脂纳米乳液的浓度为0.5g/L以上。
有益效果：相比较于现有技术，本申请具有以下优势：(1)本发明的纳米乳液仅由丁香酚、槐糖脂和水构成，不需要添加助表面活性剂，稳定性高，不仅可以使丁香酚增溶，而且槐糖脂与丁香酚起到协同功效，对一般食源性病菌有显著的抑菌效果，具有广谱性。(2)本发明操作方法简单，成本低，适应于防腐剂、抑菌涂膜、抗菌包装膜等领域中，具有广阔的市场前景。
附图说明
图1是丁香酚-槐糖脂(比例9:1)纳米乳液分别在4℃、25℃、37℃条件下储存15天的粒径变化情况；
图2是不同质量比的丁香酚和槐糖脂制备的纳米乳液对蜡样芽胞杆菌(A)、金黄色葡萄球菌(B)和志贺氏菌(C)培养24h后的抑制结果；
图3是丁香酚-槐糖脂(比例9:1)纳米乳液处理蜡样芽胞杆菌72h的生长情况；
图4是对照(未处理)组蜡样芽胞杆菌培养2h的形态；
图5是丁香酚-槐糖脂(比例9:1)纳米乳液处理组蜡样芽胞杆菌培养2h的形态；
图6是槐糖脂单一组分组蜡样芽胞杆菌培养2h的形态；
图7是丁香酚单一组分处理组蜡样芽胞杆菌培养2h的形态；
图8是阳性对照乳酸链球菌素处理组蜡样芽胞杆菌培养2h的形态。
具体实施方式
下面结合具体实施例对本申请作出详细说明。
槐糖脂是一类由酵母菌代谢生产的糖脂型生物表面活性剂，具有优越的表/ 界面活性、乳化性、稳定性、生物可降解性，安全无毒，同时具有抑菌活性、诱导肿瘤细胞凋亡和变异等生理活性，在食品、化妆品、环保、医药、材料等领域有潜在的应用价值。
酸型和内酯型槐糖脂结构式
本发明的槐糖脂可以通过发酵获得，可以是发酵液提取物或浓缩物，可以是经有机溶剂分离纯化获得的纯品，可以是液体、半固体、固体膏状或粉末状。
槐糖脂的发酵制备方法：
球拟假丝酵母经YPD培养基(g/L，葡萄糖15.0，蛋白胨20.0，酵母粉10.0) 活化，活化后的液体种子以2％接种到装有100mL种子培养基(g/L，葡萄糖20.0，酵母粉3.0，KH2PO40.3，MgSO4·7H2O 0.3，Na2HPO4 0.3)的300mL三角瓶中， 200rpm、30℃下培养10h。活化后的种子液以2％接种量接种到装有100mL发酵培养基的三角瓶中，200rpm、30℃下培养7d。发酵结束后采用等体积的乙酸乙酯萃取3次，获得槐糖脂。
槐糖脂也可以通过购买获得，CAS 148409-20-5。
丁香酚可以通过购买获得，CAS 97-53-0。
实施例1丁香酚-槐糖脂纳米乳液的制备
按质量比9:1称取丁香酚和槐糖脂，溶于氯仿，氮吹仪吹干溶剂，并真空干燥过夜，彻底去除溶剂。按照100μL逐滴逐次加蒸馏水，每加完一次，混合液在800rpm条件下涡旋混匀10min，静置平衡30min。控制加水量至丁香酚和槐糖脂混合组分的占比为0.5g/L，浊度A600为0.2，采用激光纳米粒度仪测定乳液的平均粒径为137nm。
实施例2
按质量比8:2称取丁香酚和槐糖脂，溶于氯仿，氮吹仪吹干溶剂，并真空干燥过夜，彻底去除溶剂。按照100μL逐滴逐次加蒸馏水，每加完一次，混合液在800rpm条件下涡旋混匀10min，静置平衡30min，备用。控制加水量至丁香酚和槐糖脂混合组分的占比为0.5g/L，浊度A600为0.8，采用激光纳米粒度仪测定乳液的平均粒径为175nm。
实施例3
按质量比1:9称取丁香酚和槐糖脂，溶于氯仿，氮吹仪吹干溶剂，并真空干燥过夜，彻底去除溶剂。按照50μL逐滴逐次加蒸馏水，每加完一次，混合液在 800rpm条件下涡旋混匀10min，静置平衡30min，备用。控制加水量至丁香酚和槐糖脂混合组分的占比为10g/L，浊度A600为1.0，采用激光纳米粒度仪测定乳液的平均粒径为301nm。
实施例4
按质量比9:1称取丁香酚和槐糖脂，溶于氯仿，氮吹仪吹干溶剂，并真空干燥过夜，彻底去除溶剂。按照100μL逐滴逐次加蒸馏水，每加完一次，混合液在1000rpm条件下涡旋混匀10min，静置平衡30min。控制加水量至丁香酚和槐糖脂混合组分的占比为0.5g/L，浊度A600为0.01，采用激光纳米粒度仪测定乳液的平均粒径为52nm。
实施例5
按质量比6:4称取丁香酚和槐糖脂，溶于氯仿，氮吹仪吹干溶剂，并真空干燥过夜，彻底去除溶剂。按照100μL逐滴逐次加蒸馏水，每加完一次，混合液在1000rpm条件下涡旋混匀10min，静置平衡30min，备用。控制加水量至丁香酚和槐糖脂混合组分的占比为0.5g/L，浊度A600为1.0，采用激光纳米粒度仪测定乳液的平均粒径为189nm。
丁香酚-槐糖脂纳米乳液的稳定性测定
将新鲜制备的丁香酚-槐糖脂纳米乳液分别放在4、25、37℃温度条件下储存，每隔3d测定平均粒径，评价丁香酚-槐糖脂纳米乳液的稳定性。结果如图1所示，根据图示可见，制备的丁香酚-槐糖脂纳米乳液粒径分布在200nm以下，呈透明状液体，在4、25、37℃温度条件下粒径变化不大，储存稳定性好。
实施例6抑菌方法和检测
(1)制备菌悬液
将斜面保存的微生物用接种环挑取1～2环，接种到LB培养基中，在37℃和 180r/min转速的全温摇床中培养12h，取出1mL菌液于离心管中，在8000～15000 rpm、4℃条件下离心3～5min收集菌体，再用无菌生理盐水进行洗涤、重悬至少 3次，最后用无菌的生理盐水配制成菌悬液，浓度为1×107CFU/mL。
(2)最小抑菌浓度的确定
采用二倍梯度稀释法测定纳米乳液的最小抑菌浓度。对数期的指示菌用LB 培养基梯度稀释至105CFU/mL，分别取100μL菌液于96孔板，每孔再加入100 μL纳米乳液梯度稀释液。以200μL LB培养基作为阴性空白对照，添加100μL 稀释后的菌液和100μL LB肉汤为阳性空白对照，作六个平行。混匀后将96孔板置于培养箱中37℃培养18h，取出后用酶标仪测定OD600。以纳米乳液浓度为横坐标，以OD600值为纵坐标做曲线，确定拐点为最小抑菌浓度。
(3)纳米乳液的抑菌效果
采用最小抑菌浓度的纳米乳液处理菌悬液，置于培养箱中37℃培养24h，取出后用酶标仪测定OD600。以添加100μL菌液和100μL LB肉汤培养基为空白对照，每个样品作六个平行。抑菌率计算按照：抑菌率％＝(对照组菌量-试验组菌量)/对照组菌量*100％。
采用平板涂布方法，评价抑菌时效。具体取100μL对照或纳米乳液处理后的菌液，涂布于含有肉汤培养基的平板，在37℃温度条件下培养72h，分别在 1h、2h、24h、72h取样观察菌生长情况。培养24h后的抑制结果如图2所示，根据图示可见，按照最小抑菌浓度，添加0.5g/L丁香酚-槐糖脂纳米乳液至菌液中，根据附图2(A、B、C)分别所示的蜡样芽孢杆菌、金黄色葡萄糖球菌和志贺氏菌培养24h后的OD600结果可以看出，与对照组(CK)相比，纳米乳液处理的菌液OD显著降低，说明所制备的丁香酚-槐糖脂纳米乳液对蜡样芽胞杆菌、金黄色葡萄糖球菌和志贺氏菌具有显著的抑制作用。其中，
实施例1制备的丁香酚-槐糖脂纳米乳液对蜡样芽胞杆菌的抑菌率为73.5％，对金黄色葡萄球菌抑制率为80.3％，对志贺氏菌抑制率为84.2％；
实施例2制备的丁香酚-槐糖脂纳米乳液对蜡样芽胞杆菌的抑菌率为82.9％，对金黄色葡萄球菌抑制率为67.9％，对志贺氏菌抑制率为76.0％；
实施例3制备的丁香酚-槐糖脂纳米乳液对蜡样芽胞杆菌的抑菌率为57.6％，对金黄色葡萄球菌抑制率为55.0％，对志贺氏菌抑制率为77.9％；
实施例4制备的丁香酚-槐糖脂纳米乳液对蜡样芽胞杆菌的抑菌率为85.3％，对金黄色葡萄球菌抑制率为79.4％，对志贺氏菌抑制率为82.1％；
实施例5制备的丁香酚-槐糖脂纳米乳液对蜡样芽胞杆菌的抑菌率为60.4％对金黄色葡萄球菌抑制率为64.3％，对志贺氏菌抑制率为70.6％。
培养72h的生长情况如图3所示，根据图示可见，与对照组相比，丁香酚- 槐糖脂纳米乳液处理组的菌培养1h、2h、24h或72h后，菌密度显著降低，由此可以看出丁香酚-槐糖脂纳米乳液对蜡样芽孢杆菌起效时间为1h，抑菌时效长达 72h。
采用扫描电镜确定纳米乳液对菌形态的影响。培养后的菌悬液离心后向菌体沉淀中加入2.5％(v/v)戊二醛溶液，在4℃下固定24h。固定后离心收集菌体，采用pH 7.2的1％锇酸溶液对菌体再固定1.5h。将固定好的菌体用PBS缓冲液漂洗3次，去除多余的固定液。将样品依次置于系列体积分数为30％、50％、70％、80％、90％和100％的乙醇溶液中脱水置换，每次15min。将置换后的样品用100％乙酸异戊酯漂洗两次以取代乙醇，每次15min；将菌体喷于铜网上临界点干燥，并喷金。制备好的切片在扫描电镜上观察并采集图像。结果如图4和图5所示，很明显，与对照组蜡样芽胞杆菌的菌体相态(图4)相比，图5显示的丁香酚- 槐糖脂纳米乳液处理2h后的蜡样芽孢杆菌形态破坏，发生团聚，说明丁香酚- 槐糖脂纳米乳液对蜡样芽孢杆菌具有显著的抑制作用。
对比例1单一槐糖脂对蜡样芽孢杆菌的生长抑制
配制与实施例所用相同浓度(0.05g/L)的槐糖脂乳液，按照实施例6的操作，得到结果如附图6所示。与图4和图5结果相比，单一槐糖脂对蜡样芽孢杆菌作用2h的抑菌效果明显差于丁香酚-槐糖脂纳米乳液的效果。
对比例2单一丁香酚对蜡样芽孢杆菌的生长抑制
配制与实施例所用相同浓度的丁香酚乳液(0.45g/L)，按照实施例6的操作，得到结果如附图7所示。与图4和图5相比，单一丁香酚对蜡样芽孢杆菌作用 2h的抑菌效果明显差于丁香酚-槐糖脂纳米乳液的效果。
对比例3乳酸链球菌素对蜡样芽孢杆菌的生长抑制
选用食品常用的天然抑菌剂乳酸链球菌素作为阳性对照。按照最小抑菌浓度，配制浓度为2.0g/L的乳酸链球菌素溶液，依据实施例6的操作，得到结果如附图8所示。与图4和图5相比，乳酸链球菌素对蜡样芽孢杆菌作用2h的抑菌效果明显差于丁香酚-槐糖脂纳米乳液的效果。
以上可以看出，本发明的纳米乳液，槐糖脂与丁香酚起到协同抑菌效果，对一般食源性病菌有显著的抑菌效果，适应于防腐剂、抑菌涂膜、抗菌包装膜等领域中，具有广阔的应用前景。
Claims (7)
1.一种丁香酚-槐糖脂纳米乳液在抑制微生物生长中的应用，其特征在于，所述微生物选自蜡样芽孢杆菌Bacillus cereus、志贺氏菌Shigella Castellani、金黄色葡萄球菌Staphylococcus aureus；所述丁香酚-槐糖脂纳米乳液由丁香酚、槐糖脂、蒸馏水、有机溶剂制成，每1L蒸馏水中含有的丁香酚和槐糖脂的总质量为0.5g~10g，其中，丁香酚、槐糖脂的质量比为1:9~9:1；所述纳米乳液粒径小于500 nm。
2.根据权利要求1所述的应用，其特征在于，所述的丁香酚-槐糖脂纳米乳液的制备方法包括以下步骤：
（1）取配方量的丁香酚、槐糖脂，溶于有机溶剂，吹干溶剂并干燥；
（2）逐滴加入蒸馏水，涡旋混匀即得纳米乳液。
3.根据权利要求2所述的应用，其特征在于，步骤（1）中，所述有机溶剂选自氯仿、丙酮、乙酸乙酯、甲醇、乙醇中的一种。
4.根据权利要求2所述的应用，其特征在于，步骤（1）中，采用氮吹仪吹干，氮气流速1.2~2.5 L/min，温度为20~45℃，真空干燥过夜，确保溶剂无残留。
5.根据权利要求2所述的应用，其特征在于，步骤（1）中，所述丁香酚和槐糖脂按照1.5~9:1的质量比混合。
6.根据权利要求2所述的应用，其特征在于，步骤（2）中，按照50~100uL/次逐滴加入蒸馏水，每加完一次，混合液在800~1500rpm涡旋混匀并静置，控制加水次数使混合液浊度A600为0.01~1.0，即得纳米乳液。
7.根据权利要求1所述的应用，其特征在于，所述应用包括以下步骤：丁香酚-槐糖脂纳米乳液加入培养基中，并接种蜡样芽孢杆菌Bacillus cereus、志贺氏菌Shigella Castellani、金黄色葡萄球菌Staphylococcus aureus中的至少一种微生物，起效时间1~2h，抑菌时效24~72h。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110900426.2A CN113632910B (zh) | 2021-08-06 | 2021-08-06 | 丁香酚-槐糖脂纳米乳液及其制备方法与应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110900426.2A CN113632910B (zh) | 2021-08-06 | 2021-08-06 | 丁香酚-槐糖脂纳米乳液及其制备方法与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113632910A CN113632910A (zh) | 2021-11-12 |
| CN113632910B true CN113632910B (zh) | 2023-07-25 |
Family
ID=78419834
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110900426.2A Active CN113632910B (zh) | 2021-08-06 | 2021-08-06 | 丁香酚-槐糖脂纳米乳液及其制备方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113632910B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117338795B (zh) * | 2023-10-30 | 2024-05-17 | 四川农业大学 | 和厚朴酚槐糖脂复合物及其制备方法和应用 |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101019875A (zh) * | 2007-01-29 | 2007-08-22 | 山东大学 | 槐糖脂在制备抗生素药物中的应用 |
| CN101199539B (zh) * | 2007-12-10 | 2010-04-14 | 山东大学 | 槐糖脂在制备抗皮肤癣菌药物中的应用 |
| CN101214355B (zh) * | 2007-12-29 | 2010-12-01 | 西北农林科技大学 | 一种复方丁香酚纳米乳组合物及其制备方法 |
| CN104012991A (zh) * | 2014-05-23 | 2014-09-03 | 南昌大学 | 乙醇注入-动态高压微射流制备丁香酚纳米脂质体的方法 |
| CN107751197A (zh) * | 2017-11-10 | 2018-03-06 | 江苏省农业科学院 | 一种复配增效的植物源抑菌剂 |
| JP7114922B2 (ja) * | 2018-02-13 | 2022-08-09 | 住友化学株式会社 | バイオフィルム生成抑制用組成物 |
| CN108938558A (zh) * | 2018-06-25 | 2018-12-07 | 宁波立华制药有限公司 | 一种纳米乳复合物及制备方法和应用 |
| CN109316443A (zh) * | 2018-12-03 | 2019-02-12 | 广州中医药大学(广州中医药研究院) | 姜黄素维生素e琥珀酸酯前药、纳米乳及其制备方法和用途 |
| KR20210145122A (ko) * | 2019-03-29 | 2021-12-01 | 라보라뚜아르 엠므2 | 항균 나노 에멀젼 |
| CN111202739A (zh) * | 2020-01-16 | 2020-05-29 | 山东大学 | 槐糖脂在制备抗痤疮丙酸杆菌及其生物膜的药物中的应用 |
-
2021
- 2021-08-06 CN CN202110900426.2A patent/CN113632910B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113632910A (zh) | 2021-11-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Antifungal activity of thymol and carvacrol against postharvest pathogens Botrytis cinerea | |
| Zahi et al. | Enhancing the antimicrobial activity of d-limonene nanoemulsion with the inclusion of ε-polylysine | |
| Yang et al. | Antifungal effect of cinnamaldehyde, eugenol and carvacrol nanoemulsion against Penicillium digitatum and application in postharvest preservation of citrus fruit | |
| Shahbazi | Application of carboxymethyl cellulose and chitosan coatings containing Mentha spicata essential oil in fresh strawberries | |
| Umagiliyage et al. | Antimicrobial efficacy of liposomes containing d-limonene and its effect on the storage life of blueberries | |
| Cui et al. | Inhibition of Escherichia coli O157: H7 biofilm on vegetable surface by solid liposomes of clove oil | |
| Zhang et al. | Effects of nisin on the antimicrobial activity of D-limonene and its nanoemulsion | |
| Marín et al. | Effect of different coating-forming agents on the efficacy of the biocontrol agent Candida sake CPA-1 for control of Botrytis cinerea on grapes | |
| Li et al. | Thymol nanoemulsions formed via spontaneous emulsification: Physical and antimicrobial properties | |
| Aloui et al. | Efficacy of the combined application of chitosan and Locust Bean Gum with different citrus essential oils to control postharvest spoilage caused by Aspergillus flavus in dates | |
| Wang et al. | Enhanced antibacterial activity of eugenol-entrapped casein nanoparticles amended with lysozyme against gram-positive pathogens | |
| Wu et al. | Influence of nonionic and ionic surfactants on the antifungal and mycotoxin inhibitory efficacy of cinnamon oil nanoemulsions | |
| Li et al. | Effects and possible mechanism of tea tree oil against Botrytis cinerea and Penicillium expansum in vitro and in vivo test | |
| CN113632910B (zh) | 丁香酚-槐糖脂纳米乳液及其制备方法与应用 | |
| Perez et al. | Biocontrol features of Clavispora lusitaniae against Penicillium digitatum on lemons | |
| Zhang et al. | Antimicrobial activity and action mechanism of triglycerol monolaurate on common foodborne pathogens | |
| Wang et al. | Cryoprotective effect of silver carp muscle hydrolysate on baker's yeast Saccharomyces cerevisiae and its underlying mechanism | |
| CN113317431A (zh) | 一种水包油型香芹酚纳米乳剂及其制备方法 | |
| Soltani et al. | Insights for the control of dried-fruit beetle Carpophilus hemipterus (Nitidulidae) using rosemary essential oil loaded in chitosan nanoparticles | |
| Haiyan et al. | Antibacterial effect of limonene on food-borne pathogens | |
| Ozdemir et al. | Fabrication and characterization of basil essential oil microcapsule-enriched mayonnaise and its antimicrobial properties against Escherichia coli and Salmonella Typhimurium | |
| Zamaniahari et al. | Preparation and evaluation of Mentha spicata L. essential oil nanoemulsion: physicochemical properties, antibacterial activity against foodborne pathogens and antioxidant properties | |
| Purkait et al. | Development of nanoemulsion formulation of mustard oil, its chemical characterization and evaluation against post harvest anthracnose pathogens | |
| Zhang et al. | γ-Cyclodextrin encapsulated thymol for citrus preservation and its possible mechanism against Penicillium digitatum | |
| Guo et al. | Polymethoxylated flavonoids (PMFs)-loaded citral nanoemulsion controls green mold in citrus by damaging the cell membrane of Penicillium digitatum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |