CN113208059A - 一种用于细胞培养肉的可食用的果胶壳聚糖胶原蛋白3d支架的制造方法 - Google Patents
一种用于细胞培养肉的可食用的果胶壳聚糖胶原蛋白3d支架的制造方法 Download PDFInfo
- Publication number
- CN113208059A CN113208059A CN202011466575.4A CN202011466575A CN113208059A CN 113208059 A CN113208059 A CN 113208059A CN 202011466575 A CN202011466575 A CN 202011466575A CN 113208059 A CN113208059 A CN 113208059A
- Authority
- CN
- China
- Prior art keywords
- pectin
- chitosan
- collagen
- scaffold
- cell culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000008186 Collagen Human genes 0.000 title claims abstract description 43
- 108010035532 Collagen Proteins 0.000 title claims abstract description 43
- 229920001436 collagen Polymers 0.000 title claims abstract description 43
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 42
- 229920001277 pectin Polymers 0.000 title claims abstract description 37
- 239000001814 pectin Substances 0.000 title claims abstract description 37
- 235000010987 pectin Nutrition 0.000 title claims abstract description 37
- 235000013372 meat Nutrition 0.000 title claims abstract description 29
- 238000004113 cell culture Methods 0.000 title claims abstract description 22
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 14
- PYMYPHUHKUWMLA-UHFFFAOYSA-N 2,3,4,5-tetrahydroxypentanal Chemical compound OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 20
- 235000013305 food Nutrition 0.000 claims abstract description 6
- 231100000331 toxic Toxicity 0.000 claims abstract description 6
- 230000002588 toxic effect Effects 0.000 claims abstract description 6
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 5
- 230000009881 electrostatic interaction Effects 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 230000021164 cell adhesion Effects 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 6
- 239000000017 hydrogel Substances 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 abstract description 4
- 238000004108 freeze drying Methods 0.000 abstract description 4
- 238000007710 freezing Methods 0.000 abstract description 4
- 230000008014 freezing Effects 0.000 abstract description 4
- 210000002363 skeletal muscle cell Anatomy 0.000 abstract description 4
- 231100000252 nontoxic Toxicity 0.000 abstract description 3
- 230000003000 nontoxic effect Effects 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 239000000499 gel Substances 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 210000004292 cytoskeleton Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 210000003098 myoblast Anatomy 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000446313 Lamella Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- 229920001586 anionic polysaccharide Polymers 0.000 description 1
- 150000004836 anionic polysaccharides Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- -1 cationic polysaccharide Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 235000019615 sensations Nutrition 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
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
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
-
- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
- A23L29/231—Pectin; Derivatives thereof
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/72—Chitin, chitosan
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Nutrition Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Rheumatology (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Dispersion Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明是一种用于细胞培养肉的可食用的果胶/壳聚糖/胶原蛋白3D支架的制造方法，以所述方法制造的果胶/壳聚糖/胶原蛋白3D凝胶支架可用于骨骼肌细胞的培养，为细胞培养肉的制造提供支撑，属于未来食品科技领域。本发明所述方法通过将一定浓度的果胶（高甲氧基果胶和低甲氧基果胶）、壳聚糖和胶原蛋白按照一定比例均匀混合后，于室温下充分反应2h以上，于‑80℃下冷冻过夜后，再经冷冻干燥得到果胶/壳聚糖/胶原蛋白3D支架。本发明的制造方法简单高效，在不使用有毒交联剂的情况下，通过果胶、壳聚糖、胶原蛋白之间的静电相互作用形成3D支架，得到的果胶/壳聚糖/胶原蛋白3D支架具有良好的细胞粘附性和相容性，可以进行骨骼肌细胞培养。本发明为细胞培养肉的研究提供了一种新型、无毒的3D支架的制造方法。
Description
技术领域
本发明涉及未来食品科技领域，具体涉及一种用于细胞培养肉的可食用的果胶/壳聚糖/胶原蛋白3D支架的制造方法。
背景技术
肉作为一种珍贵的动物蛋白质来源，占据了人们日常饮食的很大一部分比例。根据粮食及农业组织（FAO）最近调整的预测，到2050年，对肉的需求将增长70％，这对畜牧业系统将是巨大的挑战。近年来，人们逐渐认识到通过牲畜进行常规肉类生产可能需要替代品。这通常是基于对可持续性，环境负担和动物福利的关注。由于对牲畜业和屠宰行业的进一步紧缩，以及另一方面预计全球肉类消费量的快速增长，这些担忧日益增加。
肉类替代品要实现被大众接受和工业化的两个关键条件是拟态和效率。为了让一种新的肉类替代品被广泛采用，它需要精确地模仿甚至更好地再造传统肉类的所有物理感觉，如视觉外观、气味、质地，当然还有味道。
利用细胞培养工程和组织工程等技术，在体外培养动物肌肉组织作为食用材料，该技术被称为细胞培养肉技术。细胞培养肉技术可能是一种可持续且环保的肉类生产方式，因为一旦技术成熟，无需开发其他组织和功能结构(如骨骼和消化系统)，就可以高效地获得肉组织。总的来说，该技术是一种潜在的可持续和环保的肉类生产方式，可以缓解人口增长带来的压力，满足人们对肉类的需求。与此同时，细胞培养肉还可以控制肉制品的营养、质地和口味等，从而减少与营养相关的疾病。
三维（3D）支架对细胞培养肉的发展是必需的，是细胞附着的基本框架和代谢场所，其形态和功能直接影响细胞所构成的组织形态和功能。理想的三维支架应具有以下特点：在体内不引起炎症反应和毒性反应、可吸收性、可塑性、可成任意的三维结构、表面化学特性和表面微结构利于细胞的粘附与生长。
果胶是一类广泛存在于植物细胞壁的初生壁和细胞中间片层的杂多糖，是一种阴离子多糖，通常按其酯化度的高低分为高甲氧基果胶和低甲氧基果胶。果胶作为一种天然的植物胶体，可作为胶凝剂、稳定剂、组织形成剂、乳化剂和增稠剂广泛应用于食品工业中。壳聚糖是一种独特的碱性多糖，由甲壳素脱乙酰化而制备，是一种阳离子多糖，由于其良好的细胞相容性和生物降解性，它作为一种潜在的支架材料受到了广泛的关注。胶原蛋白是一种生物高分子，是动物结缔组织中的主要成分，也是哺乳动物体内含量最多、分布最广的功能性蛋白，因其具有良好的生物相容性、可生物降解性以及生物活性，在食品、医药、组织工程等领域获得广泛的应用。通常情况下，为了维持三维支架的稳定性，大多数利用果胶或壳聚糖制备的支架中需使用化学交联剂，例如戊二醛、氯化钙等，而这些化学交联剂大部分具有一定毒性，不适合用在可食用的3D支架中。
为了克服这些限制，并制造出适用于细胞培养肉的可食用的3D凝胶支架，我们发明了一种用于细胞培养肉的可食用的果胶/壳聚糖/胶原蛋白3D凝胶支架的制造方法：将壳聚糖和胶原蛋白溶液按照一定体积比均匀混合后，分别加入一定质量的果胶（低甲氧基果胶和高甲氧基果胶），在室温下搅拌反应2h以上，于-80℃下冷冻过夜后，再进行冷冻干燥得到3D支架。本发明的制造方法简单高效，未使用有毒交联剂，通过果胶、壳聚糖和胶原蛋白之间的静电相互作用形成水凝胶，得到的果胶/壳聚糖/胶原蛋白3D支架具有良好的细胞粘附性和相容性，可以进行骨骼肌细胞培养，用于生产细胞培养肉。
发明内容
本发明的目的在于针对国内现有细胞培养肉研发成本高、短时间内无法实现大规模生产等不足，提供一种用于细胞培养肉的可食用的果胶/壳聚糖/胶原蛋白3D凝胶支架的制造方法。
为了达到以上目的，本发明公开了一种可食用的果胶/壳聚糖/胶原蛋白3D凝胶支架的制造方法，利用果胶、壳聚糖和胶原蛋白之间的静电相互作用形成水凝胶，经冷冻干燥后得到3D支架。其中，所述胶原蛋白的浓度为0.5~5%，所述壳聚糖的浓度为0.5~3%，溶于0.5~3%乙酸溶液中，所述果胶的浓度为0.05~0.5%。
所述壳聚糖溶液和胶原蛋白溶液的体积比为1：1~5：1，所述果胶（低甲氧基果胶和高甲氧基果胶）加入壳聚糖与胶原蛋白混合溶液的质量为0.01~1g。其中，将混合溶液在室温下搅拌反应2h以上，于-80℃下冷冻过夜后，再经冷冻干燥得到3D凝胶支架。
本发明的优点：
1.本发明选用果胶、壳聚糖和胶原蛋白作为原料，三种原料均安全无毒，广泛应用于食品添加剂，已实现商业化。因此，选用这几种原料制造可食用的3D凝胶支架对细胞培养肉领域的应用具有重要价值。
2.本发明制造的3D支架分别采用了高甲氧基果胶和低甲氧基果胶与壳聚糖、胶原蛋白混合制备凝胶支架，以果胶酯化程度的高低形成对比，同时也研究了果胶酰化程度对凝胶支架性能的影响；
3.相较于其他交联方式，本发明利用果胶、壳聚糖和胶原蛋白之间的静电相互作用，在室温搅拌条件下即可制备，无任何副产物且无需加入带有毒性的交联剂，成胶条件简单绿色无毒。
附图说明
图1为果胶/壳聚糖/胶原蛋白3D支架的照片；
图2为果胶/壳聚糖/胶原蛋白3D支架的孔隙率，其中A、B、C、D分别代表不同浓度的高甲氧基果胶/低甲氧基果胶；
图3、图4、图5、图6为果胶/壳聚糖/胶原蛋白3D支架培养C2C12小鼠成肌细胞7d后细胞核和细胞骨架的染色图，分别代表不同浓度的高甲氧基果胶/低甲氧基果胶。
具体实施方式
以下结合附图和实施例，对本发明进行进一步的详细说明。
1.果胶/壳聚糖/胶原蛋白3D支架的制造：
（1）将壳聚糖、胶原蛋白分别溶于0.5~3%乙酸溶液中分别得到浓度为0.5~3%、0.5~3%的壳聚糖溶液和胶原蛋白溶液；
（2）将壳聚糖、胶原蛋白溶液以体积比1：1~5：1混合；
（3）将果胶（低甲氧基果胶和高甲氧基果胶）加入壳聚糖与胶原蛋白混合溶液的质量为0.01~1g；
（4）混合溶液于室温搅拌反应2h以上，于-80℃下冷冻过夜后，再经冷冻干燥得到3D支架。
2.在制造的果胶/壳聚糖/胶原蛋白3D凝胶支架上进行骨骼肌细胞培养：
（1）C2C12小鼠成肌细胞复苏离心并重悬于DMEM/F-12培养基中，该培养基含10％FBS和1％双抗溶液，于37°C、5% CO2培养箱中培养至细胞汇合达到约80%时，通过胰蛋白酶消化和离心获得细胞悬液。
（2）将果胶/壳聚糖/胶原蛋白3D凝胶支架用含1%双抗的PBS溶液洗涤三次后，加入培养基浸泡30min后弃去培养基，接种细胞后待细胞贴壁后补充培养基，在37°C、5% CO2培养箱中培养。
（3）培养7d后将支架进行细胞核和细胞骨架染色，并使用激光共聚焦显微镜进行观察，蓝色为细胞核，红色为细胞骨架。
Claims (5)
1.一种用于细胞培养肉的可食用的果胶/壳聚糖/胶原蛋白3D支架的制造方法，其特征在于，所述方法包括以下步骤：
将壳聚糖和胶原蛋白以一定浓度溶解于乙酸溶液中；
将壳聚糖溶液和胶原蛋白溶液以一定体积比均匀混合；
在强烈搅拌条件下将果胶（低甲氧基果胶和高甲氧基果胶）分别溶于壳聚糖和胶原蛋白的混合溶液中，通过静电相互作用形成水凝胶；
将混合后得到的溶液于室温下搅拌反应一定时间；
将反应后的混合溶液于-80℃下冷冻过夜后，再进行冷冻干燥。
2.根据权利要求1所述的方法，其特征在于，所述壳聚糖的浓度为0.5~3%，所述胶原蛋白的浓度为0.5~5%，溶于0.5~3%乙酸溶液中，所述果胶的浓度为0.05~0.5%。
3.根据权利要求1所述的方法，其特征在于，所述壳聚糖溶液和胶原蛋白溶液的体积比为1：1~5：1，所述果胶（低甲氧基果胶和高甲氧基果胶）加入壳聚糖与胶原蛋白混合溶液的质量为0.01~1g。
4.根据权利要求1所述的方法，其特征在于，所述混合溶液在室温下搅拌反应2h以上。
5.根据权利要求1所述的方法，其特征在于所述果胶、壳聚糖和胶原蛋白均为食品级物料，且已实现商业化生产；所述可食用3D支架的制造方法简单、绿色、高效，无需添加任何有毒交联剂，所形成的水凝胶具有良好的细胞粘附性和相容性，适用于细胞培养肉等组织工程领域。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011466575.4A CN113208059A (zh) | 2020-12-14 | 2020-12-14 | 一种用于细胞培养肉的可食用的果胶壳聚糖胶原蛋白3d支架的制造方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011466575.4A CN113208059A (zh) | 2020-12-14 | 2020-12-14 | 一种用于细胞培养肉的可食用的果胶壳聚糖胶原蛋白3d支架的制造方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113208059A true CN113208059A (zh) | 2021-08-06 |
Family
ID=77085842
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011466575.4A Pending CN113208059A (zh) | 2020-12-14 | 2020-12-14 | 一种用于细胞培养肉的可食用的果胶壳聚糖胶原蛋白3d支架的制造方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113208059A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215089A (zh) * | 2020-10-11 | 2021-08-06 | 西北农林科技大学 | 一种用于细胞培养肉的可食用的壳聚糖3d凝胶支架的制造方法 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200927074A (en) * | 2007-12-25 | 2009-07-01 | Univ Nat Taiwan | Colloidal frame used in tissue engineering |
| US20150079238A1 (en) * | 2013-09-13 | 2015-03-19 | Francoise Suzanne Marga | Edible and animal-product-free microcarriers for engineered meat |
| US20200140821A1 (en) * | 2017-06-07 | 2020-05-07 | Wild Type, Inc. | Ex vivo meat production |
| CN111165738A (zh) * | 2020-01-19 | 2020-05-19 | 太阳雨林（厦门）生物医药有限公司 | 一种可食用仿生动物组织 |
| WO2020123876A1 (en) * | 2018-12-12 | 2020-06-18 | Wild Type, Inc. | Synthetic food compositions |
| WO2020219755A1 (en) * | 2019-04-23 | 2020-10-29 | The Regents Of The University Of California | Methods and compositions for cell culture on heterogeneous scaffolds |
-
2020
- 2020-12-14 CN CN202011466575.4A patent/CN113208059A/zh active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200927074A (en) * | 2007-12-25 | 2009-07-01 | Univ Nat Taiwan | Colloidal frame used in tissue engineering |
| US20150079238A1 (en) * | 2013-09-13 | 2015-03-19 | Francoise Suzanne Marga | Edible and animal-product-free microcarriers for engineered meat |
| US20200140821A1 (en) * | 2017-06-07 | 2020-05-07 | Wild Type, Inc. | Ex vivo meat production |
| WO2020123876A1 (en) * | 2018-12-12 | 2020-06-18 | Wild Type, Inc. | Synthetic food compositions |
| WO2020219755A1 (en) * | 2019-04-23 | 2020-10-29 | The Regents Of The University Of California | Methods and compositions for cell culture on heterogeneous scaffolds |
| CN111165738A (zh) * | 2020-01-19 | 2020-05-19 | 太阳雨林（厦门）生物医药有限公司 | 一种可食用仿生动物组织 |
Non-Patent Citations (5)
| Title |
|---|
| A. SIONKOWSKA ET AL: "Modification of 3D materials based on chitosan and collagen blends by sodium alginate", 《MOLECULAR CRYSTALS AND LIQUID CRYSTALS》 * |
| 周光宏 等: "培养肉的研究进展与挑战", 《中国食品学报》 * |
| 尤凤健等: "胶原蛋白-壳聚糖支架的构建及其与骨髓基质干细胞复合的实验研究", 《现代生物医学进展》 * |
| 詹兴旺等: "胶原-壳聚糖支架与软骨细胞的生长", 《中国组织工程研究与临床康复》 * |
| 顾其胜 等主编: "《壳聚糖基海洋生物医用材料》", 31 January 2020, 上海科学技术出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215089A (zh) * | 2020-10-11 | 2021-08-06 | 西北农林科技大学 | 一种用于细胞培养肉的可食用的壳聚糖3d凝胶支架的制造方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113215090A (zh) | 一种用于细胞培养肉的可食用的壳聚糖/海藻酸钠/明胶3d支架的制造方法 | |
| CN101203615B (zh) | 含有细菌纤维素的制剂以及生产含有有效细菌纤维素的制剂的方法 | |
| Chiellini et al. | Ulvan: A versatile platform of biomaterials from renewable resources | |
| Mathur et al. | Chitin and chitosan, versatile polysaccharides from marine animals | |
| AU2020101687A4 (en) | Preparation Method for Dextran-Hyaluronic Acid Hydrogel for Three-Dimensional Cell Culture and Application Thereof | |
| CN106243410B (zh) | 一种羟乙基壳聚糖/透明质酸双网络水凝胶及其制备方法 | |
| Sun et al. | Covalently crosslinked hyaluronic acid‐chitosan hydrogel containing dexamethasone as an injectable scaffold for soft tissue engineering | |
| CN106084303B (zh) | 一种可生物降解高分子多孔复合材料及其制备方法 | |
| WO2018191244A1 (en) | Hydrogel for cell culture and biomedical applications | |
| CN113892643A (zh) | 一种用于细胞培养肉的可食用的采用茶多酚涂层的海藻酸钠/明胶3d凝胶支架的制造方法 | |
| CN101979636B (zh) | 一种球形细菌纤维素的制备方法 | |
| CN106117570A (zh) | 一种负载聚酰胺‑胺树状分子的海藻酸钠抗菌水凝胶及其制备方法和应用 | |
| BRPI0916850B1 (pt) | processo para a produção de complexo quitina-glucano que compreende quitina/quitosana e glucano; e polímeros contendo glicose, manose, e/ou galactose a partir de subproduto da indústria do biodiesel | |
| CN113208059A (zh) | 一种用于细胞培养肉的可食用的果胶壳聚糖胶原蛋白3d支架的制造方法 | |
| CN111172222B (zh) | 一种发酵生产透明质酸的方法及其应用 | |
| Smith et al. | Alginate hydrogels with tuneable properties | |
| KR102466561B1 (ko) | 바이오 셀룰로오스 분말을 포함하는 배양육 제조용 스캐폴드 및 이의 제조 방법 | |
| Yap et al. | Algae-based bioplastic for packaging: a decade of development and challenges (2010–2020) | |
| CN110180023A (zh) | 一种高强度生物质组织工程支架材料的制备方法 | |
| CN113215089A (zh) | 一种用于细胞培养肉的可食用的壳聚糖3d凝胶支架的制造方法 | |
| Moslemy et al. | Review in edible materials for sustainable cultured meat: Scaffolds and microcarriers production | |
| CN114438615A (zh) | 用于细胞培养肉的大豆蛋白纤维支架的规模化生产方法 | |
| Pandit et al. | Alginates production, characterization and modification | |
| Freile-Pelegrín et al. | Biodegradable polymer blends and composites from seaweeds | |
| CN100484496C (zh) | 含甲壳素的医用神经移植物的制备方法 |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210806 |