CN111264634B - 一种植物油体乳液及应用 - Google Patents
一种植物油体乳液及应用 Download PDFInfo
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- vegetable oil
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- oleosin
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Images
Classifications
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- A—HUMAN NECESSITIES
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- A23D7/02—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by the production or working-up
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- A—HUMAN NECESSITIES
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
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Abstract
本发明为一种植物油体乳液及应用,公开了黄原胶作为植物油体的稳定剂的应用;所述的植物油体为过表达oleosin的植物油体;一种植物油体乳液,是将黄原胶加入去离子水,搅拌均匀,在磁力搅拌器作用下,缓慢加入植物油体悬浮液;一种含有植物油体的食品,它添加了所述的一种植物油体乳液;按重量添加下列物质:所述的一种植物油体乳液、蔗糖酯、蔗糖、单甘脂、棕榈油、香精,余量为磷酸盐缓冲液,总重量为100%;或添加下列物质:所述的一种植物油体乳液、蛋黄、食醋、白砂糖、酱油、食用盐,余量为磷酸盐缓冲液,总重量为100%。本发明乳液平均粒径100‑300nm,稳定性好、安全性高。
Description
技术领域
本发明属于生物技术和食品技术领域,具体涉及一种植物油体乳液及应用。
背景技术
植物油料种子的油体主要含有蛋白质、多种游离脂肪酸和碳水化合物,它的开发利用程度影响着食品领域、饲料工业、美容护肤行业以及医药领域。因此提高植物油体的利用水平,进一步拓宽植物油体的应用范围。
植物油体含有高度不饱和脂肪酸, 亚油酸含量高达 80 %以上, 能够降低血脂与胆固醇而发挥医疗保健作用。油体是植物细胞中最小的细胞器之一,直径为0.5-2.5µm,大小因植物种类及品种而异,并且受营养和环境的影响。在干燥种子细胞内油体很稳定,油体之间并不会发生融合或聚合,经过长时间的储存能保持稳定。
油体在食品应用中的研究还处在起始阶段,目前的研究主要集中在提高油体在水溶液中的稳定性,以利于其在食品工业中的应用。从植物种子中分离出来的油体能为合适的食品系统提供稳定的预乳化的油体。水包油乳液的稳定性取决于液滴之间的相互作用,也就是取决于其界面组成。油体从结构上可看成一种天然的乳化粒子,其表面是一层蛋白质-磷脂膜,可将外界与核心的中性脂质隔开,膜上的蛋白称为油体蛋白。而在加热条件下,油体吸附蛋白会发生解离,并且不同加热强度会导致不同程度的解离。
黄原胶俗称玉米糖胶、汉生胶,是一种糖类(葡萄糖、蔗糖、乳糖)经由野油菜黄单孢菌发酵产生的复合多糖体。它通常是经由玉米淀粉所制造,由甘蓝黑腐病野油菜黄单胞菌以碳水化合物为主要原料,经好氧发酵生物工程技术,切断1,6-糖苷键,打开支链后,在按1,4-键合成直链组成的一种酸性胞外杂多糖。黄原胶是白色或浅黄色的粉末,并具有良好的热、酸碱稳定性,无毒,并且有益生作用。所以被广泛应用于各种食品中。众所周知,黄原胶可添加在大范围中食品产品,如压缩肉类,糕点,奶制品等等。黄原胶稳定的双螺旋结构使其具有极强的抗氧化和抗酶解能力,许多的酶类如蛋白酶、淀粉酶、纤维素酶和半纤维素酶等酶都不能使黄原胶降解。美国对黄原胶首先进行了毒理学及安全性试验。1969年黄原胶被美国食品与药物管理局(FDA)批准为食品添加剂。FDA(1994)将黄原胶列为GRAS物质,GRAS是“一般认为安全”的意思,也就是在通常的使用量下,没有安全性的担心。其后,丹麦、英国、爱尔兰、荷兰、西班牙和加拿大等国对黄原胶用于食品予以法律认可。联合国粮食与农业组织和世界卫生组织(FAO/WHO)(1994)规定,黄原胶的每日允许摄入量(ADI)不作特殊规定。1985年南开大学率先研究食品级黄原胶,对黄原胶产品进行了毒理学试验,对生产黄原胶的菌株进行毒性试验,结果表明黄原胶和菌株均没有毒性 。1988年国家卫生部批准食品级黄原胶的卫生标准,并被列入食品添加剂名单。酪蛋白酸钠亦称酪蛋白钠、酪酸钠或干酪素,是牛乳中主要蛋白质酪蛋白的钠盐,是一种安全无害的增稠剂和乳化剂,因为酪蛋白酸钠含有人体所需的各种氨基酸,营养价值很高,也可作为营养强化剂食用。酪蛋白酸钠具有很好的起泡性,这可广泛应用于冰淇淋等冷食之中,用以改善其质地和口感。另一个特点是具有很好的热稳定性,大多蛋白质分子如鸡蛋、大豆蛋白均由一疏水基团和一个亲水基团相互连接,易受热变性,而酪蛋白酸钠乳状液能在120℃高温杀菌也不会破坏其稳定性及功能性。
一种自然多糖和重要的生物高聚物等物质可以溶于冷水和热水中,具有高黏度,高耐酸、碱、盐特性、高耐热稳定性、悬浮性和触变性等,具有广阔的市场前景。多糖类物质因为具有显著性的增加体系黏度和形成弱凝胶结构的特点而经常被用于食品或其他产品,以提高O/W乳状液的稳定。近年公众的视线更倾向于无毒无害无残留、绿色环保型保鲜剂的应用。油体乳液用于饮料可有效地延长果肉饮料的悬浮时间,提高水果和巧克力饮料的口味。用于冷冻食品可通过结合自由水使其稳定,并可控制冰晶的生长速度提供理想的质构。在罐头食品中加入多糖类物质可使物料便于泵送与灌装,且易于保持产品的外观。
发明内容
本发明目的是提供一种植物油体乳液及应用。
黄原胶作为植物油体的稳定剂的应用;
所述黄原胶的浓度为0.05-0.5%;
所述的植物油体为过表达oleosin的植物油体。
一种植物油体乳液,是将黄原胶加入去离子水,搅拌均匀无气泡,使其充分溶胀,在磁力搅拌器作用下,缓慢加入植物油体悬浮液;
所述的植物油体为过表达oleosin的植物油体;
所述的油体终浓度为10%。
一种含有植物油体的食品,它添加了所述的一种植物油体乳液;
所述的一种含有植物油体的食品,按重量添加下列物质:20-60%所述的一种植物油体乳液、0.05-0.1%蔗糖酯、1-2%蔗糖,0.1-0.2%单甘脂、0.1-0.5%棕榈油、0.05-0.1%香精,余量为磷酸盐缓冲液,总重量为100%;
所述的一种含有植物油体的食品,按重量添加下列物质:20-60%所述的一种植物油体乳液、2-8%蛋黄、0.1-0.5%食醋、1-2%白砂糖、0.1-0.5%酱油、0.1-0.5%食用盐,余量为磷酸盐缓冲液,总重量为100%。
本发明提供了黄原胶作为植物油体的稳定剂的应用;所述的植物油体为过表达oleosin的植物油体;一种植物油体乳液,是将黄原胶加入去离子水,搅拌均匀,在磁力搅拌器作用下,缓慢加入植物油体悬浮液;一种含有植物油体的食品,它添加了所述的一种植物油体乳液;所述的一种含有植物油体的食品,按重量添加下列物质:20-60%所述的一种植物油体乳液、0.05-0.1%蔗糖酯、1-2%蔗糖,0.1-0.2%单甘脂、0.1-0.5%棕榈油、0.05-0.1%香精,余量为磷酸盐缓冲液,总重量为100%;或按重量添加下列物质:20-60%所述的一种植物油体乳液、2-8%蛋黄、0.1-0.5%食醋、1-2%白砂糖、0.1-0.5%酱油、0.1-0.5%食用盐,余量为磷酸盐缓冲液,总重量为100%。本发明乳液平均粒径100-300nm,稳定性好、安全性高。
附图说明;
图1 植物油体的表观;
图2 重组植物油体和野生油体显微结构对比图;A、野生油体;B、重组植物油体;C、野生油体乳液;D、野生油体乳液;
图3 重组植物油体悬浮液外观图;
图4 重组植物油体悬浮液粒径检测图;
图5 重组植物油体乳液外观图;
图6 重组植物油体乳液粒径检测图。
具体实施方式
实施例1 重组植物油体的制备
一、过表达oleosin植物的获得
构建pOBT表达载体,载体骨架pCAMBIA1301的基础上改造T-DNA区,该质粒上还有NcoI和HindIII酶切位点;将红花oleosin基因(碱基序列见序列表SEQ ID NO.1)插入pOBT表达载体,得到重组pOTB-Oleosin载体,转染农杆菌感受态细胞,以菌液为模板,进行PCR鉴定;将转染后的农杆菌,转化红花,采用农杆菌介导法:在萌发的种子还未长出真叶时,横切子叶下方生长点处,外植体的表面伤口处接触制备好的农杆菌菌液中进行侵染10min,25℃继续暗箱中培养4天。经过4天暗培养的子叶节转移到芽增殖培养基上,待子叶节伤口处长出1~3个绿色的芽点后,转移至伸长培养基,培养约4周,进行炼苗、移栽;收种后,获得T1代过表达oleosin红花植株。
二、过表达oleosin植物种子基因水平和蛋白水平检测
提取过表达oleosin的红花基因组DNA,分别以特异引物:
F:3’-5’: ATGGGTACGGTTGAAACGACA
R:3’-5’: AGCTCCGACAACCGACCAA
进行PCR检测,扩增出与oleosin蛋白大小一致的目标条带,初步确定已经插入植物基因组中;提取重组油体进行SDS-PAGE检测,最终获得T3代过表达oleosin的红花种子。
三、重组植物油体的制备
取20g T3代过表达oleosin的红花种子,清洗2~3次,吸水晾干;使用超高速粉碎机进行破碎10s;加入200 mL PBS缓冲溶液,榨汁机进行研磨,纱布粗过滤,滤液放置在50 mL离心管中,利用超速离心机4℃ 12000 r/min,15 min;上层固体物质用PBS缓冲溶液重悬,玻璃棒搅拌至无颗粒,超速离心机4℃12000 r/min,15min重复一次;上层固体物质用PBS缓冲溶液重悬,玻璃棒搅拌至无颗粒,超速离心机4℃ 12000 r/min,15 min重复2次;得到的上层白色固体即为重组植物油体(过表达oleosin植物油体),4℃储存备用。
实施例2 重组植物油体的制备
一、过表达oleosin植物的获得
构建pOBT表达载体,载体骨架pCAMBIA1301的基础上改造T-DNA区,该质粒上还有NcoI和HindIII酶切位点;将拟南芥oleosin基因(碱基序列见序列表SEQ ID NO.2)插入pOBT表达载体,得到重组pOTB-Oleosin载体,转染农杆菌感受态细胞,以菌液为模板,进行PCR鉴定;采用Flora Dip法,将转染后的农杆菌,转化拟南芥外植体:拟南芥在露白花和夹的状态时,倒扣于制备好的农杆菌菌液中侵染8-15min,暗培养2-3天,收种后,进行抗性筛选,获得T1代过表达oleosin拟南芥植株。
二、过表达oleosin植物种子基因水平和蛋白水平检测
提取过表达oleosin的拟南芥基因组DNA,分别以特异引物:
F:3’-5’:ATGGCGGATACAGC TAGAGGAA
R:3’-5’:CACCGGGTGGAGTAGTGTGCTGG;
进行PCR检测,扩增出与oleosin蛋白大小一致的目标条带,初步确定已经插入拟南芥植物基因组中;提取重组油体进行SDS-PAGE检测,最终获得T3代过表达oleosin的拟南芥种子。
三、重组植物油体的制备
取20g T3代过表达oleosin的拟南芥种子,清洗2~3次,吸水晾干;加入200 mL PBS缓冲溶液,使用研杵进行研磨破碎;纱布粗过滤,滤液放置在50 mL离心管中,利用超速离心机4℃ 12000 r/min,15 min;上层固体物质用PBS缓冲溶液重悬,玻璃棒搅拌至无颗粒,超速离心机4℃12000 r/min,15min重复一次;上层固体物质用PBS缓冲溶液重悬,玻璃棒搅拌至无颗粒,超速离心机4℃ 12000 r/min,15 min重复2次;得到的上层白色固体即为重组植物油体(过表达oleosin植物油体),4℃储存备用。
实施例3 重组油体悬浮液的制备
利用1 moL的HCl或NaOH将PBS缓冲液的pH调节为6-9,离子浓度为10mM,将重组植物油体加入PBS缓冲液中,磁力搅拌器充分搅拌均匀,无颗粒,制备重组油体悬浮液,重组油体悬浮液浓度为10%,外观如图3所示;重组油体悬浮液粒径大小在400-900nm,野生型油体悬浮液粒径大小在700-1500nm(表1,图4)。
实施例4 重组油体乳液的制备
从卡波姆、羧甲基纤维素钠、Tween-80、Tween-20,黄原胶,葡聚糖等多种稳定剂中筛选合适的稳定剂;称取适量的稳定剂加入去离子水,搅拌均匀无气泡,使其充分溶胀。在磁力搅拌器下缓慢加入植物油体悬浮液,最终制备成重组植物油体乳液,稳定剂浓度范围在0.3%,并且油体终浓度为10%;利用显微结构,粒径及电位指标的测定,最终定为黄原胶为最佳稳定剂(表2),重组植物油体乳液外观见图5。
实施例5 重组油体乳液稳定性的检测
1、将制备好的植物油体乳液(黄原胶为稳定剂)分别放置恒温水浴锅中,进行30℃、60℃、90℃的恒温处理30min,和50w-250w的高超声处理30min、60min、90min;利用显微结构,粒径及电位指标的测定,发现植物油体乳液对温度及超声有良好的稳定性。
2、耐碱实验:向植物油体乳液(黄原胶为稳定剂)加入5%氢氧化钠,仍呈乳白色油体乳液,感观无变化。
实施例6 重组植物油体饮料
按重量添加下列物质:10%重组植物油体悬浮液,添加黄原胶0.3%、蔗糖酯0.05-0.1%,蔗糖1-2%,单甘脂0.1-0.2%,棕榈油0.1-0.5%,香精0.05-0.1%,余量为磷酸盐缓冲液,总重量为100%;
具体操作为:
1)黄原胶先溶于pH7.0的0.05 mol/L 磷酸盐缓冲液中,加热至60°C,磁力搅拌2h吸水溶胀后与重组植物油体悬浮液混合,再搅拌2h,形成重组植物油体乳液;
2)蔗糖酯溶于pH7.0的磷酸盐缓冲液中,棕榈油加热至40±5°C,液化10-20min;
3)再加入蔗糖、单甘脂,混合,磁力搅拌2h后制成粗乳状液,用高速分散机在12000 r/min 下乳化1-5 min;4)用高速分散机在12000-13000r/min下均质1-3min,再用高压均质机在30MPa均质两次,加入0.05-0.1%的香精混合均匀,得到重组植物油体饮料。
实施例7 重组植物油体酱料的制备
按重量添加下列物质:20-60%重组植物油体悬浮液、0.05-0.5%黄原胶、2-8%蛋黄、0.1-0.5%食醋、1-2%白砂糖、酱油0.1-0.5%、食用盐0.1-0.5%,余量为磷酸盐缓冲液,总重量为100%;
具体操作为:
1)黄原胶先溶于pH7.0的0.05 mol/L磷酸盐缓冲液中,加热至60°C,磁力搅拌2h吸水溶胀后与重组植物油体悬浮液混合,再搅拌2h,形成重组植物油体乳液;
2)蛋黄加入少量水60±5℃水浴打散搅拌均匀,冷却;
3)冷却后,按比例加入重组植物油体乳液、白砂糖,食醋、酱油、食用盐、打搅均匀,得到重组植物油体酱料。
序列表
<110> 吉林农业大学
<120> 一种植物油体乳液及应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 447
<212> DNA
<213> 红花(Carthamus tinctorius L.)
<400> 1
atgggtacgg ttgaaacgac attcgacacg atggatcgac gacgagtgga tcaaatcact 60
ggcgagacga tgctactcgc gaccttagtc gccatagctg ttgccggacc actgatggtc 120
ctgatggtta tcagtttttg taccacattg acactgtttc tagtcagtgc tccacttttc 180
ataatcttcg gccctttgct gttgggtgcc ggttttgtgc tcacgatggt gttggtgggg 240
ttcggtatgg ccgccatgat ggcgatggcg gggcttgcta cgttgggatg ggttttcaga 300
tcgcttagag gcgacactgg gaaggtgatc agagacatgg tcgtggagtt gggggacagg 360
gttaaagatg ccaaaagcaa ttgggctatt catttgaatc agaaggtgca taatatgccg 420
gaaaattggt cggttgtcgg agcttag 447
<210> 2
<211> 731
<212> DNA
<213> 哥伦比亚型拟南芥(Arabidopsis thanliana)
<400> 2
atggcggata cagctagagg aacccatcac gatatcatcg gcagagacca gtacccgatg 60
atgggccgag accgagacca gtaccagatg tccggacgag gatctgacta ctccaagtct 120
aggcagattg ctaaagctgc aactgctgtc acagctggtg gttccctcct tgttctctcc 180
agccttaccc ttgttggaac tgtcatagct ttgactgttg caacacctct gctcgttatc 240
ttcagcccaa tccttgtccc ggctctcatc acagttgcac tcctcatcac cggttttctt 300
tcctctggag ggtttggcat tgccgctata accgttttct cttggattta caagtaagca 360
cacatttatc atcttacttc ataattttgt gcaatatgtg catgcatgtg ttgagccagt 420
agctttggat caattttttt ggtcgaataa caaatgtaac aataagaaat tgcaaattct 480
agggaacatt tggttaacta aatacgaaat ttgacctagc tagcttgaat gtgtctgtgt 540
atatcatcta tataggtaaa atgcttggta tgatacctat tgattgtgaa taggtacgca 600
acgggagagc acccacaggg atcagacaag ttggacagtg caaggatgaa gttgggaagc 660
aaagctcagg atctgaaaga cagagctcag tactacggac agcaacatac tggtggggaa 720
catgaccgtg a 731
Claims (10)
1.黄原胶在缩小过表达oleosin植物油体粒径及耐碱性方面的应用。
2.权利要求1所述的应用,其特征在于:所述的oleosin植物油体为油体悬浮液,黄原胶的浓度为0.05-0.5%。
3.一种植物油体乳液,它包括:植物油体悬浮液和黄原胶;黄原胶的浓度为0.3-0.5%;
所述的植物油体为过表达oleosin的植物油体。
4.根据权利要求3所述的一种植物油体乳液,其特征在于:所述的植物为红花或拟南芥;所述的oleosin,其碱基序列如SEQ ID NO.1或2所示。
5.根据权利要求3或4所述的一种植物油体乳液,其特征在于:是将黄原胶加入去离子水,搅拌均匀无气泡,使其充分溶胀,在磁力搅拌器作用下,缓慢加入植物油体悬浮液制备的。
6.根据权利要求5所述的一种植物油体乳液,其特征在于:所述的植物油体100-300nm。
7.根据权利要求6所述的一种植物油体乳液,其特征在于:所述的油体终浓度为10%,黄原胶的浓度为0.3%。
8.一种含有植物油体的食品,其特征在于:它添加了权利要求3所述的一种植物油体乳液。
9.根据权利要求8所述的一种含有植物油体的食品,其特征在于,按重量添加下列物质:20-60%权利要求3所述的一种植物油体乳液、0.05-0.1%蔗糖酯、1-2%蔗糖,0.1-0.2%单甘脂、0.1-0.5%棕榈油、0.05-0.1%香精,余量为磷酸盐缓冲液,总重量为100%。
10.根据权利要求9所述的一种含有植物油体的食品,其特征在于,按重量添加下列物质:20-60%权利要求3所述的一种植物油体乳液、2-8%蛋黄、0.1-0.5%食醋、1-2%白砂糖、酱油0.1-0.5%、食用盐0.1-0.5%,余量为磷酸盐缓冲液,总重量为100%。
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