Effects of Heating Temperature, pH and Ionic Strength on the Stability of Flammulina velutipes Emulsions
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摘要: 为了探究金针菇乳状液抵抗环境变化(温度、pH和离子强度)的能力,以金针菇为原料,制备1.0%和2.0%水包油型乳状液,通过测定乳状液的外观及分层指数(Creaming index,CI)、粒径分布及平均粒径、Zeta电位及热稳定性等指标及观察微观结构分析加热温度(30~90 ℃)、pH(3~11)和离子强度(0~0.7 moL/L NaCl)对乳状液稳定性的影响。结果表明:1.0%金针菇乳状液的稳定性易受加热温度影响而出现分层现象,2.0%乳状液在温度4~90 ℃保持稳定且Zeta电位绝对值维持在29.10~29.40 mV;pH的变化显著(P<0.05)影响1.0%金针菇乳状液的稳定性,当pH为3和5时分层较为明显,2.0%金针菇乳状液表现出较高的pH耐受性,特别是当pH≥7时未分层,此时乳状液平均粒径显著变小(P<0.05),微观结构图显示液滴颗粒更加均匀细小;当离子强度增加时,1.0%金针菇乳状液的CI、平均粒径增大,Zeta电位绝对值降低,热稳定性较差,而2.0%金针菇乳状液未随离子强度增加而发生分层,平均粒径保持在385.20~414.60 nm,热稳定性较高。因此,较高浓度(2.0%)金针菇乳状液具有较好的乳化稳定性,能够耐受加热温度、pH和离子强度的变化,有望作为一种食用菌源天然乳化剂应用于食品工业。Abstract: The aim of this study was to evaluate the stability of the Flammulina velutipes emulsion when the environmental factors (such as temperature, pH and ionic strength ) changes. The 1.0% and 2.0% oil-in-water emulsions were prepared using Flammulina velutipes as raw material. The effects of heating temperature (30~90 ℃), pH (3~11) and the ionic strength (0~0.7 moL/L NaCl) on emulsions stability were analyzed by measuring the appearance, creaming index (CI), particle size distribution, average particle size, Zeta potential, the thermal stability, and observing the microstructure. The results showed that the 1.0% Flammulina velutipes emulsion delaminated with the increase of temperature from 4 ℃ to 90 ℃. The 2.0% Flammulina velutipes emulsion remained stable and the absolute value of Zeta potential was maintained at 29.10~29.40 mV at 4~90 ℃. The stability of 1.0% Flammulina velutipes emulsion was significantly (P<0.05) affected by pH, with obvious delamination at pH3 and 5. The 2.0% Flammulina velutipes emulsion had higher tolerance for pH, and showed no delamination, this was attributed that the average particle size of the 2.0% emulsion significantly (P<0.05) decreased, and the droplet particles were more uniform and fine. With the increasing of the ionic strength, the CI and average particle size of 1.0% Flammulina velutipes emulsion increased, the absolute value of Zeta potential decreased, and the thermal stability deteriorated. In contrast, the 2.0% Flammulina velutipes emulsion did not delaminated with the increase of the ionic strength, and the average particle size remained at 385.20~414.60 nm, and the thermal ability was stable. Hence, the higher concentration (2.0%) of Flammulina velutipes emulsion had good emulsion stability and could resistant changes of heating temperature, pH, and ionic strength. As a rusult, the 2.0% Flammulina velutipes emulsion is expected to be used as a natural emulsifier of edible fungi in the food industry.
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Keywords:
- Flammulina velutipes emulsion /
- stability /
- temperature /
- pH /
- ionic strength
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乳液是由两种互不相溶液体组成的均匀分散体,其中一相以小液滴的形式分散到另一相中,通常需要表面活性剂或其他物质在两相的界面之间充当乳化剂来降低界面张力,提高乳状液的稳定性[1−2],乳化剂可以根据来源不同分为天然乳化剂和合成乳化剂。上世纪下半叶,食品合成乳化剂被应用于食品工业[3]。但可能对消费者健康产生不利影响[4],如阴离子乳化剂可能与人体内的蛋白质、酶和磷脂结合,改变蛋白质的结构、造成酶功能障碍等[5],羧甲基纤维素钠和吐温80可能会改变肠道微生物群组成,最终诱发小鼠炎症性肠病、肥胖或代谢综合征[6]。随着消费者对健康需求的提高以及“清洁标签”的追求,研究人员开始将研究重点转移到天然乳化剂上。
食用菌是一类既能药用又能食用的大型真菌,其营养丰富,含有多糖、蛋白等主要营养物质[7]。新鲜的食用菌含水量大约70%~95%[8−11],说明食用菌中干物质具有很高的持水性,食用菌有望作为一种良好的天然乳化剂源。金针菇又称益智菇,因其富含碳水化合物、蛋白质和维生素而受到亚洲人、特别是中国和日本人喜爱[12]。有研究发现金针菇添加到肉制品中提高了肉制品的乳化特性,如申鑫玉等[13]利用金针菇菇脚代替部分脂肪加入猪肉糜中制成肉糜凝胶,结果发现可以明显改善猪肉糜凝胶品质且提高其营养价值;Choe等[14]研究发现将金针菇粉添加到猪肉糜体系可以提高肉糜的乳化特性。因此,金针菇可能成为天然乳化剂有价值的来源之一,但其乳状液的乳化能力及乳化稳定性鲜有报道。
在实际生活中,乳液容易受到外部环境的干扰和影响,包括温度、pH、离子强度等[15]。因此,研究外部环境对乳状液的影响具有重要意义。Jia等[16]研究了环境因素对柿子果胶乳液、商业甜菜和柑橘果胶的影响,结果表明柿子果胶乳液对盐离子敏感,但比商业甜菜和柑橘果胶更耐高温。Huang等[17]研究发现以辣木籽渣蛋白为稳定剂的Pickering乳液在NaCl(<0.2 moL/L)和弱酸(pH5~7)环境中具有良好的耐受性。
本试验分别制备1.0%和2.0%金针菇乳状液,通过观察测定乳状液的外观及分层指数(Creaming index,CI)、粒径分布及平均粒径、Zeta电位、微观结构和热稳定性,探究乳状液是否能在不同温度、pH以及离子强度下保持稳定,为开发天然食用菌源乳化剂新产品提供参考。
1. 材料与方法
1.1 材料与仪器
金针菇 上海九道菇生物科技有限公司;金龙鱼大豆油 益海嘉里金龙鱼食品集团股份有限公司;其他化学试剂均为分析纯,生工生物工程(上海)股份有限公司。
KSI马弗炉 中国上海电机(集团)公司;RE-52AA旋转蒸发仪 上海亚荣生化仪器厂;101-1BS电热恒温鼓风干燥箱 力辰科技公司;FW100小型高速粉碎机 天津市泰斯特科技有限公司;HH-S4型电热恒温水浴锅 北京科伟永兴仪器有限责任公司;FM-200型高剪切均质乳化仪 上海弗鲁克流体机械制造有限公司;SP707破壁机 浙江苏泊尔股份有限公司;JY-15A磁力搅拌器 永康市江业制造有限公司;BSA-124S-CW电子天平 上海精密科学仪器公司;PB-10 pH计 梅特勒-托利多仪器有限公司;Nano-ZS90电位仪 英国Malvern公司;Stemi-508光学显微镜 德国蔡司有限公司。
1.2 实验方法
1.2.1 金针菇营养成分测定
总糖含量的测定参考GB/T 15672-2009《食用菌中总糖含量的测定》;粗蛋白质含量的测定采用凯氏定氮(N×6.25)法,参考GB/T 5009.5-2016《食品安全国家标准 食品中蛋白质的测定》;粗脂肪含量的测定采用索氏抽提法,参考GB/T 5009.6-2016《食品安全国家标准 食品中脂肪的测定》;水分含量的测定采用直接干燥法,参考GB/T 5009.3-2016《食品中水分的测定》;灰分含量测定采用总重量差法,参考GB/T 5009.4-2016《食品中灰分的测定》;粗纤维含量的测定参考GB/T 5009.10-2003《植物类食品中粗纤维的测定》。
1.2.2 不同浓度金针菇乳状液的制备
参照Zhu等[18]方法,略有改动。首先将金针菇切成小段,按质量比1:1加入蒸馏水,用破壁机打浆2 min,得到金针菇匀浆物;然后在100 mL烧杯中加入一定质量的上述匀浆物、水和油(油水比3:7),用磁力搅拌器搅拌30 min,然后用高剪切均质乳化仪12000 r/min均质4 min,制得质量分数为1.0%和2.0%的金针菇乳状液。
1.2.3 金针菇乳状液稳定性实验
将制备好的1.0%和2.0%的金针菇乳状液,置于4 ℃下贮藏,并分别在30、50、70和90 ℃下加热30 min,冷却至室温;将制备好的1.0%和2.0%的金针菇乳状液的pH分别调至范围为3.0、5.0、7.0、9.0和11.0;在1.2.2金针菇乳状液制备过程中,加入不同体积5.0 mol/L NaCl溶液,使1.0%和2.0%金针菇乳状液的NaCl浓度分别为0、0.1、0.3、0.5和0.7 mol/L。测定外观及CI、粒径分布及平均粒径、Zeta电位、热稳定性等指标,并观察微观结构来考察温度、pH及离子强度对金针菇乳状液稳定性的影响。
1.2.4 指标测定
1.2.4.1 外观及分层指数CI
取上述乳状液10 mL倒入带盖玻璃瓶中,放置于4 ℃冰箱中,分别在0、1、2、3、4、5、6、7 d时观察乳状液外观并拍照,同时用游标卡尺测量乳状液总高度Hr和水相高度Hs。
CI的计算公式如下。
CI(%)=HsHr×100 式中:Hr为乳状液总高度(cm);Hs为水相高度(cm)。
1.2.4.2 粒径及Zeta电位的测定
将乳状液用去离子水稀释100倍,取稀释液1 mL置于比色皿中,用电位仪测定乳状液的粒径分布、平均粒径和Zeta电位。
1.2.4.3 显微结构观察
取乳状液10 mL于带盖玻璃瓶中,4 ℃下放置48 h,在玻璃瓶中间位置取20 μL乳状液滴于载片上,用光学显微镜观察、拍照。
1.2.4.4 热稳定性的测定
参照刘炯娜等[19]方法,略有改动。取10 mL新鲜金针菇乳状液于带盖玻璃瓶中,沸水浴加热30 min,观察加热前后乳液的形态变化,拍照记录并测定CI。
1.3 数据处理
所有样品均进行3次平行试验,结果用平均值±标准差表示;使用Excel进行实验数据统计分析,采用Statistix8.1对数据进行差异显著性统计分析,P<0.05代表具有显著性差异,采用Origin2018软件处理绘制图像。
2. 结果与分析
2.1 营养成分分析
表1为金针菇营养成分含量。由表1可知,金针菇含水率为90.18%,总糖、蛋白、粗纤维和脂肪含量分别为4.29%、1.79%、1.35%和0.19%,表现出高碳水、高蛋白、高纤维和低脂肪的特点。这与况丹[20]的研究结果相似。表1显示总糖和粗纤维为金针菇干物质的主要组成成分,分别占到干物质的43.69%和13.75%,总糖由水溶性多糖和水不溶性多糖组成,而粗纤维包括纤维素(由β-1,4葡萄糖聚合而成的同质多糖)、半纤维素(由葡萄糖、果糖、木糖等聚合而成的异质多糖)、木质素及角质等成分。有研究表明多糖是一种天然高分子物质,具有良好的乳化、增稠等多种功能特性[21],如杏鲍菇多糖[22]、双孢蘑菇多糖[23]、香菇多糖[24]、银耳多糖[25]和金针菇多糖[26]等都被证实了有一定的乳化活性。邓伟等[27]研究发现添加银耳多糖的Pickering乳液具有良好的稳定性。因此,金针菇可能作为天然乳化剂的来源。所以,本试验以金针菇为原料制备乳状液,研究不同环境因素对金针菇乳状液稳定性的影响。
表 1 金针菇营养成分含量Table 1. Nutrient content of Flammulina velutipes主要成分 总糖(%) 蛋白质(%) 粗脂肪(%) 粗纤维(%) 灰分(%) 水分含量(%) 含量 4.29±0.06 1.79±0.08 0.19±0.01 1.35±0.01 0.81±0.01 90.18±0.05 2.2 温度对金针菇乳状液稳定性的影响
2.2.1 外观及CI
在食品生产加工过程中通常利用热处理技术提高食品的物理、化学和感官特性,防止食品腐败变质[28]。本试验研究不同温度处理30 min对低浓度(1.0%)、高浓度(2.0%)金针菇乳状液稳定性的影响,外观结果如图1所示。1.0%金针菇乳状液的外观随温度升高发生了不同程度的分层;而2.0%金针菇乳状液,在实验温度范围内(4~90 ℃)外观无明显变化,表明2.0%金针菇乳状液具有更好的稳定性。这结果与Zhu等[29]的研究结果相类似,低浓度茄肉乳状液比高浓度茄肉乳状液更易受温度影响。
CI更直观地反映了乳状液的分层情况。1.0%金针菇乳状液在4 ℃储存时,CI为32.50%,经过30~90 ℃加热处理后CI升高到33.95%~34.63%,而2.0%金针菇乳状液在实验温度范围内未发生分层。该结果表明高浓度的乳状液比低浓度的乳状液更能抵抗温度变化引起的分层。这可能是当金针菇浓度达到2.0%时,金针菇中的有效成分在油滴表面形成了排列紧密的球膜层以及在连续相中形成强大的空间位阻,这种结构在不同温度下可以保持稳定,从而保护油滴的聚集,提高乳化稳定性[30]。
2.2.2 粒径分布及平均粒径
图2为温度对金针菇乳状液粒径分布及平均粒径的影响。由图可知,两种浓度的金针菇乳状液热处理后其粒径峰型并无显著变化(P>0.05),均呈单峰分布。随着温度的增加,两种金针菇乳状液粒径分布的主峰向大粒子尺寸方向移动,1.0%金针菇乳状液主峰位置集中在120~900 nm之间,而2.0%金针菇乳状液的主峰位置集中于100~700 nm之间,可见随着金针菇浓度增大,主峰位置左移。
通过平均粒径变化(图2C)分析可知:当温度升高到50 ℃及以上时,1.0%金针菇乳状液平均粒径均显著增大(P<0.05),这可能是金针菇蛋白在较高温度时发生了变性,从而导致乳状液液滴部分聚集。余静怡[31]的研究表明,由糖基化乳清分离蛋白和壳寡糖复合物制成乳状液的平均粒径与加热温度呈正相关。对于2.0%金针菇乳状液而言,热处理温度的升高(30~90 ℃)并不能使其平均粒径发生显著变化(P>0.05)。这说明随着金针菇浓度的增加,乳状液对温度的耐受性提高。此结果与CI测定结果相一致。
2.2.3 Zeta电位
Zeta电位是判断乳液稳定性的重要指标,一定程度上能够反映乳液液滴之间相互作用的强度,其绝对值越大,液滴间的静电斥力越大,乳液越稳定。图3为温度对金针菇乳状液Zeta电位的影响。由图可知,1.0%和2.0%金针菇乳状液在4 ℃下的Zeta电位绝对值分为25.8 mV和29.5 mV,经30~90 ℃热处理后虽略有下降,但仍维持在24.8~25.43 mV和28.17~28.70 mV,可见,热处理不会显著改变金针菇乳状液液滴间的静电斥力(P>0.05)。Wang[32]的研究结果也表明壳聚糖双层乳液在整个热处理过程中(30~90℃)没有发生显著变化。2.0%金针菇乳状液Zeta电位绝对值显著高于1.0%金针菇乳状液(P<0.05),这可能是因为金针菇含有羟基,浓度越大,羟基含量越多,羟基之间存在的静电斥力使金针菇乳状液更加稳定[33]。
2.3 pH对金针菇乳状液稳定性的影响
2.3.1 外观及CI
图4为pH对金针菇乳状液外观及CI的影响。如图4所示,静置48 h后,不同pH(3、5、7、9、11)的低浓度(1.0%)乳状液均出现分层现象,特别是当pH为3和5时分层较为严重,pH为3时乳状液不仅出现分层,而且质地变稀,这可能是因为金针菇中部分多糖在酸性条件下被降解,导致连续相中多糖形成的空间网状结构被破坏[34];2.0%金针菇乳状液只有在pH为3时分层明显,pH为5时略有分层,当pH大于7时,乳状液保持稳定,这表明2.0%金针菇乳状液在pH为7以上具有更好的乳化稳定性。这与韩宇[35]的研究结果一致。
CI结果与外观的结果相一致。pH小于7时,乳状液的CI随pH的增大显著减小(P<0.05);而pH大于7时CI无显著变化(P>0.05),这表明金针菇乳状液在酸性环境下不稳定,在中性及碱性环境下稳定。这与Chen等[36]研究结果相似,碱提取的茶多糖复合物制成的乳液,在碱性条件下更为稳定。
2.3.2 平均粒径和Zeta电位
图5为pH对金针菇乳状液平均粒径和Zeta电位的影响。由图可知,pH为7、9和11的乳状液的平均粒径显著小于pH为3和5的乳状液(P<0.05),且在碱性条件下,乳状液的平均粒径没有显著变化(P>0.05),这表明金针菇乳状液的平均粒径在碱性条件下更为稳定。杨贵妃等[37]研究发现辛烯基琥珀酸酯淀粉乳液在碱性条件下可以保持稳定。
pH对金针菇乳状液Zeta电位的影响如图5B所示,金针菇乳状液的Zeta电位绝对值随pH的升高呈现先增大后平稳的趋势,当pH在3~9范围内时,2个试验组Zeta电位绝对值都随着pH的升高显著增加(P<0.05),但当pH≥9时,2个试验组Zeta电位绝对值变化平缓。当pH为11时,1.0%和2.0%金针菇乳状液Zeta电位绝对值都达到最高24.67 mV和29.77 mV。Zeta电位绝对值的增大说明液滴间的静电斥力增强,可以提高乳状液的稳定性[38]。2.0%金针菇乳状液的Zeta电位绝对值大于1.0%金针菇乳状液,结果表明2.0%金针菇乳状液更加稳定。
2.3.3 微观结构
图6为pH对金针菇乳状液显微结构的影响。由图可知,金针菇乳状液的乳滴均为球状,无论是1.0%金针菇乳状液还是2.0%金针菇乳状液均随着pH的增加,液滴颗粒数量增大、粒径变小。该结果与平均粒径的变化趋势相同。这可能是当pH较高时,液滴的净电荷量相对较多,液滴间的静电斥力较大,阻止了液滴之间的融合[39]。翟希川[39]的研究表明当pH为11时,细菌纳米纤维素Pickering乳液液滴分布均匀,呈现小液滴状。
2.3.4 热稳定性
图7为pH对不同浓度的金针菇乳状液热稳定性的影响。由图可知,经过热处理30 min后的乳状液均发生分层,但1.0%金针菇乳状液分层现象更为明显。CI随着pH的增加,呈先下降后上升的趋势,当pH为7时,金针菇乳状液的CI最低,表明此时乳液的热稳定性最高。不同浓度进行比较,2.0%金针菇乳状液的CI显著(P<0.05)低于1.0%金针菇乳状液,表明2.0%金针菇乳状液在不同pH条件下的热稳定性更强。
2.4 离子强度对金针菇乳状液稳定性的影响
2.4.1 外观及CI
食品加工过程中通常添加NaCl来改善食品的风味、口感和货架期,本试验设置了不同NaCl浓度来考察离子强度对金针菇乳状液外观及分层指数的影响。图8为离子强度对金针菇乳状液外观及CI的影响。由图可知,1.0%金针菇乳状液在不同的NaCl浓度下均发生分层,但NaCl浓度的变化(0.1~0.7 moL/L)对乳状液的外观影响不明显;对于2.0%金针菇乳状液,无论NaCl浓度如何变化均没有发生分层现象。
考察指标CI可知,对于1.0%金针菇乳状液,当NaCl浓度从0增加到0.1 moL/L时,CI显著增大到27.5%(P<0.05),当NaCl浓度从0.1 moL/L增大至0.7 moL/L时,CI维持在27.5%~31.50%,无显著变化(P>0.05),而2.0%金针菇乳状液的CI始终为0,说明NaCl浓度的变化对2.0%金针菇乳状液的CI没有影响。这与Yang等[40]研究结果相一致,在不同离子强度下制备的大豆分离蛋白-壳聚糖Pickering乳液的外观和CI没有变化。
2.4.2 平均粒径和Zeta电位
图9A表征了离子强度的变化对金针菇乳状液的平均粒径的影响。由图可知,随着NaCl浓度的增加,两种金针菇乳状液平均粒径均增大。结果表明,不添加NaCl的乳状液平均粒径最小,稳定性最好,添加NaCl会降低乳液的稳定性,2.0%金针菇乳状液比1.0%金针菇乳状液更耐盐。这可能是盐析作用导致大分子聚集,乳状液粒径增大[41]。
图9B为离子强度对金针菇乳状液Zeta电位的影响。由图可知,随着NaCl浓度的增加,金针菇乳状液的Zeta电位绝对值逐渐减小。何康慧[42]的研究也表明离子强度的增强会导致竹笋水不溶性膳食纤维乳液Zeta电位绝对值的减小。随着NaCl浓度的升高,两种金针菇乳状液Zeta电位绝对值降低(P<0.05),2.0%金针菇乳状液Zeta电位绝对值显著高于1.0%金针菇乳状液(P<0.05),并且变化趋势也相对平坦。这可能是因为2.0%金针菇乳状液液滴表面电荷相对充足,离子强度的变化不足以破坏乳状液的稳定,静电斥力足以有效地阻碍液滴聚结[43]。
2.4.3 微观结构
图10为离子强度对金针菇乳状液显微结构的影响。对于浓度为1.0%金针菇乳状液,随着NaCl浓度增加,液滴数量变少、粒径变大。杨凯麟[44]的研究也表明微晶纤维素-猪油Pickering乳液乳滴的大小会随着NaCl浓度的增加而变大。对于浓度为2.0%金针菇乳状液,随着NaCl浓度增加,液滴粒径无显著变化。
2.4.4 热稳定性
离子强度对金针菇乳状液热稳定性的影响如图11所示。由图11可知,离子强度对1.0%金针菇乳状液的热稳定性影响显著(P<0.05),乳状液加热后均会发生分层与絮凝,并且随NaCl浓度增加,乳状液絮凝程度增强,这可能是因为NaCl浓度的增加,导致金针菇蛋白质间静电斥力减弱,从而无法克服热变性[45]。CI指标的测定清晰地表明了金针菇乳状液在不同浓度NaCl条件下的热稳定性。1.0%的金针菇乳状液的CI(44.47%~47.12%)显著高于2.0%的金针菇乳状液的CI(0~9.09%),表明高浓度乳状液具有更高的热稳定性(P<0.05)。
3. 结论
本文探究了环境因素(温度、pH、离子强度)对金针菇乳状液的影响。结果表明,1.0%金针菇乳状液易受温度、pH、离子强度的影响,当温度升高、pH降低以及离子强度增强时,乳状液CI和平均粒径均增加,Zeta电位绝对值减小,乳状液容易发生分层、凝聚等不稳定现象;2.0%金针菇乳状液在温度为4~90℃、pH为7~11、离子强度为0.1~0.7 moL/L NaCl时未出现分层现象,平均粒径和Zeta电位无显著变化(P>0.05),热稳定性较高。总而言之,当金针菇浓度达到2.0%时,乳状液不易受环境因素影响,具有良好的温度、pH和离子强度稳定性。然而,金针菇中起乳化作用的有效成分尚不明确,需进一步探讨金针菇乳状液的乳化成分和乳化机理,为拓展金针菇乳状液在食品加工领域的应用提供参考。
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表 1 金针菇营养成分含量
Table 1 Nutrient content of Flammulina velutipes
主要成分 总糖(%) 蛋白质(%) 粗脂肪(%) 粗纤维(%) 灰分(%) 水分含量(%) 含量 4.29±0.06 1.79±0.08 0.19±0.01 1.35±0.01 0.81±0.01 90.18±0.05 -
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