CN113519686B - 一种婴儿乳蛋白部分水解配方食品及其制备方法 - Google Patents
一种婴儿乳蛋白部分水解配方食品及其制备方法 Download PDFInfo
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- CN113519686B CN113519686B CN202110830336.0A CN202110830336A CN113519686B CN 113519686 B CN113519686 B CN 113519686B CN 202110830336 A CN202110830336 A CN 202110830336A CN 113519686 B CN113519686 B CN 113519686B
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
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- A23C9/1522—Inorganic additives, e.g. minerals, trace elements; Chlorination or fluoridation of milk; Organic salts or complexes of metals other than natrium or kalium; Calcium enrichment of milk
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- A—HUMAN NECESSITIES
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
- A23C9/1528—Fatty acids; Mono- or diglycerides; Petroleum jelly; Paraffine; Phospholipids; Derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
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- A23C9/156—Flavoured milk preparations ; Addition of fruits, vegetables, sugars, sugar alcohols or sweeteners
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C9/00—Milk preparations; Milk powder or milk powder preparations
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Abstract
本申请提供了一种婴儿乳蛋白部分水解配方食品，其包括在酶解体系中乳化剂存在的条件下以碱性蛋白酶或复合风味蛋白酶酶解乳清蛋白获得的水解乳清蛋白。本申请的所用的水解乳清蛋白在较低水解程度下减少了过敏表位对应肽段的残留，实现了口感与低致敏性的良好平衡。
Description
技术领域
本申请属于食品，特别是婴幼儿配方食品领域，具体地，本申请提供了一种婴儿乳蛋白部分水解配方食品，其特征在于，其包括在酶解体系中乳化剂存在的条件下以碱性蛋白酶或复合风味蛋白酶酶解乳清蛋白获得的水解乳清蛋白。
背景技术
牛乳是婴幼儿替代母乳的主要食物蛋白来源，但其也是最常见的食物过敏原之一(学前儿童的牛乳过敏率可达10％左右，到成人阶段仍有3％左右)，制备防止过敏的牛乳制品或代用配方食品，对于牛乳过敏人群，特别是婴幼儿牛乳过敏人员的健康非常有意义。β-乳球蛋白占乳清蛋白总量50％左右，是乳清蛋白的主要功能活性成分。对牛乳过敏人群中80％以上属于对β-乳球蛋白过敏，消除β-乳球蛋白或其过敏原表位可以有效减少牛乳过敏发生的概率。
目前消除β-乳球蛋白或其过敏原表位的方法主要包括热加工、高压处理、发酵、辐射处理、酶解处理等。由于热加工、高压处理、发酵、辐射处理在实际效果和安全性上都存在难以克服的问题，酶解处理依然是生产低致敏乳制品/代用配方食品的主流方法。
一方面，由于β-乳球蛋白具有紧密的空间折叠结构，导致β-乳球蛋白具有耐蛋白酶水解的特性；另一方面，提高水解程度(如深度水解奶粉/乳清蛋白或酪蛋白)虽然可以降低致敏性，但也一般伴随着成本的提高和风味的下降；因此控制水解过程/程度是制备低致敏乳制品/代用配方食品中的关键步骤。本领域中已经尝试了各种蛋白酶及其组合和适合的条件，一般认为碱性蛋白酶、胰蛋白酶、胃蛋白酶、风味蛋白酶和木瓜蛋白酶等蛋白酶均有用于水解乳清蛋白的报道，单纯从酶和条件方面提高酶解效果潜力已经不大；但目前对于影响水解的其他因素如助剂的研究还有不足。
发明内容
申请人研究了酶解中加入的助剂，发现在发现在碱性蛋白酶或复合风味蛋白酶酶解过程中加入适量乳化剂，如硬脂酰乳酸钠或者硬脂酰乳酸钙可以实现在适当的水解度和风味下有效降低致敏性，实现了风味、低致敏性和食品安全性能的良好平衡，为配方食品或奶粉提供了良好的原材料。
一种婴儿乳蛋白部分水解配方食品，其特征在于，其包括在酶解体系中乳化剂存在的条件下以碱性蛋白酶或复合风味蛋白酶酶解乳清蛋白获得的水解乳清蛋白。
进一步地，所述配方食品中成分为：植物脂肪粉C 69.9％、植物脂肪粉E 24％、低聚果糖2％、胆碱1.1％、氯化钾0.7％、磷酸氢钙0.5％、矿物质0.5％、肌醇0.3％、磷酸三钙0.3％、维生素0.3％、氯化钠0.2％、碳酸钙0.1％、左旋肉碱0.05％、核苷酸0.05％；
其中植物脂肪粉C为：乳糖45％、水解乳清蛋白26％、复合植物油26.6％、磷酸氢钙1.4％、单，双甘油脂肪酸酯0.7％、L-抗坏血酸钠0.2％、抗坏血酸棕榈酸酯0.033％、磷脂0.033％、混合生育酚浓缩物0.034％；
植物脂肪粉E为：固体淀粉糖26.8％、无水奶油25％、水解乳清蛋白23％、麦芽糊精19.2％、复合植物油5％、单，双甘油脂肪酸酯0.71％、L-抗坏血酸钠0.21％、抗坏血酸棕榈酸酯0.03％、磷脂0.03％、混合生育酚浓缩物0.02％；
进一步地，所述酶解乳清蛋白为以7％的酶/底物质量比的碱性蛋白酶或者9％的复合风味蛋白酶酶解乳清蛋白。
进一步地，所述乳化剂为硬脂酰乳酸钠或者硬脂酰乳酸钙；
进一步地，所述酶解体系中的乳化剂为1％w/v的硬脂酰乳酸钠。
另一方面，本申请提供了上述婴儿乳蛋白部分水解配方食品的制备方法，其特征在于，包括在乳化剂存在的条件下以7％的酶/底物质量比的碱性蛋白酶或者9％的复合风味蛋白酶酶解在酶解体系中乳清蛋白的步骤。
进一步地，所述乳化剂为硬脂酰乳酸钠或者硬脂酰乳酸钙。
进一步地，所述酶解体系中的乳化剂为1％w/v的硬脂酰乳酸钠。
进一步地，酶解条件为pH 8.5、温度50摄氏度、时间4小时或者pH 7、温度50摄氏度、时间4小时。
具体实施方式
主要试剂、仪器和方法
乳清蛋白为申请人使用自有奶源自制，实验过程中使用相同批次的产品；
碱性蛋白酶、复合风味蛋白酶：诺维信；
木瓜蛋白酶：东恒华道；
硬脂酰乳酸钠、硬脂酰乳酸钙：河北润步生物科技有限公司；
KU812(人外周血嗜碱性白血病细胞)：购自EK-bioscience；
雌性C57BL6小鼠：购自上海加科生物科技有限公司；
β-HEX ELISA检测试剂盒：睿信生物；
液质联仪器：岛津LCMS-2020；
小鼠IgG、IgE ELISA检测试剂盒(HPR标记羊抗鼠)：上海研谨生物科技有限公司；
小鼠组胺ELISA检测试剂盒：江莱生物；
其他试剂和配料均为符合相关规定的常规国产或进口种类。
酶解方法：
以5mM的所需PBS配制10g/L的乳清蛋白溶液，加入所需酶和乳化剂后在恒温水浴锅中酶解预定时间(期间低速搅拌)，100摄氏度10分钟灭酶后进行测定或置于冰箱冷冻室中保存待用。
水解度测定：
OPA(邻苯二甲醛)法，取400微升样品或丝氨酸对照液(0.1g/L)加入3mLOPA试剂中，反应2分钟后，测定340nm吸光度后计算(详细试剂和步骤参考CHURCH F C等.，SpectrophotometricAssay Using o-Phthaldialdehyde for Determination ofProteolysis in Milk and Isolated Milk Proteins，Journal of Dairy Science，1983年，第66卷，第6期)；
此外，以SDS-PAGE电泳测定水解后乳清蛋白片段的分子量分布情况。
致敏性细胞水平测定：
通过合作医疗机构获取对牛乳β-乳球蛋白确诊过敏的患者(特异性IgE>0.8KUA/L)血清，测试所用血清池由25位患者的血清混合而成。
将购买的冻存KU812细胞复苏、传代；向培养基中加入2％牛乳β-乳球蛋白确诊过敏患者血清池血清(空白组为无菌缓冲液)后孵育12小时，使细胞处于致敏状态；加入培养基体积1％的蛋白浓度为10mg/mL的样品(阴性对照组为无菌缓冲液)，孵育6小时；TritonX-100裂解后，使用上述试剂盒检测β-HEX，并计算β-HEX释放率：β-HEX释放率(100％)＝(样品组OD值-阴性组OD值)/(阳性组OD值-阴性组OD值)×100％。HPLC-MS检测：
取水解乳清蛋白，适当稀释后进样；流动相1：0.1％甲酸水溶液、流动相2：0.1％甲酸乙腈溶液，0-60分钟内流动相2从0％-100％；电喷雾正离子模式，碰撞电压28ev；PEAKS软件对Swiss-Prot数据库进行分析。
小鼠过敏反应检测：
分组；灌胃1mL含20mg/mL样品+10微克霍乱毒素佐剂的PBS/含10微克霍乱毒素佐剂的PBS；间隔一周相同方案免疫一次；再间隔一周相同方案再免疫一次；再间隔一周相同方案激发，30分钟后采血以上述试剂盒检测组胺和特异性IgG、IgE水平。
实施例1水解乳清蛋白的制备
根据前期研究经验和实际水解实验(出于篇幅原因未在此展示)中的水解度、时间、苦味值、风味评价等数据，我们从多种可用蛋白酶及其组合中选择了碱性蛋白酶、复合风味蛋白酶单酶酶解方法(相比多种酶复合酶解的方法产品质量更均一且成本较低)以及复合酶解方法，并进一步确定了其适合的酶解条件：
碱性蛋白酶：7％酶/底物质量比、pH 8.5、温度50摄氏度、时间4小时；
复合风味蛋白酶：9％酶/底物质量比、pH 7、温度50摄氏度、时间4小时；
碱性蛋白酶+复合风味蛋白酶：5％+5％的酶/底物质量比、pH 7.5、温度50摄氏度、时间6小时；
使用上面的记载的方法制备如下样品：
未水解乳清蛋白：不进行任何水解、热处理、压力处理、辐照处理的乳清蛋白样品样品；
水解乳清蛋白1：以上述条件碱性蛋白酶酶解；
水解乳清蛋白2：以上述条件复合风味蛋白酶酶解；
水解乳清蛋白3：以上述条件加酶解体系中1％w/v的硬脂酰乳酸钠的碱性蛋白酶酶解；
水解乳清蛋白4：以上述条件加酶解体系中1％w/v的硬脂酰乳酸钠的复合风味蛋白酶酶解；
水解乳清蛋白5：以上述条件碱性蛋白酶+复合风味蛋白酶酶解；
实施例2水解乳清蛋白的基本性质研究
水解程度的测定：
通过上述OPA法测定和SDS电泳观察，水解乳清蛋白1-8的水解情况如下表所示：
表1各种水解乳清蛋白的水解情况：
结果表明，无论是否加入乳化剂，碱性蛋白酶和复合风味蛋白酶的酶解效果都类似(水解度和产物分子量均类似)，水解程度明显低于碱性蛋白酶+复合风味蛋白酶处理(不定量，简单从电泳图上看水解乳清蛋白5的水解程度接近深度水解的标准)。经过评分品评，水解乳清蛋白1-4的口感和味道普遍好于水解乳清蛋白5，反映在口感顺滑，奶香味更明显。
实施例3致敏性测定
使用上述方法对未水解乳清蛋白和水解乳清蛋白1-5的β-HEX释放率进行检测，结果如下：
表2各种水解乳清蛋白的β-HEX释放率
| 样品 | β-HEX释放率(％) |
| 未水解乳清蛋白 | 45.33 |
| 水解乳清蛋白1 | 30.53 |
| 水解乳清蛋白2 | 29.82 |
| 水解乳清蛋白3 | 22.37 |
| 水解乳清蛋白4 | 21.09 |
| 水解乳清蛋白5 | 20.22 |
结合实施例2的结果来看，加入适量的硬脂酰乳酸钠发挥乳化作用使得某些致敏关键位置暴露进而被水解。
进一步以上述HPLC-MS方法检测了水解乳清蛋白1-5中的残留肽段，在水解乳清蛋白1、2中发现β-乳球蛋白的1-9、34-40、42-60、73-82、93-101、87-94、140-149残留肽段，分别有对应的过敏表位；而在水解乳清蛋白3、4中未检测到34-40肽段(对应IgE表位21-40，Fritsche R.等，IgE-mediated rat mast cell triggering with tryptic andsynthetic peptides of bovine beta-lactoglobulin，Int Arch Allergy Immunol，2005年，第138卷，第4期)；在水解乳清蛋白5仅检测到1-9、93-101、87-94肽段。
实施例4配方食品实际产品制备及其效果
以水解乳清蛋白4和未水解的乳清蛋白实际制备配方食品，具体配方如下：
植物脂肪粉C 69.9％、植物脂肪粉E 24％、低聚果糖2％、胆碱1.1％、氯化钾0.7％、磷酸氢钙0.5％、矿物质0.5％、肌醇0.3％、磷酸三钙0.3％、维生素0.3％、氯化钠0.2％、碳酸钙0.1％、左旋肉碱0.05％、核苷酸0.05％；
其中植物脂肪粉C为：乳糖45％、水解/未水解乳清蛋白26％、复合植物油26.6％、磷酸氢钙1.4％、单，双甘油脂肪酸酯0.7％、L-抗坏血酸钠0.2％、抗坏血酸棕榈酸酯0.033％、磷脂0.033％、混合生育酚浓缩物0.034％；
植物脂肪粉E为：固体淀粉糖26.8％、无水奶油25％、水解/未水解乳清蛋白23％、麦芽糊精19.2％、复合植物油5％、单，双甘油脂肪酸酯0.71％、L-抗坏血酸钠0.21％、抗坏血酸棕榈酸酯0.03％、磷脂0.03％、混合生育酚浓缩物0.02％
矿物质包括硫酸铜、硫酸镁、硫酸亚铁、硫酸锌、硫酸锰、碘酸钾、亚硒酸钠；维生素包括醋酸视黄酯、胆钙化醇、dl-α-醋酸生育酚、植物甲萘醌、盐酸硫胺素、核黄素、盐酸吡哆醇、氰钴胺、烟酰胺、叶酸、D-泛酸钙、L-抗坏血酸钠、D-生物素；
复合植物油包括葵花籽油、大豆油、低芥酸菜籽油、椰子油、玉米油。
使用前述小鼠模型验证配方食品和市售某深度水解奶粉的效果(每组5只小鼠，取平均值)。结果如下
可见本申请的水解乳清蛋白配方食品抗敏效果明显，实现了类似深度水解奶粉的效果。
Claims (3)
1.一种婴儿乳蛋白部分水解配方食品，其特征在于，所述配方食品包括以碱性蛋白酶或复合风味蛋白酶酶解乳清蛋白获得的水解乳清蛋白；
所述酶解的酶解体系中包括7％的酶/底物质量比的碱性蛋白酶以及1％w/v的硬脂酰乳酸钠，酶解条件为pH 8.5、温度50摄氏度、时间4小时；
或者所述酶解的酶解体系中包括9％的酶/底物质量比的复合风味蛋白酶以及1％w/v的硬脂酰乳酸钠；酶解条件为pH 7、温度50摄氏度、时间4小时。
2.根据权利要求1 所述的配方食品，其中所述配方食品中成分为：植物脂肪粉C69.9％、植物脂肪粉E 24％、低聚果糖2％、胆碱1.1％、氯化钾0.7％、磷酸氢钙0.5％、矿物质0.5％、肌醇0.3％、磷酸三钙0.3％、维生素0.3％、氯化钠0.2％、碳酸钙0.1％、左旋肉碱0.05％、核苷酸0.05％；其中植物脂肪粉C为：乳糖45％、水解乳清蛋白26％、复合植物油26.6％、磷酸氢钙1.4％、单，双甘油脂肪酸酯0.7％、L-抗坏血酸钠0.2％、抗坏血酸棕榈酸酯0.033％、磷脂0.033％、混合生育酚浓缩物0.034％；植物脂肪粉E为：固体淀粉糖26.8％、无水奶油25％、水解乳清蛋白23％、麦芽糊精19.2％、复合植物油5％、单，双甘油脂肪酸酯0.71％、L-抗坏血酸钠0.21％、抗坏血酸棕榈酸酯0.03％、磷脂0.03％、混合生育酚浓缩物0.02％。
3.根据权利要求1所述的婴儿乳蛋白部分水解配方食品的制备方法，其特征在于，
所述方法包括以碱性蛋白酶或复合风味蛋白酶酶解乳清蛋白获得水解乳清蛋白的步骤；
所述酶解的酶解体系中包括7％的酶/底物质量比的碱性蛋白酶以及1％w/v的硬脂酰乳酸钠，酶解条件为pH 8.5、温度50摄氏度、时间4小时；
或者所述酶解的酶解体系中包括9％的酶/底物质量比的复合风味蛋白酶以及1％w/v的硬脂酰乳酸钠；酶解条件为pH 7、温度50摄氏度、时间4小时。
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