CN107365356A - 肉制品保水肽及其应用 - Google Patents
肉制品保水肽及其应用 Download PDFInfo
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- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical compound [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 description 1
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Meat, Egg Or Seafood Products (AREA)
Abstract
本发明公开了肉制品保水肽及其应用，属于食品生物技术领域。该肉制品保水肽以谷氨酰胺为底物和缬氨酸经酶法摩尔比为1‑2:1，在溶液中谷氨酰胺和游离缬氨酸的浓度在400‑1000mmol/L；调节反应液pH为8.0‑10.0，添加谷氨酰胺酶或γ‑谷氨酰胺转肽酶为溶液质量0.02‑1%（w/w），在25‑50℃反应3‑12小时，后用4 mol/L 盐酸回调pH至6~7之间，90℃灭酶10 min，最后经洗脱得到肉制品保水肽γ‑Glu‑γ‑Glu‑γ‑Glu‑γ‑Glu‑val。采用这一方法制备的肉制品保水肽具有较强的保水能力和较低的添加量。
Description
技术领域
本发明涉及保水剂制备的方法，更具体说，本发明涉及生产一种具有较强保水效果的肽的方法。
背景技术
保水性是指肉制品在加压、切碎、加热、冷冻、解冻以及腌制等加工或贮藏过程中，保持其水分的能力。磷酸盐对肉制品品质的改良起着重要作用，因此在肉制品加工领域应用非常广泛。国家标准GB2760－2011中规定磷酸盐添加量≤0.5％，但一些企业为了追逐经济利益而超量添加。磷酸盐过量会产生不良影响，导致产品存在金属涩味。磷酸盐摄入量过大，可能在肠道中与钙结合成难溶于水的正磷酸钙，降低身体对钙的吸收，影响身体健康。因此欧盟等地区对肉制品中的磷酸盐限量要求非常严格。另外，肉制品原料本身含有一定的磷酸盐，肉制品即使正常添加也可能面临超标的风险。针对这些问题，无磷保水剂作为一种替代品受到众多厂家的追捧，近年来占据较大市场。无磷保水剂多采用淀粉、蛋白、亲水胶体、碳酸盐、柠檬酸盐、食盐等作为传统磷酸盐保水剂的替代物，以单一或者复配的形式应用于肉制品中，从而达到提高系水力、粘结力、乳化性的作用。天津三农金科技有限公司公开了一种肉制品高效保水剂(201010511898.0)，其特征在于：由下列原料按照如下的重量配比混合制成：氯化钠30‐50份、海藻糖10‐20份、山梨醇20‐30份、甘油10‐20份、卡拉胶5‐10份。申请人宣称该产品使用安全、无毒、无害。主要用在肉制品中保水保鲜防腐、吸收水份、增加重量，加入后可以提高产品的稳定性，保持食品内部持水性，嫩化肉质，可缩短蒸煮时间，改善食品的形态、风味、口感、色泽。无锡商业职业技术学院公开了一种肉制品无磷保水剂及其使用方法(201510195136.7)。该无磷保水剂包括碳酸氢盐3～5份、甘油20～30份、单糖和/或其糖醇10～20份、食用胶5～10份、大豆磷脂5～10份、其余是水。其使用方法是：先正向盐渗透，将肉腌制成干肉，再将肉浸入无磷保水剂反向渗透。申请人宣称该无磷保水剂使用方法使无磷保水剂中的锁水物质渗透能力增强，提升无磷保水剂在肉细胞内的锁水效果。湖北兴发化工集团股份有限公司提供了一种肉制品保水护色剂(201310013650.5)，以重量计，由三聚磷酸钠10‐40份，焦磷酸一氢三钠5‐30份，六偏磷酸钠1‐10份，褐藻酸钠10‐30份，D‐异抗坏血酸钠3‐15份，食用盐5‐25份，食用葡萄糖10‐30份混匀组成。该发明采用一种新型的食品添加剂—焦磷酸一氢三钠，代替传统复配磷酸盐中的焦磷酸钠成为一种新型的保水剂和护色剂；其中性的pH不仅避免焦磷酸钠和三聚磷酸钠等高碱性磷酸盐带来的皂味，而且更有利于肉制品自然腌制色的形成，避免高碱性磷酸盐对腌制色形成的延缓作用和带来的颜色不均，对肉制品的护色和改善口感具有明显的效果。
发明内容
本发明的目的在于克服现有技术存在的上述不足，提供肉制品保水肽及其应用。
本发明提供的肉制品保水肽，氨基酸序列为γ-Glu-γ-Glu-γ-Glu-γ-Glu-val。
所述肉制品保水肽的应用，具体是在肉制品中添加量为1000mg/kg以上的肉制品保水肽γ-Glu-γ-Glu-γ-Glu-γ-Glu-val。
制备所述肉制品保水肽的方法：谷氨酰胺与游离缬氨酸，摩尔比为1-2:1，在溶液中谷氨酰胺和游离缬氨酸的浓度在400-1000mmol/L。调节反应液pH为8.0-10.0，添加谷氨酰胺酶或γ-谷氨酰胺转肽酶为溶液质量0.02-1％(w/w)，在25-50℃反应3-12小时，后用4mol/L盐酸回调pH至6～7之间，90℃灭酶10min，得到富含寡肽γ-Glu-γ-Glu-γ-Glu-γ-Glu-val的反应液。
采用XSelect HSS T3 5μm 4.6x 250mm分析柱，流动相A液为0.1％(V/V)甲酸-水溶液；流动相B液为0.1％(V/V)甲酸-乙腈溶液，柱温为40℃，流速为1mL/min，反应液的进样量10μL，采用高效液相色谱对反应液进行梯度洗脱，出峰时间为5.619min的峰即为γ-Glu-γ-Glu-γ-Glu-γ-Glu-val。
本发明与现有技术相比，具有如下优点：
(1)本发明制备的肉制品保水肽在添加量仅1000mg/kg的时候，就有较好的保水效果。
(2)本发明制备的肉制品保水肽即使在添加量为2-4％的时候，也不会对肉制品的风味产生不良风味。
附图说明
图1为实例中保水肽γ-Glu-γ-Glu-γ-Glu-γ-Glu-val的液相色谱分离图。
图2为保水肽γ-Glu-γ-Glu-γ-Glu-γ-Glu-val的二级质谱图。
具体实施方式
以下结合实例和附图对本发明的具体实施作进一步说明，但本发明的实施和保护不限于此，需指出的是，以下若有未特别详细说明之过程，均是本领域技术人员可参照现有技术理解或实施的。
实例中相关量的计算：
式中:m1为腌制后肉重(g)，m2为腌制前肉重(g)，m3为油炸前肉重(g)，m4为油炸后肉重(g)，m5为解冻前肉重(g)，m6为解冻后肉重(g)。
腌制吸水率：根据实验要求，准确称取食盐等调味料以及保水剂，一组保水剂海藻糖、复合磷酸盐、的添加量均为0.1％％(按原料肉重计)，一组添加量依次为0.4％，另设一组空白对照，不添加任何保水剂，然后将配料在冰水中进行充分溶解，待用。
滚揉及腌制工艺：将配制好的腌制液和鸡肉一起放入真空滚揉机中(真空度为-0.08MPa)，采用间歇滚揉方式(滚揉30min静置10min)，总共滚揉1.5h，冷库温度控制在8℃以下，低温腌制24h称重。
解冻失水率:将肌肉先速冻，然后在-18℃条件下进行冷冻储存2d后，测定解冻失水率。
油炸失水率:开启油炸设备，待油温升至180℃后，放入添加了保水剂的鸡胸肉，油炸时间为1.5min，油炸后沥油、冷却称重，测定油炸失水率。
实施例1
将300mM谷氨酰胺与300mM缬氨酸溶于100g水中，调节溶液的pH值至10.0，加入0.4g谷氨酰胺酶(日本天野株式会社，活力为100GTU/g)，在37℃分别反应12h，后用4mol/L盐酸回调pH至7.0后，90℃灭酶10min，得富含γ-glu-γ-glu-γ-glu-γ-glu-val的肉制品保水肽。
HPLC液相对反应产物进行定量分析：分析柱：XSelect HSS T3 5μm 4.6x250mm；流动相：A液：0.1％(V/V)甲酸-水溶液；B液：0.1％(V/V)甲酸-乙腈溶液；柱温：40℃，流速：1mL/min。进样量10μL。其液相色谱图见图1。流动相进行梯度变化如表1所示。
表1
| Time/min | 0 | 5 | 10 | 15 | 20 |
| A/％ | 90 | 85 | 20 | 90 | 90 |
图1中,出峰时间为5.619对应的峰7为γ-glu-γ-glu-γ-glu-γ-glu-val，对峰7进行收集，冷冻干燥得到γ-glu-γ-glu-γ-glu-γ-glu-val，并进行UPLC-MS/MS鉴定。
UPLC-MS/MS对反应产物进行定性分析：包括：An Agilent 1290series UPLCsystem(Agilent Technologies)用于分离各γ-Glu-peptides,带有an elec-trosprayionization(ESI)的质谱系统(Q-TOF MS/MS，Bruker Daltonics)进行定性分析。色谱柱为：Agilent ZORBAX RRHD SB-C18(2.1x 50mm,1.8μm)。进样量为5μL。液相条件为：溶液A为0.1％甲酸-水溶液，和溶液B为0.1％甲酸-乙腈溶液，梯度洗脱程序为：0–10％B,0-5.0min；10-15％B,5.0-10.0min；然后100％B,10.0-12.0min。MS条件：Ionization(离子化)：正离子模式；Drying gas(干燥气体)：350℃下10L/min；Nebulizer(喷雾器压力)：25psig；Fragmentor(毛细管电压)：30V。采用UPLC-MS/MS对峰7，其检测结果如图2。由图2可知，其一级结构为γ‐glu‐γ‐glu‐γ‐glu‐γ‐glu‐val。
肉制品保水肽的效果评价见表2‐表4。
表2(％为重量百分比)
表3
| 解冻失水率 | 添加量0.1％ | 解冻失水率 | 添加量0.4％ |
| 实施例1 | 4.5％ | 实施例1 | 3.5％ |
| 复合磷酸盐 | 7.8％ | 复合磷酸盐 | 3.6％ |
| 海藻糖 | 8.6％ | 海藻糖 | 5.1％ |
| 卡拉胶 | 8.4％ | 卡拉胶 | 5.6％ |
| 空白 | 9.5％ | 空白 | 9.5％ |
表4
| 油炸失水率 | 添加量0.1％ | 油炸失水率 | 添加量0.4％ |
| 实施例1 | 37.5％ | 实施例1 | 33.5％ |
| 复合磷酸盐 | 42.6％ | 复合磷酸盐 | 34.6％ |
| 海藻糖 | 41.4％ | 海藻糖 | 35.8％ |
| 卡拉胶 | 40.6％ | 卡拉胶 | 35.5％ |
| 空白 | 43.5％ | 空白 | 35.9％ |
由表2，表3和表4，可见，实施例1所制备的肉制品保水肽在腌制吸水率、解冻失水率和油炸失水率这三个指标上均优于复合磷酸盐、海藻糖、卡拉胶等食品添加剂。
实施例2
将解淀粉芽孢杆菌(Bacillus amyloliquefaciens)SWJS22(中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.8425，已在申请号为201410349296.8的中国专利申请中保藏)以1％的接种量接种于发酵培养基(发酵培养基的组成为：4％豆粕、2％麸皮、2％玉米粉)中，37～39℃发酵培养30h。将发酵液于4℃，10000r/min条件下高速离心10min，过滤得上清液，即谷氨酰胺酶粗酶液。
将600mM谷氨酰胺和300mM缬氨酸溶于100g水中，分别调节溶液的pH值至9.0，加入1g粗酶液，在40℃分别反应12h后，用4mol/L盐酸回调pH至7.0后，90℃灭酶10min，得到γ-glu-γ-glu-γ-glu-γ-glu-val的反应液。采用XSelect HSS T3 5μm 4.6x 250mm分析柱，流动相A液为0.1％(V/V)甲酸-水溶液；流动相B液为0.1％(V/V)甲酸-乙腈溶液，柱温为40℃，流速为1mL/min，反应液的进样量10μL，采用高效液相色谱对反应液进行梯度洗脱，第7个峰即为寡肽γ-Glu-γ-Glu-γ-Glu-γ-Glu-Val。
Claims (3)
1.肉制品保水肽，其特征在于氨基酸序列为γ-Glu-γ-Glu-γ-Glu-γ-Glu-val。
2.根据权利要求1所述的肉制品保水肽，其特征在于由如下方法制得：谷氨酰胺与游离缬氨酸，摩尔比为1-2:1，在溶液中谷氨酰胺和游离缬氨酸的浓度在400-1000mmol/L；调节反应液pH为8.0-10.0，添加谷氨酰胺酶或γ-谷氨酰胺转肽酶为溶液质量0.02-1%（w/w），在25-50℃反应3-12小时，后用4 mol/L 盐酸回调pH至6~7之间，90℃灭酶10 min，得到富含寡肽γ-Glu-γ-Glu-γ-Glu-γ-Glu-val的反应液；采用XSelect HSS T3 5μm 4.6x 250mm分析柱，流动相A 液为0.1％（V/V） 甲酸-水溶液；流动相B 液为0.1％(V/V) 甲酸-乙腈溶液，柱温为40℃，流速为1 mL/min，反应液的进样量 10 μL，采用高效液相色谱对反应液进行梯度洗脱，出峰时间为5.619min的峰即为γ-Glu-γ-Glu-γ-Glu-γ-Glu-val。
3.权利要求1所述肉制品保水肽在肉制品中的应用，其特征在于在肉制品中的添加量为1000mg/kg以上。
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