牛乳蛋白质酶改性的研究与液态婴儿乳的研制
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摘要
牛乳因其来源广泛、营养丰富、且与人乳成分较为接近而被长期用作母乳代用品。然而由于牛乳与人乳在蛋白质成分上具有较大差异，使得牛乳蛋白质不易被婴儿消化吸收，部分婴儿对牛乳会产生过敏反应。这种蛋白质的差异主要表现在：酪蛋白与乳清蛋白含量比例的差异；牛乳中含有大量的α s-酪蛋白，而母乳中几乎没有；牛乳清蛋白中含有β-乳球蛋白，而母乳中不含有。本实验通过对牛乳蛋白的水解，调整酪蛋白与乳清蛋白含量的比例，去除或减少α s-酪蛋白和β-乳球蛋白，从而制备出利于婴儿消化的母乳代用品的乳基原料。
本实验通过对安全、来源丰富的几种酶的比较筛选，最终确定以胰蛋白酶为水解用酶，对牛乳蛋白质进行改性实验。
采用单因素的实验设计方法，以体外消化率为指标，选择胰蛋白酶水解牛乳蛋白的最佳温度、最佳pH值。然后以苦味的产生为标准，确定最佳水解时间。可以得出：在温度45℃、酶浓度0.5％时具有最佳的体外消化率，当水解时间为20分钟时，水解液不产生苦味。
体外消化率的测定方法是采用体外模拟婴儿胃内的消化过程，以体外三小时模拟体内六小时发生的变化。以凝乳酶和胃蛋白酶对牛乳进行连续的体外消化，分别在消化至15分钟、45分钟和两小时分别增加酶用量，以三氯乙酸作用，可溶性蛋白与总蛋白含量的比值即为蛋白质的体外消化率。
对水解液、母乳及牛乳进行体外实验，以模拟婴儿胃内的絮凝状态，调节牛乳温度40℃、pH4.0。观察样品的絮凝状态，并进行拍照。可以观察到，母乳呈微凝状态，而牛乳的凝块粗大、坚硬，水解液的絮凝状态介于二者之间，接近于母乳。
对三种母乳样品进行氨基酸含量的测定，其结果与牛乳蛋白水解液的氨基酸含量进行比较分析，可作为调节水解液氨基酸含量接近母乳的依据。
测定了以胰蛋白酶水解牛乳蛋白在最适条件下的水解度及体外消化率，当反应初期的30分钟，二者的增加较快，随着时间的延长，反应速度的增加趋于平缓。
采用凝胶柱层析，测定水解液分子量的分布范围。对水解液的稳定性进行测定，结果表明水解液的初始稳定性较好，放置24小时后，水解液稳定性下降。对水解液的感官质量进行评定，水解液基本无苦味，但稍有杂味。
为解决水解液的稳定性及味觉的变化，将牛乳与水解液进行适当比例的混合，结果表明，当牛乳：水解液为5：5时，无不良风味感，且稳定性好，可作为易于婴儿消化的母乳代用品的乳基原料。
Cow ed has been used as infan fonnula for its uinversality of source' bodance of
nutrition and the comParability betWen cow miili and hUman ndlk. However, Ther are Still
some imPortan difornces betWen them, which make the cow milk protein not be easily
digested and absorbed, pat of infants will be caused allerg1c phenomenon by cow milk PrOtein.
The dries between hUman ndlk and cow ndlk include f the thee of the Protein
content Proponion between the tWo kind of milk, there are large amoun of Protein conen of
a s-casem in cow milk, while alInost no a s-casein m hUInan ndlk, 0-lactoglobulin existS in
cow milk but not in human milk. Through hydrolyZmg the cow milk protein, the Proponion of
casein to whey protein was adjusted and, at the same trie, a s-casem and 0-1actoglobulin
were modified. rus Product can be used as Inilk raw material for infan formula and be
faVorable to infan digest
Aner compmp and chOos1ng five kinds of safe and abUndan enZymes, the eapenment
detennined pancreatin as hydrolyzing emp to moop ndlk protein.
Usmg single factor exPchmere and taking the vitro digest raho as the index, the eapenment
detenmned the ophmum temPeratUIe and pH. Then taken the bitterness extent as standard- the
oPtimum hydrolyzing hme was detedned. The result ls f under the condition of temperature
45t, Wme concentration 0.5%, the oPtimum vitro digest ration was acqUired. When
hydrOlyzing for was 20 Ininates, the hydIolysate was no bitter.
The way to detennine digest ratio in infan vivo is tO simulate infan stomach digest Process
in vbo. Three hOurs in vitro could be used tO shmulate 6 hOurs in vivo. Continuously aPpend
rennet and pePsin after hydrolyZing l5 ndnutes, 45 ndnutes and 2 hOurs. TCA was used tO
dePosit protein. The ratio of solvable protCin to the tOtal protein was the vitro digest ratio.
Using vitr exPerimen to simulate the state of curd in infan stomach.. Adusted
temPeratUre 40'C, pH4.0, observed and phoned the flocculahon. The state of human mllk was
mlld, but the curd of cow milk was bulky and snd The state of hydrolysate was evaluated, it
was more lilie hUInan Iinfa.
han acid (AA) content was determined by tbIee kinds of human mIIk. ComPartri
research had been done betWeen human milk and hvdrolvsate. The differences can be used to
detennine how to adjust AA conten in hydrolysate more lilie hUman milk
,
*fl% HJdftg 2002
The DH and vitro digest ratio were determined under the optimum condition. In the first 30 minutes, both increase at a high speed, with the proceeding of hydrolyzing, the increase of the reaction speed was imperceptible.
The sensor index was assessed, Molecule mass of hydrolysate was measured and stability was mensured, too. All of which were close to human milk.
In order to weeken the bad taste and enhance stability of hydrolysate, the experiment mix cow milk and hydrolysate at all kinds of proportion. The result showed that when the ratio of cow milk:hydrolysate is 5:5, the hydrolysate has no ill flavor and has good stability. The mixture can be used as material of infant milk.
Postgraduate: Liu Fang
Major: Food Science
Supervisor: Prof. Huo Guicheng
本实验通过对安全、来源丰富的几种酶的比较筛选，最终确定以胰蛋白酶为水解用酶，对牛乳蛋白质进行改性实验。
采用单因素的实验设计方法，以体外消化率为指标，选择胰蛋白酶水解牛乳蛋白的最佳温度、最佳pH值。然后以苦味的产生为标准，确定最佳水解时间。可以得出：在温度45℃、酶浓度0.5％时具有最佳的体外消化率，当水解时间为20分钟时，水解液不产生苦味。
体外消化率的测定方法是采用体外模拟婴儿胃内的消化过程，以体外三小时模拟体内六小时发生的变化。以凝乳酶和胃蛋白酶对牛乳进行连续的体外消化，分别在消化至15分钟、45分钟和两小时分别增加酶用量，以三氯乙酸作用，可溶性蛋白与总蛋白含量的比值即为蛋白质的体外消化率。
对水解液、母乳及牛乳进行体外实验，以模拟婴儿胃内的絮凝状态，调节牛乳温度40℃、pH4.0。观察样品的絮凝状态，并进行拍照。可以观察到，母乳呈微凝状态，而牛乳的凝块粗大、坚硬，水解液的絮凝状态介于二者之间，接近于母乳。
对三种母乳样品进行氨基酸含量的测定，其结果与牛乳蛋白水解液的氨基酸含量进行比较分析，可作为调节水解液氨基酸含量接近母乳的依据。
测定了以胰蛋白酶水解牛乳蛋白在最适条件下的水解度及体外消化率，当反应初期的30分钟，二者的增加较快，随着时间的延长，反应速度的增加趋于平缓。
采用凝胶柱层析，测定水解液分子量的分布范围。对水解液的稳定性进行测定，结果表明水解液的初始稳定性较好，放置24小时后，水解液稳定性下降。对水解液的感官质量进行评定，水解液基本无苦味，但稍有杂味。
为解决水解液的稳定性及味觉的变化，将牛乳与水解液进行适当比例的混合，结果表明，当牛乳：水解液为5：5时，无不良风味感，且稳定性好，可作为易于婴儿消化的母乳代用品的乳基原料。
Cow ed has been used as infan fonnula for its uinversality of source' bodance of
nutrition and the comParability betWen cow miili and hUman ndlk. However, Ther are Still
some imPortan difornces betWen them, which make the cow milk protein not be easily
digested and absorbed, pat of infants will be caused allerg1c phenomenon by cow milk PrOtein.
The dries between hUman ndlk and cow ndlk include f the thee of the Protein
content Proponion between the tWo kind of milk, there are large amoun of Protein conen of
a s-casem in cow milk, while alInost no a s-casein m hUInan ndlk, 0-lactoglobulin existS in
cow milk but not in human milk. Through hydrolyZmg the cow milk protein, the Proponion of
casein to whey protein was adjusted and, at the same trie, a s-casem and 0-1actoglobulin
were modified. rus Product can be used as Inilk raw material for infan formula and be
faVorable to infan digest
Aner compmp and chOos1ng five kinds of safe and abUndan enZymes, the eapenment
detennined pancreatin as hydrolyzing emp to moop ndlk protein.
Usmg single factor exPchmere and taking the vitro digest raho as the index, the eapenment
detenmned the ophmum temPeratUIe and pH. Then taken the bitterness extent as standard- the
oPtimum hydrolyzing hme was detedned. The result ls f under the condition of temperature
45t, Wme concentration 0.5%, the oPtimum vitro digest ration was acqUired. When
hydrOlyzing for was 20 Ininates, the hydIolysate was no bitter.
The way to detennine digest ratio in infan vivo is tO simulate infan stomach digest Process
in vbo. Three hOurs in vitro could be used tO shmulate 6 hOurs in vivo. Continuously aPpend
rennet and pePsin after hydrolyZing l5 ndnutes, 45 ndnutes and 2 hOurs. TCA was used tO
dePosit protein. The ratio of solvable protCin to the tOtal protein was the vitro digest ratio.
Using vitr exPerimen to simulate the state of curd in infan stomach.. Adusted
temPeratUre 40'C, pH4.0, observed and phoned the flocculahon. The state of human mllk was
mlld, but the curd of cow milk was bulky and snd The state of hydrolysate was evaluated, it
was more lilie hUInan Iinfa.
han acid (AA) content was determined by tbIee kinds of human mIIk. ComPartri
research had been done betWeen human milk and hvdrolvsate. The differences can be used to
detennine how to adjust AA conten in hydrolysate more lilie hUman milk
,
*fl% HJdftg 2002
The DH and vitro digest ratio were determined under the optimum condition. In the first 30 minutes, both increase at a high speed, with the proceeding of hydrolyzing, the increase of the reaction speed was imperceptible.
The sensor index was assessed, Molecule mass of hydrolysate was measured and stability was mensured, too. All of which were close to human milk.
In order to weeken the bad taste and enhance stability of hydrolysate, the experiment mix cow milk and hydrolysate at all kinds of proportion. The result showed that when the ratio of cow milk:hydrolysate is 5:5, the hydrolysate has no ill flavor and has good stability. The mixture can be used as material of infant milk.
Postgraduate: Liu Fang
Major: Food Science
Supervisor: Prof. Huo Guicheng
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