CN100497613C - Method for producing lactase of neutral liquid - Google Patents
Method for producing lactase of neutral liquid Download PDFInfo
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- CN100497613C CN100497613C CNB200610010516XA CN200610010516A CN100497613C CN 100497613 C CN100497613 C CN 100497613C CN B200610010516X A CNB200610010516X A CN B200610010516XA CN 200610010516 A CN200610010516 A CN 200610010516A CN 100497613 C CN100497613 C CN 100497613C
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Abstract
This invention relates to a method for producing neutral liquid lactase. The method comprises: (1) performing deep liquid fermentation to culture mature wall-crisp Kluyveromyce cell in large scale; (2) centrifuging in a high speed, washing with sterilized water, and preparing cell bacteria suspension with sterilized water; (3) adding glucanase, cellulose and protease, and keeping the temperature; (4) taking out and rapidly freezing; (5) melting to obtain freeze-thaw cell suspension; (6) secondarily breaking the walls in a high-speed mixer or an ultrasonic cell crusher to obtain active cell structure-free lactase solution; (7) centrifuging or filtering to obtain active lactase solution without strain cell chips; (8) adding sodium chloride and sucrose to obtain neutral liquid lactase. The obtained neutral liquid lactase can be used to treat fresh milk, sterilized milk or reduced milk and produce liquid milk and milk powder with low lactose content.
Description
(1) technical field
That the present invention relates to is a kind of preparation method of organized enzyme, specifically a kind of preparation method of active lactose enzyme.
(2) background technology
The enzyme that can reduce lactose among the human small intestine has Sumylact L (lactase) and beta-galactosidase enzymes, and (β-galactosidase), Sumylact L is positioned at the mucous membrane of small intestine brush border, and beta-galactosidase enzymes is positioned at lysosome, can not decompose the lactose in the enteric cavity.Lactase deficiencies is meant that the membrane bound enzyme of intestinal brush border chorionic cells is that lactase activity is low.Lactase deficiencies has three types: (1) congenital lactase deficiency: being meant the low or shortage of body lactase activity when being born, is on the body euchromosome due to the recessive gene, and this type is rarely found.(2) secondary lactase deficiency: be meant that the temporary lactase activity that causes the small intestine epithelium damage to cause owing to a variety of causes is low, as: diseases such as infectious diarrhea, immunoglobulin deficiency, celiac disease, regional ileitis, malnutrition, can after the organism disease rehabilitation, recover normal.(3) primary lactase deficiencies (claiming the adult type lactase deficiencies again): be modal a kind of, not by due to the influence of disease, mainly be to cause because lactase activity reduced gradually with the age, time of origin because of race with different from different places, behind weaning baby, just begin to take place, begin to occur during 20 years old left and right sides having.Population of China is many to be taken place in the time of 7～8 years old.It is generally acknowledged that lactase deficiencies is relevant with the different genetic gene mutations that cause of food habits that form from generation to generation.The lactase deficiencies incidence changes with country variant, different ethnic populations' variation.Europe crowd's 30% above lactase deficiencies, the asian population lactase deficiencies accounts for 75%～100%, the white American 12%, Black people 70%, Africa 90%～100%; Japan 100%, 3～13 years old children's lactase deficiencies incidence of China is 87%.
After lactose in the cow's milk entered small intestine, because the shortage of Sumylact L, lactose can not be broken down into glucose and semi-lactosi is absorbed, and is called lactose maldigestion and lactose malabsorption.After lactose enters colon, generated short chain organic acid such as acetic acid, propionic acid, butyric acid etc. and gas such as methane, H by fermentation using bacteria
2, CO
2Deng, the lactose fermentation process can cause borborygmus, stomachache, rectum gas and osmotic diarrhea, is called lactose intolerance when having these symptoms, serious lactose maldigestion or malabsorption are many to be taken place to a few hours after taking in a certain amount of lactose in 30 minutes.Lactose intolerance is bigger to infant's influence, semi-lactosi is the important substance of brain normal development, Sumylact L can not be decomposed into glucose with lactose and semi-lactosi is absorbed owing to lack, therefore, children's growth retardation, brain development also there is certain influence, and can be simultaneously with phenomenons such as vomitings, the adult is sometimes with the reaction of feeling sick.Have the people about 20% to have the lactose intolerance symptom among the lactase deficiencies person, and lactose intolerance symptom individual difference is very big.The lactose yield of what and severity of intolerance shape and the interior lactase activity of small intestine, absorption and whether to take in other based food simultaneously relevant, so produce low lactose milk and have crucial meaning.
(3) summary of the invention
The object of the present invention is to provide a kind of production method that fresh cow milk, sterilization breast or recombined milk can be changed into the neutral liquid Sumylact L of low lactose liquid milk and low lactose powdered milk.
The object of the present invention is achieved like this:
1, the cultivation of Kluyveromyces fragilis cell: according to weight ratio is the proportional arrangement substratum of the water of whey powder 0.5-25%, yeast extract paste 0.1-20%, peptone 0.1-20%, ammonium sulfate 0.1-10%, dipotassium hydrogen phosphate 0.01-2% and surplus, pH7.2,105 ℃ of sterilising treatment 15 minutes, according to inoculum size is the ratio inoculation Kluyveromyces fragilis cell of 1%-25%, feeds the sterile air stir culture 10-72 hour;
2, the preparation of Kluyveromyces fragilis bacteria suspension: will cultivate sophisticated Kluyveromyces fragilis cell through the 10000r/min high speed centrifugation, obtain fresh active somatic cells, fresh active somatic cells is washed 1-2 time with sterilized water, be mixed with the cell bacteria suspension with sterilized water then, the weight ratio concentration of cell is 1.0%-40.0%;
3, the pre-broken wall treatment of enzyme: get above-mentioned bacteria suspension 500mL, add beta-glucanase, cellulase and proteolytic enzyme, cellulase, proteolytic enzyme consumption are respectively the 0.1-150.0u/g wet thallus, beta-glucan enzyme dosage 0.1-150.0u/g wet thallus, pH5.5-7.5,20 ℃-65 ℃ are incubated 1-6 hour;
4, freeze thawing broken wall: cell suspension after enzyme is handled is taken out, place under-20 ℃ of--50 ℃ of temperature freezingly, again the refrigerated cell is placed 20 ℃ of-75 ℃ of thawings, obtain the frozen-thawed cell suspension;
5, high pressure homogenizer, ultimate broken wall: place the liquid shear shredder assembly to carry out the secondary broken wall cell suspension of freeze thawing, the temperature maintenance of ingress is at 18-22 ℃, the temperature in exit is less than 50 ℃, and pressure maintains 30MPa-120MPa, obtains the active lactose enzyme solution of no cell structure;
6, carry out the centrifugal or filtration treatment of 3500-15000r/min then, obtain the active lactose enzyme solution of no somatic cells fragment, carry out concentrate under reduced pressure at low temperature again to 10-70% concentration, add the sucrose of 5-30% sodium-chlor and 5-30%, promptly obtain neutral liquid Sumylact L.
Handle fresh cow milk, sterilization breast or recombined milk with the resulting neutral liquid Sumylact L of method of the present invention, at the pH nature, temperature 4-55 ℃, the reaction times is 0.5-10 hour, can produce low lactose liquid milk and low lactose powdered milk.With this understanding, if add the ratio of 0.05-20mL fluid milk carbohydrase in the 100mL liquid milk, lactose-content can reduce 40%-100%.
(4) embodiment
Embodiment one
1, the cultural method of Kluyveromyces fragilis cell: according to weight ratio is the proportional arrangement substratum of the water of whey powder 0.5-25%, yeast extract paste 0.1-20%, peptone 0.1-20%, ammonium sulfate 0.1-10%, dipotassium hydrogen phosphate 0.01-2% and surplus, pH7.2,105 ℃ of sterilising treatment 15 minutes, according to inoculum size is the ratio inoculation Kluyveromyces fragilis cell of 1%-25%, feeds the sterile air stir culture 10-72 hour.
2, the preparation method of Kluyveromyces fragilis bacteria suspension: will cultivate sophisticated Kluyveromyces fragilis cell through the 10000r/min high speed centrifugation, obtain fresh active somatic cells, somatic cells is washed 1-2 time with sterilized water, be mixed with the cell bacteria suspension with sterilized water then, cell concn is 1.0%-40.0%.
3, the pre-broken wall treatment method of enzyme: get above-mentioned bacteria suspension 500mL, add beta-glucanase, cellulase and proteolytic enzyme, cellulase, proteolytic enzyme consumption are respectively the 0.1-150.0u/g wet thallus, beta-glucan enzyme dosage 0.1-150.0u/g wet thallus, pH5.5-7.5,20 ℃-65 ℃ are incubated 1-6 hour.
4, freeze thawing wall-breaking method: the cell suspension after enzyme is handled is taken out, place quick freezing under-20 ℃ of--50 ℃ of temperature, it is thoroughly freezed, again the refrigerated cell is placed 20 ℃ of-75 ℃ of thawings, obtain the frozen-thawed cell suspension.
5, the ultimate broken wall of high pressure homogenizer: place the liquid shear shredder assembly to carry out the secondary broken wall cell suspension of freeze thawing, the temperature maintenance of ingress is at 20 ℃, the temperature in exit is not higher than 50 ℃, and pressure maintains 30MPa-120MPa, obtains the active lactose enzyme solution of no cell structure.Described liquid shear shredder assembly can be high pressure homogenizer or ultrasonic cell-break machine.
6, carry out the centrifugal or filtration treatment of 3500-15000r/min then, obtain the active lactose enzyme solution of no somatic cells fragment, carry out concentrate under reduced pressure at low temperature again to 10-70% concentration, add the sucrose of 5-30% sodium-chlor and 5-30%, promptly obtain neutral liquid Sumylact L.
The ratio that adds 0.05-20mL fluid milk carbohydrase according to the 100mL liquid milk, neutral liquid Sumylact L is added in fresh cow milk, sterilization breast or the recombined milk, at the pH nature, under temperature 4-55 ℃ the condition, reacted 0.5-8 hour, and can obtain low lactose liquid milk and low lactose powdered milk.
Embodiment two
1, substratum preparation and yeast cell are cultivated: the water of whey powder 6.0%, yeast extract paste 1.0%, peptone 1.5%, ammonium sulfate 1.0%, dipotassium hydrogen phosphate 0.1% and surplus, pH7.2, sterilized 15 minutes for 105 ℃, according to inoculum size is 10% ratio inoculation Kluyveromyces fragilis cell, feeds the sterile air stir culture 36 hours.
2, will cultivate sophisticated Kluyveromyces fragilis cell through the 10000r/min high speed centrifugation, obtain fresh active somatic cells, then, somatic cells with sterilized water washing 1 time, is mixed with the cell bacteria suspension with sterilized water then, cell concn is 5.0%, add beta-glucanase, cellulase and proteolytic enzyme again, cellulase, proteolytic enzyme consumption are respectively the 10u/g wet thallus, beta-glucan enzyme dosage 50u/g wet thallus, pH6.8,45 ℃ are incubated 2 hours.
3, place quick freezing under-20 ℃ of temperature again after the taking-up, it is thoroughly freezed, the refrigerated cell is placed under 40 ℃ of temperature melt again, obtain the frozen-thawed cell suspension.
4, the frozen-thawed cell suspension is placed liquid shear shredder assembly high pressure homogenizer or ultrasonic cell-break machine to carry out the secondary broken wall, temperature is not higher than 50 ℃ again, and pressure maintains 80MPa, obtains the active lactose enzyme solution of no cell structure.
5, carry out the centrifugal or filtration treatment of 3500-15000r/min then, obtain the active lactose enzyme solution of no somatic cells fragment, carry out concentrate under reduced pressure at low temperature to 65% concentration again, add the sucrose of 8% sodium-chlor and 10%, promptly obtain liquid active lactose enzyme.
6, handle fresh cow milk, sterilization breast or recombined milk with neutral liquid Sumylact L, the pH nature, temperature 4-55 ℃, the reaction times is 0.5-5 hour, can produce low lactose liquid milk and low lactose powdered milk.
Claims (3)
1, a kind of neutral liquid method for producing lactase is characterized in that:
(1) cultivation of Kluyveromyces fragilis cell: according to weight ratio is the proportional arrangement substratum of the water of whey powder 0.5-25%, yeast extract paste 0.1-20%, peptone 0.1-20%, ammonium sulfate 0.1-10%, dipotassium hydrogen phosphate 0.01-2% and surplus, pH7.2,105 ℃ of sterilising treatment 15 minutes, according to inoculum size is the ratio inoculation Kluyveromyces fragilis cell of 1%-25%, feeds the sterile air stir culture 10-72 hour;
(2) preparation of Kluyveromyces fragilis bacteria suspension: will cultivate sophisticated Kluyveromyces fragilis cell through the 10000r/min high speed centrifugation, obtain fresh active somatic cells, fresh active somatic cells is washed 1-2 time with sterilized water, be mixed with the cell bacteria suspension with sterilized water then, the weight ratio concentration of cell is 1.0%-40.0%;
(3) the pre-broken wall treatment of enzyme: get above-mentioned bacteria suspension 500mL, add beta-glucanase, cellulase and proteolytic enzyme, cellulase, proteolytic enzyme consumption are respectively the 0.1-150.0u/g wet thallus, beta-glucan enzyme dosage 0.1-150.0u/g wet thallus, pH5.5-7.5,20 ℃-65 ℃ are incubated 1-6 hour;
(4) freeze thawing broken wall: cell suspension after enzyme is handled is taken out, place under-20 ℃ of--50 ℃ of temperature freezingly, again the refrigerated cell is placed 20 ℃ of-75 ℃ of thawings, obtain the frozen-thawed cell suspension;
(5) cell suspension with freeze thawing places the liquid shear shredder assembly to carry out the secondary broken wall, the temperature maintenance of ingress is at 18-22 ℃, the temperature in exit is less than 50 ℃, pressure maintains 30MPa-120MPa, obtain the active lactose enzyme solution of no cell structure, the liquid shear shredder assembly is high pressure homogenizer or ultrasonic cell-break machine;
(6) carry out the centrifugal or filtration treatment of 3500-15000r/min then, obtain the active lactose enzyme solution of no somatic cells fragment, carry out concentrate under reduced pressure at low temperature again to 10-70% concentration, add the sucrose of 5-30% sodium-chlor and 5-30%, promptly obtain neutral liquid Sumylact L.
2, neutral liquid method for producing lactase according to claim 1 is characterized in that:
(1) substratum preparation and yeast cell are cultivated: according to weight ratio is the proportional arrangement substratum of the water of whey powder 6.0%, yeast extract paste 1.0%, peptone 1.5%, ammonium sulfate 1.0%, dipotassium hydrogen phosphate 0.1% and surplus, pH7.2, sterilized 15 minutes for 105 ℃, according to inoculum size is the ratio inoculation Kluyveromyces fragilis cell of 1%-25%, feeds the sterile air stir culture 36 hours;
(2), will cultivate sophisticated Kluyveromyces fragilis cell through the 10000r/min high speed centrifugation, then, somatic cells is washed 1 time with sterilized water, be mixed with the cell bacteria suspension with sterilized water then, cell concn is 5.0%, adds beta-glucanase, cellulase and proteolytic enzyme again, cellulase, proteolytic enzyme consumption are respectively the 10u/g wet thallus, beta-glucan enzyme dosage 50u/g wet thallus, pH6.8,45 ℃ are incubated 2 hours;
(3) will place quick freezing under-20 ℃ of temperature again after the cell suspension taking-up after enzyme is handled, and the refrigerated cell be placed under 40 ℃ of temperature melt again, obtain the frozen-thawed cell suspension;
(4) place the liquid shear shredder assembly to carry out the secondary broken wall frozen-thawed cell suspension again, pressure maintains 80MPa, obtains the active lactose enzyme solution of no cell structure;
(5) carry out the centrifugal or filtration treatment of 3500-15000r/min then, obtain the active lactose enzyme solution of no somatic cells fragment, carry out concentrate under reduced pressure at low temperature to 65% concentration again, add the sucrose of 8% sodium-chlor and 10%, promptly obtain liquid active lactose enzyme.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015132349A1 (en) * | 2014-03-05 | 2015-09-11 | Dsm Ip Assets B.V. | Liquid lactase compositions |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073452B (en) * | 2014-06-13 | 2016-09-28 | 天津科技大学 | The lactic acid bacteria culturing medium of fermenting and producing Lactose enzyme |
| EP3568023B1 (en) | 2017-01-13 | 2021-07-07 | Novozymes A/S | Sterile filtered lactase preparation comprising salt with monovalent cation and preparation thereof |
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2006
- 2006-09-08 CN CNB200610010516XA patent/CN100497613C/en active Active
Non-Patent Citations (4)
| Title |
|---|
| Kluyveromyces fragilis LFS_8611 β－D－半乳糖苷酶的摇瓶培养条件. 刘建福等.无锡轻工业大学学报，第23卷第4期. 2004 |
| Kluyveromyces fragilis LFS_8611 β－D－半乳糖苷酶的摇瓶培养条件. 刘建福等.无锡轻工业大学学报，第23卷第4期. 2004 * |
| 酵母细胞破壁技术研究与应用进展. 杨翠竹等.食品科技，第07期. 2006 |
| 酵母细胞破壁技术研究与应用进展. 杨翠竹等.食品科技，第07期. 2006 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015132349A1 (en) * | 2014-03-05 | 2015-09-11 | Dsm Ip Assets B.V. | Liquid lactase compositions |
| US10201178B2 (en) | 2014-03-05 | 2019-02-12 | Dsm Ip Assets B.V. | Stable liquid lactase compositions |
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