CN104561148B - The method of the fixed permeability lactic acid bacteria conversion rape oil heel production CLA of absorption - Google Patents
The method of the fixed permeability lactic acid bacteria conversion rape oil heel production CLA of absorption Download PDFInfo
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- CN104561148B CN104561148B CN201410728157.6A CN201410728157A CN104561148B CN 104561148 B CN104561148 B CN 104561148B CN 201410728157 A CN201410728157 A CN 201410728157A CN 104561148 B CN104561148 B CN 104561148B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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Abstract
A kind of method of attached fixed permeability lactic acid bacteria conversion rape oil heel production CLA, comprises the following steps：（1）Adsorb the pretreatment of immobilization carrier；（2）Prepare permeability lactic acid bacteria；（3）The fixed permeability lactic acid bacteria of absorption；（4）The emulsification of rape oil heel, enzymolysis processing；（5）The fixed permeability lactic acid bacteria conversion rape oil heel emulsification of absorption, enzymolysis liquid.The recycling of the achievable agricultural product castoff rape oil heel of the present invention and waste ceramic, it is low with cost of material, transformed cells density is high, conversion ratio is up to 40% 60%, relatively free permeability lactic acid bacteria conversion is high by 20% or so, and conversion process is not easy microbiological contamination, and interfering material is few, the advantages that product is easily separated, and thalline can reuse.
Description
Technical field
The invention belongs to agricultural product castoff to utilize technical field.
Background technology
CLA（Conjugated linoleic acid, CLA）Be one kind mainly from ruminant animal fat and ox
The natural active matter found in dairy products, it is a kind of octadecadienoic acid containing conjugated double bond（Linoleic acid）Isomers mixes
Thing, particularly c-9, t-11 CLA and t-10, c-12 CLA, are considered as most bioactivity, wherein c-9, and t-11 CLA are
The conjugated linoleic acid isomers of phospholipid layer can be uniquely absorbed into by zooblast, and t-10, c-12 CLA are in anticancer, anti-congee arteries and veins
Played an important role in terms of dynamic sample hardening, regulation immunologic function.Generally use sunflower oil, safflower seed oil etc. are expensive at present
Raw material is that substrate produces CLA.Rape oil is one of China's main edible oil, total output account for the 1/3 of vegetable oil total output with
On, its caused accessory substance --- rape oil heel is cheap and easy to get, and containing 20% or so linoleic acid, using rape oil heel
It is not only cost-effective as generation CLA substrate, can also solve the problem of environmental pollution that Rapeseed Oil Residual material strip comes.
The linoleate isomerase that lactic acid bacteria is produced can realize biological synthesis process conversion linoleic acid generation CLA.Traditional biological
There is fermentation method cell to be extremely difficult to high density, and growth cycle length, conversion ratio is low, easy microbiological contamination, and product is complicated, it is difficult to which separation carries
Take, the problems such as thalline can not reuse.Lactic acid bacteria permeabilized treatment can strengthen cell membrane and membrane passage, weaken
Shielding action of the cell membrane to isomerase on cell membrane, makes linoleate isomerase be more easy to combine with linoleic acid, improves CLA yield.
Then short with the transformation period using adsorption of immobilization permeability lactic acid bacteria direct bioconversion, it is convenient that conversion medium liquid is prepared, reaction
Interfering material is few in system, is not easy microbiological contamination, and product is more single, and cell density is high, and thalline can reuse, and product can be easily separated
The advantages that, so as to reach high efficiency, inexpensive production purpose.
The content of the invention
Permeability lactic acid bacteria is fixed by the use of discarded ceramics as carrier adsorption object of the present invention is to provide a kind of,
Realize that agricultural product castoff rape oil heel Efficient Conversion produces CLA（CLA）Method.
The present invention is achieved by the following technical solutions.
The present invention comprises the following steps.
（1）Adsorb the pretreatment of immobilization carrier：Waste ceramic is broken into pieces, obtains a diameter of 4-6 mm porous ceramics
Grain, after clear water rinses, 24-48 h first are soaked with 0.1 mol/L HCl solutions, then 24- is soaked with 0.1 mol/L NaOH solutions
48 h, finally it is washed with water to neutrality, dries, sterilizing, obtains the fixed cellular ceramic substrate of sterile absorption.
（2）Prepare permeability lactic acid bacteria：From picking 1-3 ring lactic acid bacterias on fresh slant medium, 50-200 mL are inoculated into
In MRS culture mediums, 35-37 DEG C of Anaerobic culturel 20-26 h, as lactic acid bacteria bacteria suspension, bacteria suspension is taken to continue in MRS culture mediums
Expand culture.By bacteria suspension, 4 DEG C are collected by centrifugation somatic cells, somatic cells are placed at -20 DEG C after freezing and taken out, at room temperature
Melt, multigelation 2-4 times, produce permeability lactic acid bacteria.
（3）The fixed permeability lactic acid bacteria of absorption：By step（2）Permeability lactic acid bacteria add contain step（1）Sterile suction
In the reaction tube of attached fixed cellular ceramic substrate, sterile saline submergence carrier is then added, adds 0.02%-0.05%'s
The starch of alum and 0.3%-0.6%, permeability lactobacillus cell concentration reach 0.3-0.6 g/mL, inhaled under the conditions of 4 DEG C
After attached fixed 30-120 min, supernatant is removed in 4 DEG C of centrifugations, produces the fixed permeability lactic acid bacteria of absorption.
（4）The emulsification of rape oil heel, enzymolysis processing：By rape oil heel, Tween 80, Span-80 according to 1:（0.4-
0.9）:（0.2-0.6）Ratio mixes, and 45-80 DEG C of water-bath 5-15min, adds appropriate amount of deionized water to rape oil heel final concentration
For 80-100 mg/mL, it is well mixed with blender, 110-121 DEG C of sterilizing 20-30 min, as rape oil heel emulsion；Take
Rape oil heel emulsion, adds the citric acid-sodium citrate buffer solution of pH value 8.0, and addition Mucor javanicus lipase powder is dense to end
Spend for 200-350 U/mL, 40-60 DEG C, 120-150 rpm enzymolysis 30-50 min, produce emulsification, the enzymolysis liquid of rape oil heel.
（5）The fixed permeability lactic acid bacteria conversion rape oil heel emulsification of absorption, enzymolysis liquid：By step（4）Rape oil heel
The 0.1 mol/L citric acid-sodium citrate buffer solutions that emulsification, enzymolysis liquid and pH are 6.0-6.8 add step（3）It is solid containing absorption
Determine the reaction tube of permeability lactic acid bacteria so that rape oil heel final concentration of 60-80 mg/mL, 35-38 DEG C of conversion 19-22 h, from
After heart separation, supernatant is conversion fluid, is extracted the CLA in conversion fluid with n-hexane.The fixed permeability lactic acid of absorption
Bacterium is repeatable to be utilized.
The recycling of the achievable agricultural product castoff rape oil heel of the present invention and waste ceramic, has cost of material
Low, transformed cells density is high, and transformation efficiency is high（Conversion ratio is high up to 40%-60%, the conversion of relatively free permeability lactic acid bacteria
20% or so）, the advantages that conversion process is not easy microbiological contamination, and interfering material is few, and product is easily separated, and thalline can reuse.
Brief description of the drawings
Fig. 1 is the scanning electron microscopic picture of the fixed lactobacillus acidophilus of porous ceramic surface absorption in the embodiment of the present invention 1.
Embodiment
The present invention is elaborated by way of example and in conjunction with the accompanying drawings.
Embodiment 1.
（1）Adsorb the pretreatment of immobilization carrier：Waste ceramic is broken into pieces, obtains a diameter of 4-6 mm porous ceramics
Grain, after clear water rinses, 24 h first are soaked with 0.1 mol/L HCl solutions, then 24 h are soaked with 0.1 mol/L NaOH solutions, most
After be washed with water to neutrality, dry, sterilizing, obtain the fixed cellular ceramic substrate of sterile absorption.
（2）Prepare permeability lactic acid bacteria：From the ring lactobacillus acidophilus CGMCC1.1854 of picking on fresh slant medium 3, connect
Kind is into 150 mL MRS culture mediums, 37 DEG C of h of Anaerobic culturel 20, as lactic acid bacteria bacteria suspension, takes bacteria suspension to continue to train in MRS
Support and expand culture in base.By 4 DEG C of bacteria suspension, 5000 rpm centrifuge 20 min, remove supernatant, somatic cells are placed in into -20 DEG C
Take out after lower freezing, melt at room temperature, multigelation 2 times, produce permeability lactic acid bacteria.
（3）The fixed permeability lactic acid bacteria of absorption：By step（2）Permeability lactic acid bacteria add contain step（1）Sterile suction
In the reaction tube of attached fixed cellular ceramic substrate, sterile saline submergence carrier is then added, adds 0.03% aluminum sulfate
Potassium and 0.5% starch, permeability lactobacillus cell concentration reach 0.4 g/mL, are put into after the absorption of 4 DEG C of refrigerators fixes 60 min,
4000 rpm, 4 DEG C of 10 min of centrifugation, remove supernatant, produce the fixed permeability lactic acid bacteria of absorption, and porous ceramic surface absorption is solid
The scanning electron microscopic picture for determining lactobacillus acidophilus is shown in accompanying drawing 1.
（4）The emulsification of rape oil heel, enzymolysis processing：By rape oil heel, Tween 80, Span-80 according to 1:0.8: 0.4
Ratio mixes, and 60 DEG C of min of water-bath 5, adds appropriate amount of deionized water to final concentration of 80 mg/mL of rape oil heel, uses blender
It is well mixed, 121 DEG C of 30 min of sterilizing, as rape oil heel emulsion；Rape oil heel emulsion is taken, adds the lemon of pH value 8.0
Lemon acid-sodium citrate buffer solution, addition Mucor javanicus lipase powder to final concentration of 300 U/mL, 40 DEG C, 150 rpm enzymolysis 45
Min, produce emulsification, the enzymolysis liquid of rape oil heel.
（5）The fixed permeability lactic acid bacteria conversion rape oil heel emulsification of absorption, enzymolysis liquid：By step（4）Rape oil heel
The 0.1 mol/L citric acid-sodium citrate buffer solutions that emulsification, enzymolysis liquid and pH are 6.5 add step（3）It is fixed saturating containing absorption
Property lactic acid bacteria reaction tube so that final concentration of 75 mg/mL of rape oil heel, 37 DEG C conversion 20 h, after centrifugation, supernatant
Liquid is conversion fluid, and n-hexane extracts the CLA in conversion fluid.The fixed permeability lactic acid bacteria of absorption is repeatable to be utilized.Through
Detection and analysis, it is 1885 μ g/mL to adsorb CLA contents in conversion fluid obtained by fixed permeability lactobacillus acidophilus conversion, conversion ratio
Reach 60%, and CLA contents are 1478 μ g/mL in conversion fluid obtained by free permeability lactobacillus acidophilus conversion, conversion ratio reaches
To 47%.
Embodiment 2.
（1）Adsorb the pretreatment of immobilization carrier：Waste ceramic is broken into pieces, obtains a diameter of 4-6 mm porous ceramics
Grain, after clear water rinses, 36 h first are soaked with 0.1 mol/L HCl solutions, then 24 h are soaked with 0.1 mol/L NaOH solutions, most
After be washed with water to neutrality, dry, sterilizing, obtain the fixed cellular ceramic substrate of sterile absorption.
（2）Prepare permeability lactic acid bacteria：From the ring lactobacillus acidophilus CGMCC1.1854 of picking on fresh slant medium 2, connect
Kind is into 100 mL MRS culture mediums, 35 DEG C of h of Anaerobic culturel 24, as lactic acid bacteria bacteria suspension, takes bacteria suspension to continue to train in MRS
Support and expand culture in base.By 4 DEG C of bacteria suspension, 5000 rpm centrifuge 20 min, remove supernatant, somatic cells are placed in into -20 DEG C
Take out after lower freezing, melt at room temperature, multigelation 3 times, produce permeability lactic acid bacteria.
（3）The fixed permeability lactic acid bacteria of absorption：By step（2）Permeability lactic acid bacteria add contain step（1）Sterile suction
In the reaction tube of attached fixed cellular ceramic substrate, sterile saline submergence carrier is then added, adds 0.05% aluminum sulfate
Potassium and 0.6% starch, permeability lactobacillus cell concentration reach 0.4 g/mL, are put into after the absorption of 4 DEG C of refrigerators fixes 60 min,
4000 rpm, 4 DEG C of 10 min of centrifugation, remove supernatant, produce the fixed permeability lactic acid bacteria of absorption.
（4）The emulsification of rape oil heel, enzymolysis processing：By rape oil heel, Tween 80, Span-80 according to 1:0.4: 0.6
Ratio mixes, and 80 DEG C of min of water-bath 15, adds appropriate amount of deionized water to final concentration of 90 mg/mL of rape oil heel, uses blender
It is well mixed, 115 DEG C of 30 min of sterilizing, as rape oil heel emulsion；Rape oil heel emulsion is taken, adds the lemon of pH value 8.0
Lemon acid-sodium citrate buffer solution, addition Mucor javanicus lipase powder to final concentration of 240 U/mL, 50 DEG C, 120 rpm enzymolysis 30
Min, produce emulsification, the enzymolysis liquid of rape oil heel.
（5）The fixed permeability lactic acid bacteria conversion rape oil heel emulsification of absorption, enzymolysis liquid：By step（4）Rape oil heel
The 0.1 mol/L citric acid-sodium citrate buffer solutions that emulsification, enzymolysis liquid and pH are 6.2 add step（3）It is fixed saturating containing absorption
Property lactic acid bacteria reaction tube so that final concentration of 75 mg/mL of rape oil heel, 35 DEG C conversion 22 h, after centrifugation, supernatant
Liquid is conversion fluid, is extracted the CLA in conversion fluid with n-hexane.The fixed permeability lactic acid bacteria of absorption is repeatable to be utilized.
Analyze after testing, it is 1762 μ g/mL to adsorb CLA contents in conversion fluid obtained by fixed permeability lactobacillus acidophilus conversion, conversion
Rate reaches 56%, and CLA contents are 1521 μ g/mL in conversion fluid obtained by free permeability lactobacillus acidophilus conversion, conversion ratio
Reach 49%.
Claims (1)
1. a kind of method for adsorbing fixed permeability lactic acid bacteria conversion rape oil heel production CLA, it is characterized in that including with
Lower step：
（1）Adsorb the pretreatment of immobilization carrier：Waste ceramic is broken into pieces, obtains a diameter of 4-6 mm porous ceramic particles, clearly
After water rinses, 24-48 h first are soaked with 0.1 mol/L HCl solutions, then 24-48 h are soaked with 0.1 mol/L NaOH solutions,
Finally it is washed with water to neutrality, dries, sterilizing, obtains the fixed cellular ceramic substrate of sterile absorption；
（2）Prepare permeability lactic acid bacteria：From picking 1-3 ring lactic acid bacterias on fresh slant medium, 50-200 mL MRS are inoculated into
In culture medium, 35-37 DEG C of Anaerobic culturel 20-26 h, as lactic acid bacteria bacteria suspension, bacteria suspension is taken to continue to expand in MRS culture mediums
Big culture；By bacteria suspension, 4 DEG C are collected by centrifugation somatic cells, somatic cells are placed at -20 DEG C after freezing and taken out, melted at room temperature
Change, multigelation 2-4 times, produce permeability lactic acid bacteria；
（3）The fixed permeability lactic acid bacteria of absorption：By step（2）Permeability lactic acid bacteria add contain step（1）Sterile absorption consolidate
In the reaction tube for determining cellular ceramic substrate, sterile saline submergence carrier is then added, adds 0.02%-0.05% sulfuric acid
The starch of aluminium potassium and 0.3%-0.6%, permeability lactobacillus cell concentration reach 0.3-0.6 g/mL, adsorbed under the conditions of 4 DEG C solid
After determining 30-120 min, supernatant is removed in 4 DEG C of centrifugations, produces the fixed permeability lactic acid bacteria of absorption；
（4）The emulsification of rape oil heel, enzymolysis processing：By rape oil heel, Tween 80, Span-80 according to 1:（0.4-0.9）:
（0.2-0.6）Ratio mixes, and 45-80 DEG C of water-bath 5-15min, adds appropriate amount of deionized water to the final concentration of 80- of rape oil heel
100 mg/mL, are well mixed with blender, 110-121 DEG C of sterilizing 20-30 min, as rape oil heel emulsion；Take Rapeseed Oil Residual
Expect emulsion, add the citric acid-sodium citrate buffer solution of pH value 8.0, addition Mucor javanicus lipase powder is to final concentration of
200-350 U/mL, 40-60 DEG C, 120-150 rpm digest 30-50 min, produce emulsification, the enzymolysis liquid of rape oil heel；
（5）The fixed permeability lactic acid bacteria conversion rape oil heel emulsification of absorption, enzymolysis liquid：By step（4）Rape oil heel emulsification,
Enzymolysis liquid and the 0.1 mol/L citric acid-sodium citrate buffer solutions that pH is 6.0-6.8 add step（3）It is fixed saturating containing absorption
Property lactic acid bacteria reaction tube so that the final concentration of 60-80 mg/mL of rape oil heel, 35-38 DEG C conversion 19-22 h, centrifugation point
From rear, supernatant is conversion fluid, is extracted the CLA in conversion fluid with n-hexane.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1772911A (en) * | 2005-11-07 | 2006-05-17 | 浙江工商大学 | Process of preparing conjugate linolic acid with lactic acid bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1772911A (en) * | 2005-11-07 | 2006-05-17 | 浙江工商大学 | Process of preparing conjugate linolic acid with lactic acid bacteria |
Non-Patent Citations (2)
| Title |
|---|
| 嗜酸乳杆菌静息细胞转化菜籽油生成共轭亚油酸的研究;李垚;《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》;20140215(第02期);第50页第6段至第51页第1段及第58页第1段至59页第2段 * |
| 无机-有机高分子复合絮凝剂研究与应用;周春琼等;《化工进展》;20041231;第23卷(第12期);全文 * |
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