CN1772911A - Process of preparing conjugate linolic acid with lactic acid bacteria - Google Patents
Process of preparing conjugate linolic acid with lactic acid bacteria Download PDFInfo
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Abstract
The present invention relates to biological process of preparing conjugate linolic acid with lactobacillus. Lactobacillus plantarum HSC 235 CCTCC No. M204051 of Lactobacillus can convert linolic acid into conjugate linolic acid by means of isomerization in culture liquid or buffering liquid. The new lactobacillus strain is used alone or together with Propionibacterium shermannii CCIC 10019 to convert linolic acid or linolic acid containing matter into conjugate linolic acid, and the converted conjugate linolic acid is extracted with chloroform, reaching conversion rate up to 81 % and yield of 2.1 mg/ml.
Description
(1) technical field the present invention relates to a kind of new microbial strains that screens, and this new bacterial classification has the ability that linolic acid generates conjugated linolic acid that transforms; The invention still further relates to and utilize this new microorganism to prepare the method for conjugated linolic acid;
(2) background technology conjugated linolic acid (Conjugated linoleic acid, be called for short CLA) be a series of at 9,11 or 10,12 linoleic position and geometrical isomers of carbon with two keys, a large amount of scientific researches prove, that conjugated linolic acid has is antitumor, anti-oxidant, reduce animal and human's body cholesterol and triglyceride level and low density lipoprotein cholesterol, atherosclerosis, raising immunizing power, improve skeleton density, prevent and treat multiple important physiological function such as diabetes.
Before 1997 (2004 the 6th phase P4-6 raise pigs), the synthetic of conjugated linolic acid mainly is to use linolic acid to synthesize by chemical process in the laboratory, but this method synthetic quantity and efficient are all very low, have limited the application of conjugated linolic acid in animal produces.Had now a lot of companies large-scale, specialization can synthesize CLA in the world, but this chemical industry synthetic CLA contains not saponifiable matter of the unknown of 1%～6%, thereby limited its application in food and feed additive.
The linolic acid bio-transformation is that the enzyme of conjugated linolic acid is mainly linoleate isomerase.The bacterium of having reported that following several genus are arranged: Lactobacterium acidophilum, lactobacillus bulgaricus, plant lactobacillus, propionibacterium etc.In recent years, reported that cud bacterium, bacterium acidi propionici, milk-acid bacteria can be converted into linolic acid conjugated linolic acid (food information and technology o. 11th P22 in 2004), yet its CLA output was all lower.Utilize biological process to produce conjugated linolic acid, advantage with the synthetic biological activity CLA of selectivity, and milk-acid bacteria is more convenient aspect cultivation, therefore if can obtain having higher conversion LA is the milk-acid bacteria of CLA ability and is applied to functional dairy product, will have very large application prospect.In the research of reporting with biotransformation method production conjugated linolic acid:
People such as the Zhou Yan of Northeast Agricultural University have studied seed selection of conjugated linolic acid superior strain and fermentation condition (2004 30 volumes of food and fermentation industries the 6th phase P28-31) thereof, with Lactobacillus acidophilus PB1 (Lactobacillus acidophilus) is starting strain, through ultraviolet ray, nitrosoguanidine individual curing and Combined Processing, obtain a strain CLA superior strain U-N-f34, the substratum of employing is MRS substratum, 12% degreasing milk medium.Optimize by the fermentation condition that single-factor and orthogonal test are produced conjugated linolic acid to this bacterial strain, the fermentation condition of optimizing is: 43 ℃ of culture temperature, pH value 7.5, incubation time 48h, under optimal conditions, U-N-f34 can produce conjugated linolic acid and reach 42.05 μ g/mL.
The people such as Zhou Linghua of Southern Yangtze University's lactobacillus-fermented technology and key lab of the food safety the Ministry of Education have carried out the screening and the evaluation (2004 23 volumes of Wuxi Light Industry Univ.'s journal the 5th phase P53-57) of biosynthesizing conjugated linolic acid bacterial classification, from traditional pickles and raw milk, filter out strains of lactic acid bacteria ZS2058 energy biosynthesizing conjugated linolic acid, be accredited as plant lactobacillus (Lactobacillus plantarurn) through the API system.This bacterial strain is converted into conjugated linolic acid with the linolic acid (1.024mg/mL) of massfraction 11.57% in MRS substratum (pH6.5), confirm c9 through gas chromatographic analysis, and t11-18:2 accounts for 75.9%, t10, and c12-18:2 accounts for 24.1%.Its fermentation process:, activate 2 times each 24h continuously in 37 ℃ with the inoculum size access seed culture medium of each bacterial strain with volume fraction 1%; Insert in the seed culture medium with volume fraction 1% inoculum size the bacterial classification after the activation again and add the linolic acid that mass concentration is 0.5mg/mL, cultivate 24h, the centrifugal 10min of 6000r/min collects thalline, with mass concentration 8.5g/L NaCl solution washing, again through centrifugal collection thalline, add the 10mL fermention medium and add the linolic acid of 1.024mg/mL, cultivate under the 24h conditions at 37 ℃, the LA that ZS2058 can transform massfraction 11.57% is CLA (120.56 μ g/mL).Fermented liquid Virahol and normal hexane extraction CLA.
The people such as Zhang Zhongyi of China Agricultural University's Food science and nutrition engineering college have carried out producing screening and product analysis (2004 9 volumes of China Agricultural University's journal the 3rd phase P5-8 of conjugated linolic acid milk-acid bacteria, industrial microorganism 2004 volume 34 the 2nd phase P5-9), from sauerkraut juice, filter out 1 strain CLA generative capacity stronger milk-acid bacteria.The CLA growing amount is 267.5 μ g/mL in the fermented liquid.This strain is a Gram-positive bacillus, the catalase feminine gender, and 45 ℃ of growths are not down grown for 15 ℃ substantially.To the bacterial classification evaluation, based on the situation of utilizing of biochemical test and medium component, be accredited as plant lactobacillus Lactobacillus plantarum through API system and IBIS software system, degree of confidence is 96.1%.Little oxygen condition can improve the output of CLA, and the enzyme that catalysis linolic acid (LA) generates CLA is being subjected to inducing of LA.37 ℃ of cell growth and CLA generate the most favourable.Logarithmic phase is 6h～12h, enters stationary phase behind the 18h.At 14h～22h, the CLA growing amount increases fast, reaches maximum during 24h.The culture of this bacterium has carried out the gas-chromatography separation after extraction, esterification, this bacterial classification synthetic CLA product is c9, t11/t9, c11-CLA and t10, the mixture of c12-CLA.
People such as Lee (Biotechnology Letters, 2003,25:935-938) adopt Lactobacillusreuterizai in the MRS substratum that adds 0.9g/L LA, to cultivate 24h and produce 300mg/L CLA, the CLA isomer of its generation is through being accredited as c9, t11-18:2 (comprises t9, c11-18:2) (massfraction 59%) and c10,2 kinds of isomer of t12-18:2 (massfraction 41%).
Lin (Food Chemistry, 2003,83:27-31) extract the thick enzyme of isomery, and be used for the conversion of CLA from Lactobacterium acidophilum CCRC 14079 bacterial strains.With 25,50 and the thick enzyme reaction of 75mg, condition is 50 ℃ to the 50-75mg linolic acid respectively, 10min, and pH 5.The transformation efficiency of CLA reaches 58%.
Pariza (United states Patent, No.5856149,1999.1.) feed for a long time and get intestinal tissue after linoleic experiment white mouse kills, soak 30 minutes with 50ml sodium phosphate buffer (0.05M, pH 7.0) after, this suspended substance is on MRS and potato agar substratum, 37 ℃, cultivate under anaerobism, the aerobic two kinds of conditions, isolate several bacterium colonies, isolated bacterium colony obtains tens kinds of bacterial strains once more with same culture medium culturing, separation.But the experiment proved that to have only the bacterial strain of turning out under the anaerobic condition in the four strain MRS substratum to have and transform the ability that LA becomes CLA.Wherein, a strain is named as the bacterial strain that grows among the MRS of PYR8, has the ability of the highest conversion CLA.The conformation that transforms the CLA that generates is 9c, the 11t-form.
PYR8 is in the MRS substratum, and behind the cultivation certain hour, the centrifugal cell that obtains changes in the Tris-HCl damping fluid (pH=8.55) of 0.1M under 37 ℃ of conditions, and (15mg/ml1 3-propanediol) cultivates in back 4 ℃ of cold houses to add the LA repertory.The output of discovery CLA increases with the growth of cellular biomass; Intracellular fatty acid content increases with the prolongation of incubation time, cultivates the amount maximum of the cell output CLA that obtains after 36 hours early stage.
To induce the cell transformation that obtained in 36 hours to produce CLA, detect with GC after the esterification, the result shows: the climax appears in the output linear growth since 0 minute CLA in the time of 3 hours.Carry out between 4 hours to 24 hours in reaction, the isomeric forms of CLA changes, and in the time of 3 hours, 9c, 11t-CLA account for 96.23% of total CLA, after this, 9c, the concentration of 11t-CLA reduces and 9t, and the concentration of 11t-CLA raises.But 9c, the concentration of 11c-CLA is constant, and 9t is described, and 11t-CLA is by 9c, and 11t-CLA is transformed.
(Food Chemistry such as Lin, 1999,67:1-5 Lactobacterium acidophilum Lactobacillusacidophilus, L.delbruechii subsp.bulgaricus etc., cultivate after 12 hours under 37 ℃ of conditions in the MRS substratum, change in 12% the degreasing milk medium similarity condition over to and cultivated 24 hours down.Get this culture and change in the substratum, add linolic acid, cultivated 48 hours for 37 ℃.Lipid acid in the nutrient solution being extracted, detects, find to have the CLA of suitable content to generate, is under the condition of 1000 μ g/ml at the linolic acid addition, and the detected level of CLA is 52.0-94.5 μ g/ml.
(Journal of American Oil Chemistry Society such as Kishino, 2002,79:159-163) plant lactobacillus (Lactobacillus plantarum AKU1009a) is cultivated in being added with linoleic MRS substratum and is collected thalline, reacts certain hour under 37 ℃ of anaerobic conditions in containing linoleic reaction solution.Lipid acid extracts in the pair cell liquid, check and analysis after the esterification.Finding to remove in the product has 9c, and 11t-CLA and 9t outside 11t-CLA generates, also have two kinds of intermediate products to generate.But this method is produced CLA higher output, reaches as high as 1.6mg/ml.
Above-mentioned report carries out preliminary research to adopting bacterium to transform linolic acid generation conjugated linolic acid, and groundwork is general process prescription.Key distinction part of the present invention serves as to produce bacterial strain with the bacterial strain that obtains from row filter; and relate to selectivity, protectiveness and the presentation mode of conversion process substrate; transform high-density inducing culture and conversion, and adopt milk-acid bacteria and bacterium acidi propionici associating immobilized cell to transform and produce conjugated linolic acid with bacterium.Shang Weijian has open report.
(3) summary of the invention task of the present invention provides and a kind ofly can effectively transform the new microorganism strains that linolic acid is a conjugated linolic acid; Next provides a kind of method of utilizing this new microbial transformation reaction to generate conjugated linolic acid.
New microorganism strains provided by the invention belongs to the plant lactobacillus (Lactobacillus plantarum HSC 235) of lactobacillus (Lactobacillus), this bacterial strain is that separation screening obtains in the Pickle of Haining, be preserved in Chinese typical culture collection center on July 16th, 2004, be called for short CCTCC, preservation CCTCC No.M 204051.
The new bacterial strain that the present invention's separation screening from the Pickle of Haining obtains, its bacterial characteristics is as follows:
(1) form:
1) thin rod shape, 0.5 micron～0.8 micron of diameter, long 1 micron～5 microns;
2) cultivating the starting stage, cell is elongated rod shape;
3) mobility: do not move;
4) sporulation: do not form spore;
5) gramstaining: the positive;
6) acid resistance: the positive.
(2) growth conditions (30 ℃) on various substratum:
1) MRS Agar Plating: poor growth, bacterium colony is rounded, and is irregular, smooth surface, oyster white;
2) MRS agar slant culture-medium: poor growth, tarnish, oyster white;
3) MRS liquid nutrient medium: well-grown, the liquid muddiness has precipitation.
(3) physiological characteristic:
1) wood sugar :+
2) trehalose :+
3) sucrose :+
4) sorbyl alcohol :+
5) saligenin :+
6) ribose :+
7) rhamnosyl :-
8) raffinose :+
9) close disaccharides :+
10) seminose :+
11) N.F,USP MANNITOL :+
12) maltose :+
13) lactose :+
14) gluconate :+
15) semi-lactosi :+
16) fructose :+
17) polychrom :+
18) cellobiose :+
19) pectinose :+
20) amygdaloside :+
21) lactic acid opticity: DL
22) catalase :-
23) produce the indoles ability :-
24) decompose casein :-
According to above bacterial characteristics, and this bacterial strain HSC235 culture can be defined as the plant lactobacillus Lactobacillusplantarum of lactobacillus (Lactobacillus) based on its situation of utilizing to substrate.
The substratum that is used for new bacterial strain plant lactobacillus (Lactobacillus plantarum HSC235) of the present invention:
(1) this new bacterial strain HSC235 preservation on the MRS substratum, usually used substratum composition contains: carbon source (for example glucose, lactose, whey), nitrogenous source is (as peptone, the white peptone of pancreas, phytone, extractum carnis), and organic nutrient substance (as yeast extract, peptone, the white peptone of pancreas, phytone, extractum carnis, corn steep liquor), inorganic nutrients composition (as phosphoric acid salt, magnesium, potassium, zinc, iron, cobalt and manganese), and as the linolic acid of inductive substance, perhaps contain linoleic other material, perhaps linolic acid analog etc.
(2) culture condition: anaerobism is cultivated, pH4.0 to 7.7,20 ℃ to 40 ℃ of temperature, be preferably in 28 ℃～37 ℃, this bacterial classification was cultivated 1～3 day under anaerobic condition on the MRS substratum that contains linolic acid or linoleic acid material, and the gained cell has and transforms linolic acid or linoleic acid material for gripping linoleic ability altogether.
Another key character of the present invention is a method of utilizing new microbial transformation linolic acid of the present invention to produce conjugated linolic acid, comprising:
(1) preparation fermention medium, each components contents is percent weight in volume, i.e. g/L:
Tryptones: 8-15
Extractum carnis: 10
Yeast extract: 5
Glucose: 20-30
Tween 80: 1-5ml
K
2HPO
4：2
Sodium acetate, anhydrous: 5
MgSO
4·7H
2O：0.2
MnSO
4·H
2O：0.05
Dibasic ammonium citrate: 2
Linolic acid: 8-50
(2) will cultivate 1～2 day in the above-mentioned substratum of bacterial strain CCTCC No.M 204051 accesses, 28 ℃ of culture temperature obtain nutrient solution;
(3) with above-mentioned nutrient solution, or from above-mentioned nutrient solution isolated cells (thalline), or with isolating thalline immobilization, add linolic acid, or be rich in the linoleic acid material, or carry out bioconversion reaction in the solution of linolic acid triester matter, pH value in reaction is controlled at 4.5～7.7;
(4) with above-mentioned conversion fluid, use chloroform extraction.
Linoleic acid material as substrate has very big toxicity to biological catalyst, and therefore, the adding mode of linoleic acid material is wanted may command, both can disposablely add, and also can add in batches, and the linoleic acid material that conversion fluid adds is no more than 50g/L.
The certain pH of control helps the linoleic conversion of substrate in the conversion reaction process, and therefore preferably constantly add sodium hydroxide or in advance damping fluid is added in this system, be 4.5～6.5 with control reaction optimal pH.
The linoleic acid material adopts tensio-active agent such as tween, glycerine to carry out pre-treatment: the linoleic acid material at first with tween, or glycerine mixes by 1: 4 mass ratio, and the emulsification homogeneous.
The linoleic acid material is a linolic acid, or is rich in the linoleic acid material, or linolic acid triester matter.
Be rich in the linoleic acid material, or linolic acid triester matter is Thistle oil, sunflower seed oil.
Bioconversion reaction:
With the nutrient solution after cultivating, with linoleic acid material [linolic acid, or be rich in the linoleic acid material, or linolic acid triester matter] solution be substrate, carry out bioconversion reaction, the linoleic acid material that adds in the reaction solution is 8g/L～50g/L, and temperature of reaction is with 20 ℃～40 ℃, and suitable scope is 28 ℃～37 ℃; Optimum temperuture is reacted pH scope 4.0～7.7 at 37 ℃, and optimal pH is 4.5～6.5, reaction times 24h～72h.The conversion fluid of reaction substrate and acquisition all adopts vapor-phase chromatography and ultraviolet spectrophotometry qualitative and quantitative analysis.
Perhaps, isolated cells from culture (fermented liquid freezes centrifugal through 4 ℃, collect thalline), make suspension with phosphoric acid salt, or carry out bioconversion reaction with the suspension that physiological saline is made as the enzyme source, reaction conditions is: phosphate buffered saline buffer pH7.7,37 ℃ of temperature of reaction, time 24h.
Perhaps, isolated cells from culture with the sodium alginate fixation method, is fixed in mentioned microorganism wherein, gets stable particle and carries out bioconversion reaction.
Perhaps, bacterial strain CCTCC No.M 204051 and propionic acid bacterial strain (Propionibacteriumshermannii, CCIC 10019) are inserted in the above-mentioned substratum jointly, cultivated 1～2 day, 28 ℃ of culture temperature obtain nutrient solution; Utilize nutrient solution or isolated cells (thalline) immobilization from above-mentioned nutrient solution, add linolic acid, perhaps contain linoleic other material, perhaps the linolic acid analog carries out bioconversion reaction, bacterium glue ratio according to 1: 50, be the wet thallus quality and the ratio of 3% sodium alginate soln volume, mix, slowly with the outstanding bacterium liquid 1%CaCl of sodium alginate with 3% sodium alginate soln
2Solution, the immobilized cell particle of formation 2～3mm, immobilization particle was placed in calcium chloride solution 10 hours, so that immobilization particle and calcium chloride solution fully react, made immobilization particle stablize moulding, poured out CaCl
2Solution, 4 ℃ of preservations of adding physiological saline are stand-by.
Propionic acid bacterial strain is commercial the purchase, propionic acid bacterial strain, i.e. and Xie Shi propionibacterium, Propionibacteriumshermannii, CCIC 10019, Chinese industrial microbial strains preservation administrative center, Beijing, CICC.
Grip linoleic extraction process in the reaction product altogether: the mixed solution of adding chloroform and methyl alcohol (chloroform: methyl alcohol=2: 1), fully mixing leaves standstill, centrifuging and taking bottom chloroform layer, and vacuum concentration, nitrogen dries up promptly under 30 ℃ of conditions.
Grip linoleic mensuration process altogether, operating process and experiment condition are pressed following document: (1) Wu Jihua, Qiu Aiyong. Chinese oil 2002,27:65-67; (2) Shigeneobu Kishino, Jun Ogawa.Journal of American Oil Chemistry Society.2002,79:159-163; (3) John AG, Roach M, Mossoba M.Analytica Chmica Acia.2002,465:207-226.), gained result and they are in full accord.Detailed process is as follows:
Add 1M NaOH/CH in the products therefrom
3OH solution is handled 15min in 100 ℃ of water-baths, behind the cooling 15min, adds 4% HCl/CH then under the room temperature
3OH solution, 20min methylates in 60 ℃ of water-baths.Fatty acid methyl ester is with n-hexane extraction.Capillary chromatography (HP6890) condition is as follows: detector: FID, column temperature: 60 ℃, detector and sample introduction temperature: 270 ℃, carrier gas: nitrogen, pressure: 27Kpa, combustion gas: hydrogen, flow: 30cm/sec, sampling volume: 1uL, pillar: silica gel capillary post 60cm * 0.22mm (BP * 70, SGE, Melboure, Australia).Take temperature programming, keep 5min in the time of 60 ℃, the temperature rise rate with 13 ℃/min rises to 170 ℃ with temperature then, keeps this temperature 55min.Press before the post: 100kPa; Injector temperature: 220 ℃; Detector temperature: 230 ℃; Air pressure: 50kPa; Hydrogen pressure: 60kPa; Nitrogen pressure: 400kPa.The determined by ultraviolet spectrophotometry employing UV-2800 of Toshiba ultraviolet spectrophotometer (Zhang Yagang, Fan Li etc. the journal .2002 of Xinjiang petroleum College, 14:59-64), measure its absorption value with normal hexane as reference at the 233nm place.
This shows, utilize microorganism of the present invention can transform the linolic acid preparation and grip linolic acid altogether.
The present invention has following characteristics: with the linoleic acid material is substrate, can use 4 batches continuously through cell high-density inducing culture, milk-acid bacteria and bacterium acidi propionici associating immobilized cell, and activity of conversion remains on 70%, and transformation efficiency reaches 81%, productive rate 2.1mg/mL.
(4) description of drawings
Fig. 1 is the ultraviolet-visible light spectral scan collection of illustrative plates (UV-2800 of Toshiba ultraviolet/visible spectrophotometer) of embodiment 1 product conjugated linolic acid;
Fig. 2 is the gas chromatogram of embodiment 1 product conjugated linolic acid.
(5) embodiment is intended to illustrate rather than limit the scope of the invention below the specific embodiments.[except that specifying, used bacterial classification is the plant lactobacillus (Lactobacillus plantarum HSC235) that new microorganism strains provided by the invention belongs to lactobacillus (Lactobacillus) in the example]
Embodiment 1:
(1) culture medium prescription (with the L metering): Tryptones 8g, extractum carnis 10g, yeast extract 5g, glucose 20g, tween 80 1ml, K
2HPO
42g, sodium acetate, anhydrous 5g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, linolic acid 8g, pH6.2, with distilled water preparation, 121 ℃ of moist heat sterilizations 20 minutes;
(2) preparation CCTCC No.M 204051 strain cells: CCTCC No.M 204051 is inserted above-mentioned containing in the linoleic liquid nutrient medium, shaking culture is 48 hours under 28 ℃ of conditions, also use the phosphate buffered saline buffer (pH7.7) of 0.05M to wash it again with the centrifugal separation transfer nutrient solution, CCTCC No.M 204051 strain cells of repetitive operation to prepare;
(3) bioconversion reaction: linolic acid mixes with the mass ratio of tensio-active agent tween by 1: 4, and the emulsification homogeneous, makes 25% linolic acid mother liquor;
With the phosphate buffered saline buffer (pH7.7) of the above-mentioned cell suspension that makes in 0.05M, add the linolic acid mother liquor then in two batches, the linolic acid amount that adds in the system is no more than 10g/L; Control 37 ℃ of bio-transformation system temperatures simultaneously.Vibration makes it mixing, is convenient to transform, and vibrates 24 hours;
(4) extract: add chloroform extraction, be evaporated to dried being no more than 35 ℃.Referring to Fig. 1 and Fig. 2, with vapor-phase chromatography and ultraviolet spectrophotometry qualitative and quantitative analysis, be accredited as conjugated linolic acid through esterification.
Embodiment 2:
(1) fermentative medium formula following (with the g/L metering): Tryptones 10g, extractum carnis 10g, yeast extract 5g, glucose 25g, tween 80 3ml, bovine serum 2g, K
2HPO
42g, sodium acetate, anhydrous 5g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, linolic acid 12g, pH 6.2, with distilled water preparation, 121 ℃ of moist heat sterilizations 20 minutes;
(2) preparation CCTCC No.M 204051 bacterial strain seed liquor: through MRS slant medium activatory bacterial classification CCTCC No.M204051, insert in the MRS seed culture medium and cultivate, 37 ℃ of culture temperature, shaking speed 120rpm, incubation time 1 day, obtain seed culture fluid, thalline optical density(OD) OD value is 2.0 in the seed culture fluid;
(3) bioconversion reaction: linolic acid mixes with the mass ratio of tensio-active agent glycerine by 1: 4, and the emulsification homogeneous, makes 25% linolic acid mother liquor;
In fermentor tank, liquid amount is equivalent to 87% of fermentor tank cumulative volume, inserts CCTCC No.M 204051 seed liquor after sterilizing by above-mentioned fermentative medium formula preparation 10L fermented liquid, and inoculum size is 10% of a fermented liquid, i.e. the 1L seed liquor.Fermenting process is regulated pH6.0 with sodium hydroxide.Cultivate and begin stream after 8 hours and add glucose (flow acceleration 0.55g/h), carry out high-density culture and the effect of linolic acid inducedvelocity (be added with in the substratum linolic acid be induce), in 24 hours, add glucose, temperature is controlled at 28 ℃, incubation time 24 hours, linoleic add-on is controlled at 12g/L; Temperature to 37 ℃ is adjusted in the back, and stream adds the linolic acid mother liquor and make the linolic acid total concn be controlled at 30g/L, continues to cultivate 20 hours;
(4) extract: add chloroform extraction then.Chloroform layer is evaporated to dried being no more than 35 ℃.Through esterification vapor-phase chromatography and ultraviolet spectrophotometry qualitative and quantitative analysis, conjugated linolic acid transformation efficiency 53%, productive rate 1.4mg/ml.
Embodiment 3:
(1) fermentative medium formula following (with the g/L metering): Tryptones 15g, extractum carnis 10g, yeast extract 5g, glucose 30g, tween 80 5ml, bovine serum 2g, K
2HPO
42g, sodium acetate, anhydrous 5g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, linolic acid 20g, pH 6.2, with distilled water preparation, 121 ℃ of moist heat sterilizations 20 minutes;
(2) preparation CCTCC No.M 204051 bacterial strains and bacterium acidi propionici (Propionibacteriumshermannii) CCIC 1001 strain-combined immobilized cells: CCTCC No.M 204051 and bacterium acidi propionici (Propionibacterium shermannii) CCIC 1001 bacterial strains are inserted the above-mentioned mixed culture in the linoleic liquid nutrient medium that contains together, after cultivating 30 hours under 28 ℃ of conditions, the gained nutrient solution freezes centrifugal at 4 ℃, collect thalline, the thalline of collecting with the washing of aseptic phosphoric acid buffer three times, adopt the sodium alginate immobilization to prepare the associating immobilized cell of lactic-acid bacteria cells and bacterium acidi propionici cell, immobilization process at last:
According to 1: 50 bacterium glue ratio, promptly the ratio of wet thallus quality and 3% sodium alginate soln volume mixed with 3% sodium alginate soln, slowly with the outstanding bacterium liquid 1%CaCl of sodium alginate
2Solution, the immobilized cell particle of formation 2～3mm, immobilization particle was placed in calcium chloride solution 10 hours, so that immobilization particle and calcium chloride solution fully react, made immobilization particle stablize moulding, poured out CaCl
2Solution, 4 ℃ of preservations of adding physiological saline are stand-by, transform in the MRS substratum: the consumption 15% of immobilized cell (ratio of g/ volume L), pH6.5,30 ℃, reaction times 72h;
(3) bioconversion reaction: linolic acid mixes with the mass ratio of tensio-active agent glycerine by 1: 4, and the emulsification homogeneous, makes 25% linolic acid mother liquor;
Stream adds glucose and carries out high-density culture (flow acceleration 0.75g/h), linolic acid is induced (linolic acid concentration is 20g/L), temperature is controlled at 30 ℃, add in 20 hours time,, be suspended in the MRS substratum after the dark washing of stroke-physiological saline solution through the immobilized cell pearl behind the high-density inducing culture gained, the total add-on of linolic acid is controlled at 50g/L, 30 ℃, reaction times 72h;
(4) extract: add chloroform extraction then, chloroform layer is evaporated to driedly being no more than 35 ℃, uses the ultraviolet spectrophotometry qualitative and quantitative analysis after esterification, transformation efficiency 81%, productive rate 2.1mg/ml.Immobilized cell can use 4 batches continuously, and activity of conversion remains on more than 50%.
Embodiment 4:
(1) fermentative medium formula following (with the g/L metering): Tryptones 15g, extractum carnis 10g, yeast extract 5g, glucose 30g, tween 80 5ml, bovine serum 2g, K
2HPO
42g, sodium acetate, anhydrous 5g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, linolic acid 15g, pH 6.2, with distilled water preparation, 121 ℃ of moist heat sterilizations 20 minutes;
(2) preparation CCTCC No.M 204051 bacterial strain immobilized cells: CCTCC No.M204051 bacterial strain is inserted above-mentioned containing in the linoleic liquid nutrient medium, 28 ℃ cultivate 30 hours after, the gained nutrient solution freezes centrifugal at 4 ℃, collect thalline, the thalline of collecting with the washing of aseptic phosphoric acid buffer three times, adopt the sodium alginate immobilization to obtain the milk-acid bacteria immobilized cell at last, immobilization process together
Embodiment 3;
(3) bioconversion reaction: linolic acid mixes with the mass ratio of tensio-active agent glycerine by 1: 4, and the emulsification homogeneous, makes 25% linolic acid mother liquor;
Stream adds glucose and carries out high-density culture (flow acceleration 0.45g/h), the linolic acid mother liquor is induced (linolic acid concentration is 20g/L), temperature is controlled at 28 ℃, 20 hours time, temperature to 37 ℃ is adjusted in the back, and divides three streams to add linolic acid, and the total add-on of linolic acid is controlled at 50g/L, 37 ℃, 24 hours reaction times;
(4) extract: add chloroform extraction then, chloroform layer is evaporated to dried being no more than 35 ℃, after esterification, use the ultraviolet spectrophotometry qualitative and quantitative analysis, transformation efficiency 57%, productive rate 1.63mg/ml, immobilized cell behind high-density inducing culture gained can use 5 batches continuously, and activity of conversion remains on 52%.
Embodiment 5:
(1) culture medium prescription (with the L metering): Tryptones 8g, extractum carnis 10g, yeast extract 5g, glucose 20g, tween 80 2ml, K
2HPO
42g, sodium acetate, anhydrous 5g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, linolic acid 12g, pH6.2, with distilled water preparation, 121 ℃ of moist heat sterilizations 20 minutes;
(2) preparation CCTCC No.M 204051 strain cells: CCTCC No.M 204051 is inserted in the above-mentioned liquid nutrient medium that contains the linoleic acid material, shaking culture is 48 hours under 28 ℃ of conditions, also use the phosphate buffered saline buffer (pH7.7) of 0.05M to wash it again with the centrifugal separation transfer nutrient solution, CCTCC No.M 204051 strain cells of repetitive operation to prepare;
(3) bioconversion reaction: with the phosphate buffered saline buffer (pH7.7) of the above-mentioned cell suspension that makes in 0.05M, add Thistle oil or rapeseed oil then, controlled temperature is 37 ℃ simultaneously, and makes Thistle oil in the system, perhaps the add-on of rapeseed oil is 12g/L, reacts 48 hours;
(4) extract: add chloroform extraction then.Be no more than 35 ℃ be evaporated to dried, through esterification with vapor-phase chromatography and ultraviolet spectrophotometry analysis, conjugated linolic acid transformation efficiency 6.3%, productive rate 0.756mg/ml.
Claims (8)
1. method of utilizing milk-acid bacteria to prepare conjugated linolic acid, it is characterized in that described milk-acid bacteria is the plant breast Bacterium lacticum (Lactobacillus plantarum HSC235) of lactobacillus (Lactobacillus), be CCTCC No.M204051, this bacterial strain has the linolic acid of conversion for gripping linoleic ability altogether, described plant lactobacillus (Lactobacillus plantarum HSC 235) is in the substratum that contains linoleic acid material, carbon source, nitrogenous source, inorganic salt, carry out bio-transformation, linolic acid is gripped in purification altogether, comprising:
(1) preparation fermention medium, each components contents is percent weight in volume, i.e. g/L:
Tryptones: 8-15
Extractum carnis: 10
Yeast extract: 5
Glucose: 20-30
Tween 80: 1-5ml
K
2HPO
4：2
Sodium acetate, anhydrous: 5
MgSO
4·7H
2O：0.2
MnSO
4·H
2O：0.05
Dibasic ammonium citrate: 2
Linolic acid: 8-50
(2) with bacterial strain CCTCC No.M204051, or bacterial strain CCTCC No.M204051 and bacterium acidi propionici (Propionibacterium shermannii) CCIC 10019 insert together in the above-mentioned substratum and cultivate 24h～48h, and 28 ℃ of culture temperature obtain nutrient solution;
(3) utilize above-mentioned nutrient solution, or utilize isolated cells from above-mentioned nutrient solution, maybe will isolated cells immobilization from above-mentioned nutrient solution, add in the solution of linoleic acid material and carry out bioconversion reaction, pH value in reaction is controlled at 4.5～6.5;
(4) with above-mentioned conversion fluid, use chloroform extraction.
2. method according to claim 1, it is characterized in that in the step (3), directly utilize above-mentioned nutrient solution, solution with the linoleic acid material is substrate, carry out bioconversion reaction, the linoleic acid material that adds in the reaction solution is 8g/L～50g/L, and the condition of bioconversion reaction is: 28 ℃～37 ℃ of temperature of reaction, pH value in reaction 4.5～7.7, reaction times 24h～72h.
3. method according to claim 1, it is characterized in that in the step (3), utilization is isolated cells from above-mentioned nutrient solution, be the thalline that nutrient solution is collected through frozen centrifugation, make suspension, add the linoleic acid material as the enzyme source and carry out bioconversion reaction with phosphoric acid salt, linoleic acid material add-on is 8g/L～50g/L, reaction conditions is: phosphate buffered saline buffer pH7.7,37 ℃ of temperature of reaction, time 24h.
4. method according to claim 1, it is characterized in that in the step (3), isolated cell from above-mentioned culture, promptly nutrient solution is through the thalline of frozen centrifugation collection, with the sodium alginate fixation method, thalline is fixed in wherein, gets stable particle, carry out bioconversion reaction as the enzyme source, immobilization process: according to 1: 50 bacterium glue ratio, be the wet thallus quality and the ratio of 3% sodium alginate soln volume, mix, slowly with the outstanding bacterium liquid 1%CaCl of sodium alginate with 3% sodium alginate soln
2Solution, the immobilized cell particle of formation 2～3mm, immobilization particle was placed in calcium chloride solution 10 hours, so that immobilization particle and calcium chloride solution fully react, made immobilization particle stablize moulding, poured out CaCl
2Solution, 4 ℃ of preservations of adding physiological saline are stand-by, and transform in the MRS substratum: the consumption of immobilized cell is generally controlled the ratio 15% of weight g/ volume L, pH6.5; Reaction conditions is: stream adds glucose and carries out high-density culture, the linoleic acid material is induced, 28～37 ℃ of temperature controls, in 20 hours time, added,, after the dark washing of stroke-physiological saline solution, be suspended in the MRS substratum through the immobilized cell pearl behind the high-density inducing culture gained, add the linoleic acid material, bioconversion reaction time 72h, total add-on is controlled at 50g/L;
5. method according to claim 1, the method that it is characterized in that adding the linoleic acid material in the step (3) is for adding in batches, and the ratio that makes the linolic acid total concn that joins in the reaction solution be no more than weight g/ volume L is 50g/L.
6. according to the described method of one of claim 2～5, it is characterized in that the linoleic acid material adopts tensio-active agent such as tween, glycerine to carry out pre-treatment: the linoleic acid material at first with tween, or glycerine mixes by 1: 50 mass ratio, and the emulsification homogeneous.
7. according to the described method of one of claim 2～5, it is characterized in that the linoleic acid material is a linolic acid, or be rich in the linoleic acid material, or linolic acid triester matter, be rich in the linoleic acid material, or linolic acid triester matter is Thistle oil, or sunflower seed oil.
8. method according to claim 6 is characterized in that the linoleic acid material is a linolic acid, or is rich in the linoleic acid material, or linolic acid triester matter, is rich in the linoleic acid material, or linolic acid triester matter is Thistle oil, or sunflower seed oil.
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