CN104388482A - Method for producing conjugated linoleic acid by utilizing resting lactic acid bacteria technology to convert rapeseed oil sediment - Google Patents

Abstract

The invention relates to a method for producing conjugated linoleic acid by utilizing a resting lactic acid bacteria technology to convert rapeseed oil sediment. The method comprises the following steps: extracting oil, emulsifying, performing enzymolysis, preparing resting lactic acid bacteria, converting the rapeseed oil sediment by the resting lactic acid bacteria to extract oil enzymatic hydrolysate to generate conjugated linoleic acid, extracting the conjugated linoleic acid and the like. By adopting the method provided by the invention, resource utilization of waste of agricultural products, namely the rapeseed oil sediment, can be realized, and the method has the advantages of low raw material cost, high density of transformed cells, high transformation rate of 20%-40%, effect of being less prone to bacterial infection in the transformation process, few interfering substances, easiness in separation of a product and the like.

Description

A method for producing conjugated linoleic acid by transforming rapeseed oil residues using resting lactic acid bacteria technology

technical field

The invention relates to a method for producing conjugated linoleic acid by using the vegetable oil leftovers of agricultural product waste as raw materials and adopting static lactic acid bacteria technology.

Background technique

At present, the preparation of conjugated linoleic acid (CLA) mostly adopts chemical method. This method uses safflower oil or sunflower oil with a price of 30,000/T as raw material, and adopts high temperature, high pressure, and alkali method for isomerization. Isomerization of linoleic acid in oil to generate conjugated linoleic acid has the disadvantages of high cost, and the t, t isomers produced during synthesis are harmful to the human body. The production of CLA by microbial cell fermentation uses edible oil as the raw material for production, and microorganisms (such as lactic acid bacteria, eubacteria, butyric vibrio, etc.) It is difficult to achieve high-density transformation, long growth cycle, easy to infect bacteria, complex products and other problems. the

The technology of resting cell conversion to produce CLA has the following advantages: (1) The cells are in a dormant state and can be used as a natural dialysis bag for linoleic acid isomerase, eliminating the tedious process and high cost of enzyme extraction and purification, and the product CLA is free The form accumulates outside the cell and is easy to extract; (2) The generated reaction products are c9,t11-CLA and t10,c12-CLA with physiological activity; (3) The reaction only needs to be carried out under the appropriate medium system conditions, and the conversion products Single, CLA can accumulate at a high concentration; (4) Resting cells can reach a very high density, which is more than ten times higher than the cell density of the fermentation method.

my country is the largest rapeseed producer in the world, and its annual output has already exceeded 10 million tons. During the processing of rapeseed oil, the annual discharge of oil waste is as high as 200,000 tons. Oil feet are very easy to ferment and deteriorate, rancid and smelly, and are an important source of environmental pollution in my country's urban oil factories. Vegetable oil leftovers contain 25%-30% neutral oil, 25%-35% phospholipids, and the rest are proteins, amino acids, carbohydrates such as sugar, mineral elements and pigments, so the utilization and development of oily feet has become an environmental management and the research hotspots of biological resources reuse. At present, vegetable oil leftovers are mainly used to prepare biodiesel, feed, agricultural fertilizers, etc., but it is extremely rare to extract neutral fatty acids from them and further process them to obtain high value-added products CLA.

Contents of the invention

The object of the present invention is to provide a method for producing conjugated linoleic acid (CLA) by utilizing resting lactic acid bacteria to realize the transformation of agricultural product waste and vegetable oil offal.

The present invention is achieved through the following technical solutions.

The process steps of the present invention include.

(1) Oil extraction: Weigh the vegetable oil scraps into a beaker, place them in a water bath at 60-90°C and heat them for 20-40 min with stirring, adding a small amount of slightly boiling distilled water during the process; centrifuge the heat-treated vegetable oil scraps at 4000-6000 rpm 10-30 min, after centrifugation, the upper layer is an oil layer (neutral oil), the middle layer is a water layer, and the lower layer is impurity solids such as phospholipids, and the upper layer oil is poured out, which is the neutral oil extracted from the vegetable oil residue.

(2) Emulsification: Mix the neutral oil obtained in step (1) with Tween 80 at a ratio of 1:0.8-1.7, add an appropriate amount of distilled water, and perform ultrasonic emulsification treatment (500 W, ultrasonic 10 s, intermittent 5 s, action 10 -20 times), sterilized at 110-121°C for 20-30 min to obtain oil emulsion.

(3) Enzymatic hydrolysis: Take the oil emulsion in step (2) into a conical flask, add citric acid-sodium citrate buffer solution with a pH value of 7.0, and add Aspergillus niger lipase powder to a final concentration of 150-270 U /mL, 40-45°C, 120-150 rpm for 30-50 min to obtain an enzymatic hydrolyzate of oil.

(4) Preparation of resting lactic acid bacteria: Pick lactic acid bacteria from fresh slant culture medium, inoculate them into MRS medium, and culture them anaerobically at 37°C for 20-26 hours to obtain seed liquid. The seed solution was inoculated into MRS medium supplemented with linoleic acid emulsion at an inoculum amount of 3%-5% to induce proliferation, and cultured at 37°C for 20 hours; the culture solution was centrifuged at 4000-6000 rpm for 15 hours at 4°C. -30 min, remove the supernatant, wash the bottom cells with sterile saline for 2-3 times, collect by centrifugation, and prepare a suspension of resting lactic acid bacteria.

(5) Resting lactic acid bacteria transform vegetable oil scraps to extract oil enzymatic hydrolysis solution to generate CLA: take the oil enzymatic hydrolysis solution in step (3) into the reaction tube, add the resting lactic acid bacteria suspension in step (4), and add an appropriate amount of 0.1 mol /L of citric acid-sodium citrate buffer solution, so that the final concentration of the extracted oil is 70-80 mg/mL, 32-36 ° C, 90-110 rpm for 22-26 h, to obtain the CLA conversion solution.

(6) Extraction of CLA: Take the CLA conversion solution in step (5) and mix it with n-hexane at a ratio of 1:2-5, mix quickly for 4-6 minutes, centrifuge at 3800-5000 rpm for 4-6 minutes, and divide into three layers (fat phase, emulsified layer and water phase), remove the lowermost water phase, then add an appropriate amount of distilled water, mix for 4-6 min, wash the water-soluble substances, remove the lower water phase by centrifugal layering, pass the fat phase layer through anhydrous sulfuric acid Sodium filtration, rotary evaporation to remove n-hexane to obtain CLA.

The invention can realize resource utilization of agricultural product waste vegetable oil leftovers, has the advantages of low cost of raw materials, high density of transformed cells, high transformation efficiency (the transformation rate can reach 20%-40%), the transformation process is not easily contaminated with bacteria, and there are few interfering substances. The product is easy to separate and so on.

Description of drawings

Fig. 1 is the ultraviolet spectrum scanning figure of the lactic acid bacteria conversion extract and the extracted oil in the embodiment of the present invention.

Detailed ways

The present invention will be described in detail below through embodiments and in conjunction with the accompanying drawings.

Embodiment: implement by following technological process.

(1) Extract neutral fatty acid from vegetable oil residues: Weigh 20 g of vegetable oil residues into a 200 mL beaker, place in a water bath at 85°C, stir and heat for 30 minutes, during which a small amount of slightly boiling distilled water is added, and after 30 minutes , transfer the vegetable oil scraps into a 50 mL centrifuge tube, centrifuge at 5000 rpm for 30 min, take it out, you can see that the content of the centrifuge tube is divided into 3 layers, the upper layer is the oil layer (neutral oil), the middle layer is the water layer, and the lower layer is phospholipids, etc. For impurity solids, pour out the upper layer of oil and weigh it. The obtained neutral oil in the upper layer was lighter in color and better in transparency than the vegetable oil bottoms, and the extraction rate could reach 21.76%. Through gas chromatography detection and analysis, it can be seen that the unsaturated fatty acid in the extracted oil reaches more than 90%, of which the content of linoleic acid is 15.5%, the content of oleic acid is 18.90%, and the content of linolenic acid is about 10%.

(2) Preparation of extracted oil emulsion: Mix the extracted oil in step (1) with Tween 80 at a ratio of 1:1.2, put it into a 50 mL Erlenmeyer flask, add an appropriate amount of distilled water, and perform ultrasonic emulsification treatment (500 W, ultrasonic 10 s, 5 s interval, 20 times of action), sterilized at 121°C for 30 min to extract the oil emulsion, and put it at 4°C for later use.

(3) Preparation of oil extraction enzymatic solution: take a certain amount of oil emulsion in step (2) into a 50 mL Erlenmeyer flask, add citric acid-sodium citrate buffer solution with a pH value of 7.0, and add Aspergillus niger lipase powder To a final concentration of 240 U/mL, hydrolyze at 40°C and 150 rpm for 45 minutes to obtain the hydrolyzate for oil extraction.

(4) Preparation of resting lactic acid bacteria: Pick a ring of Lactobacillus plantarum CGMCC1.0557 and Lactobacillus acidophilus CGMCC1.1854 from fresh slant medium, inoculate them into 150 mL MRS medium respectively, and culture them anaerobically at 37°C for 22 h , which is the seed liquid. According to the inoculum amount of 3%-5%, they were respectively inoculated into MRS medium supplemented with linoleic acid emulsion for induced proliferation culture, and cultured statically at 37°C for 20 h. The culture solution was centrifuged at 5000 rpm for 20 min at 4°C, the supernatant was removed, the bacteria at the bottom were washed twice with sterile saline, collected by centrifugation, and 0.25 g/mL resting lactic acid bacteria suspensions were prepared respectively.

(5) Resting lactic acid bacteria transform rapeseed oil residues to extract oil enzymatic hydrolysis solution to generate CLA: Add 5 mL of the resting Lactobacillus plantarum suspension and resting Lactobacillus acidophilus in step (4) to two 50 mL centrifuge tubes respectively Then add 15 mL of 0.1 mol/L citric acid-sodium citrate buffer solution and 3 mL of oil enzymatic solution from step (3), so that the final concentration of the extracted oil is 75 mg/mL. Transform at 100 rpm for 24 h to obtain the CLA transformation solution.

(6) Extraction and detection of CLA: Take 3 mL of the CLA conversion solution in step (5) and add it to a 15 mL centrifuge tube, add 8 mL of n-hexane, mix quickly for 5 min, centrifuge at 4000 rpm for 5 min, and divide into three layers ( Lipid phase, emulsified layer and water phase), use a pipette gun to remove the lower water phase, then add 3 mL of distilled water, mix for 2 min, wash the water-soluble substances, centrifuge to separate the lower water phase, and pass the lipid phase layer through Filtrate with anhydrous sodium sulfate, and dilute to a 25 mL volumetric flask to obtain the conversion solution to be tested, and measure its absorbance at 233 nm. Through conversion, it can be seen that the CLA content in the transformation solution obtained by Lactobacillus acidophilus transformation is 1130.17 μg/mL, and the conversion rate reaches 36.11%; the CLA content in the transformation solution obtained by Lactobacillus plantarum transformation is 869.33 μg/mL, and the conversion rate reaches 27.77 %. Linoleic acid has a maximum absorbance between 200-205 nm, while CLA has a maximum absorbance between 233-234 nm. Comparing the spectral scanning diagrams of the extract after 24 hours of conversion of lactic acid bacteria and the oil enzymolysis solution extracted from rapeseed oil residues (see accompanying drawing 1), it can be seen that the absorbance of the extract at 205 nm has decreased after 24 hours of conversion, and the absorbance at 233 nm has decreased. The light absorption value increased, indicating that part of the linoleic acid in the oil extracted from the vegetable oil leftovers was transformed into CLA under the action of lactic acid bacteria.

Claims (1)

1. utilize tranquillization lactic acid bacteria technique to transform a method for Rapeseed Oil Residual material production conjugated linolic acid, it is characterized in that comprising the steps:

(1) grease is extracted: take Rapeseed Oil Residual and expect in beaker, be placed on 60-90 DEG C of stirring heating 20-40 min in water-bath, period adds micro-distilled water that boils on a small quantity; By the centrifugal 10-30 min of Rapeseed Oil Residual material 4000-6000 rpm after thermal treatment, incline and upper strata oil, obtain neutral grease;

(2) emulsification: the neutral grease that step (1) obtains is mixed according to 1:0.8-1.7 ratio with Tween 80, add appropriate distilled water, carry out ultrasonic emulsification process: 500 W, ultrasonic 10 s, intermittently 5 s, effect 10-20 time, 110-121 DEG C of sterilizing 20-30 min, obtains grease emulsifying liquid;

(3) enzymolysis: the grease emulsifying liquid getting step (2) puts into Erlenmeyer flask, add the citric acid-sodium citrate damping fluid of pH value 7.0, adding lipase from Aspergillus Niger powder is 150-270 U/mL, 40-45 DEG C to final concentration, 120-150 rpm enzymolysis 30-50 min, obtains grease enzymolysis solution;

(4) prepare tranquillization milk-acid bacteria: picking milk-acid bacteria from fresh slant medium, be inoculated in MRS substratum, 37 DEG C of Anaerobic culturel 20-26 h, obtain seed liquor; Seed liquor is seeded to by the inoculum size of 3%-5% in the MRS substratum being added with linolic acid emulsion and carries out proliferative induction cultivation, 37 DEG C of quiescent culture 20 h; Nutrient solution 4 DEG C, the centrifugal 15-30 min of 4000-6000 rpm, removes supernatant liquor, stroke-physiological saline solution washing 2-3 time of bottom thalline, and collected by centrifugation, obtains tranquillization milk-acid bacteria bacteria suspension;

(5) tranquillization lactic acid bacteria tranformation Rapeseed Oil Residual material extracts grease enzymolysis solution and generates conjugated linolic acid: the grease enzymolysis solution getting step (3) puts into reaction tubes, add the tranquillization milk-acid bacteria bacteria suspension of step (4), add the citric acid-sodium citrate damping fluid of appropriate 0.1 mol/L, making to extract grease final concentration is 70-80 mg/mL, 32-36 DEG C, 90-110 rpm transforms 22-26 h, obtains conjugated linolic acid conversion fluid;

(6) extraction of conjugated linolic acid: the conjugated linolic acid conversion fluid getting step (5) mixes with the ratio of normal hexane in 1:2-5, quick mixing 4-6 min, the centrifugal 4-6min of 3800-5000 rpm, is divided into three layers (fat phase, emulsion layer and aqueous phases), removes orlop aqueous phase, then appropriate distilled water is added, mixing 4-6 min, washing water soluble substance, lower floor's aqueous phase is removed in centrifugal layering, filtered by anhydrous sodium sulphate by fat phase layer, rotary evaporation is removed normal hexane and is namely obtained conjugated linolic acid.