CN107099574B - Compound microbial agent for improving yield of polyunsaturated fatty acid - Google Patents
Compound microbial agent for improving yield of polyunsaturated fatty acid Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- 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
Abstract
The invention discloses a compound microbial agent for improving the yield of polyunsaturated fatty acid, which consists of three active thallus cells of Mortierella pusilla, Mortierella alpina and Mucor circinelloides and a liquid culture medium; wherein the total cell number of three active bacteria in each ml of compound microorganism bacterial agent is not less than 1 × 108The ratio of the number of the somatic cells of the three active bacteria is (1-5) to (1-5); the formula of the liquid culture medium is as follows: the tap water contained 80g/L glucose, 2ml/L soybean oil, 1g/L corn starch, 4g/L yeast extract, 2g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 1g/L dipotassium hydrogen phosphate, 2g/L sodium citrate, and 2g/L magnesium sulfate per liter. Experiments prove that compared with the traditional single strain fermentation, after the composite microbial agent disclosed by the invention is used for fermenting for the same days, the biomass and the lipid production rate are improved to different degrees, the fermentation yield of polyunsaturated fatty acid is obviously improved, and the industrial application prospect is wide.
Description
Technical Field
The invention relates to a compound microbial inoculum for improving the fermentation yield of a microbial secondary metabolite, in particular to a liquid compound microbial inoculum for improving the yield of polyunsaturated fatty acid, and belongs to the technical field of microbial fermentation.
Background
Polyunsaturated fatty acids (PUFAs) refer to a class of fatty acids having greater than or equal to 18 carbon atoms and containing two or more double bonds. Polyunsaturated fatty acids are generally classified into 3 classes according to the position of the first double bond: n-3PUFAs, polyunsaturated fatty acids such as docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) at the 3 rd carbon position counting the 1 st double bond from the methyl end; n-6PUFAs, meaning that the first double bond is located at the 6 th carbon position from the methyl end, such as arachidonic acid (20:4n-6, AA) and gamma-linolenic acid (18:3n-6, GLA); in addition, n-9 type PUFAs in which the 1 st double bond is at the 9 th carbon position are also included.
Most of PUFAs are precursors of important bioactive substances, and have various medicinal values and nutritional values. PUFAs has physiological functions of stabilizing cell membrane, reducing cholesterol and triglyceride content in blood, reducing blood viscosity, improving blood microcirculation, resisting cardiovascular diseases, promoting organism growth and development, enhancing memory and thinking ability, etc., and has more double bonds, higher unsaturated degree and higher nutritive value. At present, PUFAs are mainly derived from deep sea fish oil and certain vegetable oil. However, fish and vegetable oils have limited resources, low PUFAs content, are susceptible to environmental conditions such as climate, production area, etc., and are expensive to purify and are easily hydrogenated during processing. One of the current research directions is to find microorganisms such as bacteria, fungi and algae that are capable of metabolizing to produce PUFAs.
Currently, PUFAs-producing microorganisms are diverse and include bacteria (Lactobacillus acidophilus, Rhodococcus cloudiness, Vibrio, etc.), yeasts (Candida curvata, Cryptococcus albidus, Rhodotorula mucilaginosa, Lipomyces starkeyi, Lipomyces oleosus, etc.), molds (Mortierella pusilla, Mortierella alpina, Mucor circinelloides, Aspergillus oryzae, Aspergillus terreus, Cladosporium elegans, Blakeslea trispora), and algae (Dunaliella salina, Chlorella pyrenoidosa, Isochrysis galbana, Phaeodactylum crescentella, etc.). Due to the low bacterial yield, the current production of PUFAs is mainly focused on fungi and algae. In particular to fungi, which has the advantages of short growth cycle, simple culture, stable product quality, no limitation of raw materials, climate and producing area, easy large-scale and industrialized production and the like. The fungi include Mortierella alpina (Moritella alpina), Mortierella elongata (Mortierella elongata), Saprolegnia (Saprolegnia), Verticillium (Diasporium), Phytophthora cinnamomea (Phytophthora cinmamomij), Mucor (Mucor), and Cucoircillinus parvum (Cucoircinelloid), which contain EPA and AA, and the strains include GLA. Bacteria such as Mortierella elongata, Pythium irregulare, Thraustochytrium aunemum, Pythium ultimum, and Cephalosporium cephalospora contain EPA and DHA. The cultivation of algae is greatly influenced by external conditions, and the production of PUFAs by using the algae is difficult to realize. Fungi are widely distributed in nature and are easy to culture, so that the seeking of high-yield strains from the fungi is a concern of a plurality of researchers at present and has a promising prospect. However, the research on the production of polyunsaturated fatty acids by fungal fermentation is mostly focused on the fermentation by a single strain, and the research on the production of polyunsaturated fatty acids by the fermentation of a complex microbial inoculum prepared by mixing functional strains is not reported.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide the compound microbial agent for improving the yield of the polyunsaturated fatty acid so as to achieve the aims of quickly improving the fermentation yield of the polyunsaturated fatty acid and reducing the production cost.
The compound microbial agent for improving the yield of the polyunsaturated fatty acid is in a liquid state, and the polyunsaturated fatty acid refers to palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), α -linolenic acid (C18:3), gamma-linolenic acid (C18:3), arachidic acid (C20:0), arachidonic acid (C20:1), arachidonic acid (C20:2) and/or arachidonic acid (C20: 4);
the method is characterized in that:
the compound microbial agent consists of three active thallus cells of Mortierella pusilla, Mortierella alpina and Mucor circinelloides and a liquid culture medium; wherein the total cell number of three active bacteria in each ml of compound microorganism bacterial agent is not less than 1 × 108The ratio of the number of the somatic cells of the three active bacteria is (1-5) to (1-5); the formula of the liquid culture medium is as follows: each liter of tap water contains 80g/L glucose, 2ml/L soybean oil and 1g/L cornStarch, 4g/L of yeast extract, 2g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of sodium citrate and 2g/L of magnesium sulfate.
In the compound microbial agent for increasing the yield of polyunsaturated fatty acids: the ratio of the number of the three active bacteria cells of the mortierella flava, the mortierella alpina and the mucor circinelloides in each milliliter of the compound microbial agent is preferably 5:1: 2.
The preparation method of the compound microbial agent for improving the yield of the polyunsaturated fatty acid comprises the following steps: respectively culturing the Mortierella pusilla, the Mortierella alpina and the Mucor circinelloides in a liquid culture medium for 42-45 h according to a culture mode that the inoculation volume ratio is 5%, the temperature is 26 ℃ and the rotating speed is 180 r/min; respectively detecting absorbance values of the three bacterial liquids under the condition of 600nm, and calculating the respective bacterial cell concentrations; the total cell number of three active bacteria in the compound microbial agent is not less than 1 × 108And mixing the three active microbial agents according to the ratio of (1-5) to prepare the compound microbial agent for improving the yield of the polyunsaturated fatty acid.
In the above method for preparing a complex microbial inoculant for increasing the yield of polyunsaturated fatty acids, the total number of cells of three active thalli in each milliliter of the complex microbial inoculant is preferably not less than 1 x 108And mixing the three active microbial cells in a ratio of 5:1:2 to prepare the compound microbial agent for improving the yield of the polyunsaturated fatty acid.
Compared with the traditional single strain fermentation, after the composite microbial agent disclosed by the invention is used for fermenting for the same days, the biomass and the lipid yield are improved to different degrees, the fermentation yield of the polyunsaturated fatty acid is obviously improved, and the industrial application prospect is wide.
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FIG. 1 is a gas chromatography spectrum.
Detailed Description
The complex microbial inoculant and the fermentation method thereof provided by the invention are described and illustrated in detail below by combining specific examples. The described examples are only a few examples of the present disclosure and not all examples. The content is to be interpreted as illustrative of the invention and not to limit the scope of the invention.
Example 1: analysis of lipid-producing ability of functional Strain
(1) Activation of bacterial strains
Activating Mortierella pusilla (No. 3.341, purchased from China general microbiological culture Collection center), Mortierella alpina (No. 2557, purchased from China Industrial microbiological culture Collection center) and Mucor circinelloides (No. 2632, purchased from China Industrial microbiological culture Collection center) on a PDA test tube slant culture medium (200 ml/L potato leachate, 20g/L glucose and 15g/L agar, preparing with tap water, performing pH naturalis and sterilizing at 115 ℃ for 30min), and culturing at 28 ℃ for 5-7 days in an incubator to form deep yellow conidia.
(2) Preparation of seed liquid
Digging the activated Mortierella pusilla, Mortierella alpina and Mucor circinelloides in the step (1) by using a sterile shovel, respectively inoculating the excavated Mortierella pusilla, Mortierella alpina and Mucor circinelloides into a liquid medium (80g/L glucose, 2ml/L soybean oil, 1g/L corn starch, 4g/L yeast extract, 2g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 1g/L dipotassium hydrogen phosphate, 2g/L sodium citrate and 2g/L magnesium sulfate, preparing the activated Mortierella pusilla, Mortierella alpina and Mucor circinelloides by using tap water, adjusting pH to 6.8 +/-0.2, sterilizing at 115 ℃ for 30min), and culturing at 26 ℃ and 180r/min for 42 h.
(3) Shake flask fermentation culture
Transferring the cultured seed liquid into 100ml of fresh liquid culture medium according to the volume ratio of 5% for fermentation at the rotation speed of 180r/min at the temperature of 28 ℃ for 8 days. The mycelium was recovered by filtration through 4 layers of sterile gauze.
(4) Determination of biomass
And (4) washing the mycelium collected in the step (3) with tap water, drying at 60 ℃ and weighing. The biomass was calculated from g dry biomass/L fermentation broth.
(5) Measurement of crude lipid of cells
Weighing dried mycelium, putting into a mortar, adding a proper amount of quartz sand, and grinding to obtain particles. And (3) folding the filter paper sheets into paper bags, and then transferring the paper bags into an oven to dry for 2 hours at the temperature of 105 +/-2 ℃. Taking out and putting into a dryer to cool to room temperature. The filter paper pack was filled with the ground mycelium and the total weight thereof was weighed. Putting the filter paper bag with the sample into an extraction cylinder by using long tweezers, and adding petroleum ether to completely immerse the sample bag. Connecting all parts of the extractor, connecting condensed water flow to extract in a constant temperature water bath at 70-80 ℃, refluxing for about 7 times per hour, and extracting for 7 hours. After extraction, the filter paper bag is taken out by using long tweezers, and petroleum ether is volatilized at a ventilated place. And (3) placing the filter paper bag in a 105 +/-2 ℃ oven for drying for 2h, and placing the filter paper bag in a dryer for cooling until the weight is constant. And weighing the filter paper bag filled with the sample again, wherein the reduced weight is the weight of the crude bacteria lipid. The crude lipid weight/cell weight is the lipid production rate (%).
The result shows that the biomass of the Mortierella pusilla is 18.8g/L, the fat yield is 6.43g/L and the fat yield is 34.2% after the fermentation is finished under the above conditions; the biomass of the mortierella alpina is 19.4g/L, the fat yield is 4.98g/L, and the fat yield is 25.6%; the biomass of the mucor circinelloides is 20.4g/L, the fat yield is 4.19g/L, and the fat yield is 20.5%.
Example 2: optimization of mixing ratio of functional strains
In order to prepare the compound microbial agent suitable for improving the yield of the polyunsaturated fatty acid, the dosage proportion of the Mortierella pusilla, the Mortierella alpina and the Mucor circinelloides is L9(34) And (4) orthogonal experiment optimization. The experimental factors and levels were set as shown in table 1.
TABLE 1 orthogonal experiment factor horizon
The fermentation and lipid production analysis of the functional strains were the same as in example 1.
The results of the orthogonality experiment are shown in table 2.
TABLE 2 results of orthogonal experiments
As can be seen from Table 2, when the biomass of the Mortierella pusilla, Mortierella alpina and Mucor circinelloides is 1:5:5, the maximum biomass is 30.95g/L, and the fat yield of the composite microbial agent is 16.79%; secondly, when the ratio of the biomass of the Mortierella pusilla to the Mortierella alpina to the Mucor circinelloides is 5:5:2, the biomass reaches 29.98g/L, and the fat yield is 36.63%. Although the fat production rate of the composite microbial agent reaches the maximum when the ratio of the Mortierella pusilla to the Mortierella alpina to the Mucor circinelloides is 2:1:2 and 5:1:5, which are 50.86% and 50.76%, respectively, the biomass and oil production in the fermentation broth are also low. The worst analysis on the biomass and the lipid production rate shows that the mortierella alpina is the most influential to the biomass, and then the mucor circinelloides and the mortierella fulvescens are sequentially arranged, and the preferred scheme is B3C2A 3; the most influential on the fat production rate was mortierella alpina, followed in turn by mortierella isabellina and mucor circinelloides, the preferred embodiment being B1A3C 2.
After the shake flask fermentation is carried out on the preferred schemes B3C2A3 and B1A3C2, the results show that the biomass of the compound microbial inoculant in the previous group with the optimized proportion is 29.98g/L, the fat yield is 10.98g/L, and the fat yield is 36.63%; the biomass of the compound microbial inoculum with the optimized proportion in the last group is 32.87g/L, the fat production amount reaches 16.94g/L, and the fat production rate is 51.54%. The scheme B1A3C2 is superior to the scheme B3C2A3, so the selected optimization scheme is B1A3C2, namely the ratio of the number of the active cells of the Mortierella pusilla, the Mortierella alpina and the Mucor circinelloides in the composite microbial agent is preferably 5:1: 2.
Example 3 preparation of Complex microbial Agents
(1) The activation of Mortierella pusilla, Mortierella alpina and Mucor circinelloides was performed according to the method in example 1, and a seed liquid was prepared.
(2) According to the volume ratio of 5 percent, respectively inoculating the Mortierella pusilla, the Mortierella alpina and the Mucor circinelloides to 50ml of fresh liquid culture medium (80g/L glucose, 2ml/L soybean oil, 1g/L corn starch, 4g/L yeast extract, 2g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 1g/L dipotassium hydrogen phosphate, 2g/L sodium citrate and 2g/L magnesium sulfate, prepared by tap water, the pH value is 6.8 +/-0.2, and the mixture is sterilized at 115 ℃ for 30min) and cultured for 42h under the conditions of 26 ℃ and 180 r/min.
(3) And respectively detecting the absorbance values of the Mortierella pusilla, Mortierella alpina and Mucor circinelloides bacterial solutions under the condition of 600nm, and calculating the respective bacterial cell concentrations.
(4) The total cell number of three active bacteria in the compound microbial agent is not less than 1 × 108And mixing the three active microbial cells in a ratio of 5:1:2 to prepare the compound microbial agent for improving the yield of the polyunsaturated fatty acid.
Example 4 fermentation assay of Complex microbial Agents
(1) The compound microbial agent prepared in example 3 was selected for fermentation.
(2) The fermentation of the complex microbial agent and the extraction of lipids from the cells were performed in the same manner as in example 1. A liquid culture medium without inoculated compound microbial inoculum is used as a control group, and 3 parallel control groups and experimental groups are respectively set.
(3) Gas chromatography analysis of polyunsaturated fatty acid components
Methyl esterification of fatty acid: taking 0.05g of lipid sample, placing the lipid sample in a 10mL test tube, adding 1mL of 28g/L potassium hydroxide-methanol solution, saponifying in a 60 ℃ water bath for 15min, cooling, adding 1mL of 14% boron trifluoride-methanol solution, oscillating in a 60 ℃ water bath for 2min for methyl esterification, cooling, accurately adding 1mL of n-hexane, oscillating, adding 1mL of saturated sodium chloride, and taking supernatant.
Gas chromatography conditions: fatty acid methyl esterification samples were analyzed by GC-2010(Shimadzu Co., Japan) on a column DB-Waxetr (30 m.times.0.32 mm,. phi.0.25. mu.m). Detecting by a hydrogen flame ion detector, wherein the temperatures of a vaporization chamber and the detector are 250 ℃ and 260 ℃, the sample injection is carried out in a split-flow mode by 1 mu l, the split-flow ratio is 10:1, and the carrier gas is nitrogen. Temperature programming: the initial temperature of 120 ℃ is maintained for 3min, the temperature is increased to 190 ℃ at 5 ℃/min, then the temperature is increased to 210 ℃ at 1 ℃/min, and the temperature is maintained for 15 min.
Polyunsaturated fatty acid standards were purchased from shanghai screening biotechnology limited.
Comparing the retention time of the peaks on the gas chromatogram of the sample and the standard (figure 1), the content of unsaturated fatty acid in the complex microbial inoculant fermentation broth calculated by area normalization is respectively 1.83g/L of palmitic acid (C16:0), 1.68g/L of stearic acid (C18:0), 1.96g/L of oleic acid (C18:1), 2.72g/L of linoleic acid (C18:2), 0.34g/L of α -linolenic acid (ALA, C18:3), 0.28g/L of gamma-linolenic acid (GLA, C18:3), 0.058g/L of arachidic acid (C20:0), 0.023g/L of arachidonic acid (C20:1), 0.017g/L of arachidic acid (C20:2) and 1.94g/L of arachidonic acid (C20: 4).
The results show that the compound microbial agent has obvious promotion effect on the improvement of the yield of fermentation products, improves the biomass and the lipid production rate to different degrees, obviously improves the fermentation yield of polyunsaturated fatty acid, and has industrial application prospect.
Claims (2)
1. A compound microbial agent for improving the yield of polyunsaturated fatty acids, wherein the compound microbial agent is in a liquid state, and the polyunsaturated fatty acids refer to palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), α -linolenic acid (C18:3), gamma-linolenic acid (C18:3), arachidic acid (C20:0), arachidonic acid (C20:1), arachidonic acid (C20:2) and/or arachidonic acid (C20: 4);
the method is characterized in that:
the compound microbial agent consists of three active thallus cells of Mortierella pusilla, Mortierella alpina and Mucor circinelloides and a liquid culture medium; wherein the total cell number of three active bacteria in each ml of compound microorganism bacterial agent is not less than 1 × 108The ratio of the number of the somatic cells of the three active bacteria is 5:1: 2; the formula of the liquid culture medium is as follows: the tap water contained 80g/L glucose, 2ml/L soybean oil, 1g/L corn starch, 4g/L yeast extract, 2g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 1g/L dipotassium hydrogen phosphate, 2g/L sodium citrate, and 2g/L magnesium sulfate per liter.
2. The method for preparing the complex microbial inoculant for improving the yield of polyunsaturated fatty acids as claimed in claim 1, comprising the following steps: respectively culturing the Mortierella pusilla, the Mortierella alpina and the Mucor circinelloides in a liquid culture medium for 42-45 h according to a culture mode that the inoculation volume ratio is 5%, the temperature is 26 ℃ and the rotating speed is 180 r/min; respectively detecting absorbance values of the three bacterial liquids under the condition of 600nm, and calculating the absorbance values respectivelyThe cell concentration of the cells of (a); the total cell number of three active bacteria in the compound microbial agent is not less than 1 × 108And mixing the three active microbial cells in a ratio of 5:1:2 to prepare the compound microbial agent for improving the yield of the polyunsaturated fatty acid.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101631870A (en) * | 2007-01-15 | 2010-01-20 | 三得利控股株式会社 | Method for production of polyunsaturated fatty acid and lipid containing the polyunsaturated fatty acid |
| CN104087512A (en) * | 2014-05-23 | 2014-10-08 | 中国科学院青岛生物能源与过程研究所 | Mortierella alpine producing polyunsaturated fatty acids and application thereof |
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| CN101631870A (en) * | 2007-01-15 | 2010-01-20 | 三得利控股株式会社 | Method for production of polyunsaturated fatty acid and lipid containing the polyunsaturated fatty acid |
| CN104087512A (en) * | 2014-05-23 | 2014-10-08 | 中国科学院青岛生物能源与过程研究所 | Mortierella alpine producing polyunsaturated fatty acids and application thereof |
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