CN1157851A - A kind of mycelium rich in aliphatic acid - Google Patents
A kind of mycelium rich in aliphatic acid Download PDFInfo
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- CN1157851A CN1157851A CN 95119312 CN95119312A CN1157851A CN 1157851 A CN1157851 A CN 1157851A CN 95119312 CN95119312 CN 95119312 CN 95119312 A CN95119312 A CN 95119312A CN 1157851 A CN1157851 A CN 1157851A
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Abstract
A mycelium rich in fatty acids is prepared by fermentation process, where Mortierellaceac is used in culture, fermentation and production. Said mycelium contains myristic acid, palmic acid, palmitoleic acid, stearic acid, oleic acid, linolic acid and gamma-linolenic acid, and can be used to extract different fatty acids or as additive or main component of health-care products, beverages or medicines.
Description
What the present invention relates to is a kind of mycelium that is rich in lipid acid of fermentative Production, and method.
Fatty acid composition of the prior art is to come from natural animals and plants mostly, gets through refinement; But some lipid acid, for example gamma-linolenic acid is evening primrose seed to be carried out cold press extract in traditional method, and its equipment is simple, less investment, operation is convenient, but the gained oil quality is poor, and oil yield is low, and Oenothera oil content instability, be subjected to the place of production, climatic influences again, and refining cost height, can not satisfy the demand of society.
Utilize Production by Microorganism Fermentation to be rich in the mycelium of lipid acid, the mycelium that especially is rich in gamma-linolenic acid is the new way of opening up both at home and abroad in recent years.
In the cell of part bacterium, fungi and microalgae, the lipid acid that contains higher amount, biological study institute of Japan Onacls Cement company and Tokyo Agricultural Univ.'s biotechnology engineering department carry out illumination cultivation to the Lan Sizao in the fresh seawater and chlorella all can obtain to contain 26.25% total fatty acids, wherein contains 10% the gamma-linolenic acid of having an appointment.
The objective of the invention is to utilize Chinese Shanghai industrial microorganism institute by the mutagenic obtained bacterial classification of relevant bacterial screening under the mucorales, be rich in the mycelium of lipid acid through fermentative preparation.This bacterial classification is a kind of of zygomycetes (Zygomycetes) mucorales (Mucorales) Mortierellaceaes (Mortierellaceae) genus mortierella (Mortierilla Coemans), separates from the Jilin Anguo, and basic thing is a lead fungi.This bacterial strain has following good production performance:
1, has higher bacterial strain coefficient (the thalline gram number that generates when consuming 100 gram carbon sources) and lipid coefficient (the gram number of the lipid that generates when consuming 100 gram carbon sources).For example: thalline coefficient and lipid coefficient are respectively 30 and 15 during big jar of production, all obtain very high value.
2, be suitable for high-density culture, thalline separates easily.
3, fatty acid content height contains the fat more than or equal to 30% in mycelium; Gamma-linolenic acid content is also high in the described fat, and by suitable culture condition and suitable medium, gamma-linolenic acid content in the lipid of bacterial strain surpasses 4-21%, and (and gamma-linolenic acid content is about 67% in the evening primrose seed oil.)
The mycelial concrete technology that fermentative Production provided by the invention is rich in lipid acid is as follows:
One, culture medium prescription:
Potato substratum (doing the inclined-plane):
Potato 20-30%
Agar 1.5-2%
Glucose 2-3%
2, seed culture medium:
Glucose 6-8%
Urea 0.5-1%
(NH4)2?SO4 0.5-1％
Wort 0.1%
Yeast extract paste 0.05-0.1%
KH2?PO4 0.1-0.2％
MgSO4 0.04-0.08mg/L
Trisodium citrate 0.4-0.6%
3, fermention medium:
Glucose 8-10%
KH2?PO4 0.2-0.3％
NH4?NO3 0.2-0.3％
MgSO4 0.03-0.05％
Yeast powder 0.03-0.05%
Fe2+(FeSO4) 10-30mg/L
Ca(CaCL2) 10-30mg/L
Cu(CuSO4) 0.4-0.8％
Zn(ZnSO4) 1-3mg
Mn(MnCL2) 1-3mg
Trisodium citrate 1.0-1.2%
Citric acid 0.3-0.6%
Prepare described mycelial technical process and see Figure 11.
To forward seed bottle (including seed culture medium) to from the inclined-plane through the strain AS.3410 of slant culture in the potato substratum, last shaking table, 28-30 ℃, 24hr, the shaking table frequency is 45-50HZ; When treating that growth is vigorous in the seed bottle, receive 50L first class seed pot (interior dress seed culture medium), 28-30 ℃ with 10% inoculum size, stir and ventilate, cultivate 24hr, 500L secondary seed jar (interior dress seed culture medium) is changeed in the back, stir and ventilate, cultivate 24hr, temperature is 28-30 ℃; Forward 5 tons of fermentor tanks (including fermention medium) then to, 72-96hr is cultivated in the 28-30 ℃ of stirring of ventilating.Last seeding tank, during the fermentor tank, every the 4hr inspection by sampling, measure pol, pH value, the mycelium weight in wet base, and microscopic examination thalli growth situation, nutrient solution keeps treating that the fermented liquid pol drops to about 1% between the PH3.7-4.1, when mycelium weight in wet base 30% is above, put jar, carry out centrifugation again, promptly obtain the gamma-linolenic acid mycelium, clean mycelium with distilled water again, shred mycelium with slicing machine then, with the oven dry of vacuum low humidity moisture eliminator, about vacuum tightness 0.1 normal atmosphere, temperature 45-50 ℃, drying time 4hr obtains water content at the mycelium that is rich in lipid acid below 5%.
Be description of drawings below,, can better understand the present invention by being described in detail of accompanying drawing and back thereof, specific as follows:
Accompanying drawing 1 is a linolenic acid formicester standard infrared spectrogram;
Accompanying drawing 2 is for mycelium separates, the infrared spectrogram of esterification product;
Accompanying drawing 3 is for mycelium separates, the ultraviolet spectrogram of esterification product;
Accompanying drawing 4 is linolenic acid formicester standard H nucleus magnetic resonance figure;
Accompanying drawing 5 is for mycelium separates, the H nucleus magnetic resonance figure of esterification product;
Accompanying drawing 6 is a gamma-linolenic acid formicester standard mass spectrum;
Accompanying drawing 8 is high resolution mass spectrometry figure;
Accompanying drawing 9 is for mycelium separates, the gas chromatogram of esterification product;
Accompanying drawing 10 is for mycelium separates, the liquid chromatogram of esterification product;
The mycelium that aforesaid method is made is with its formicester isolated in form, and identified that separation method is as follows:
Gamma-linolenic acid formicester molecular formula: C19 H32 O2
Molecular weight: 292.2402
Structural formula: CH3 (CH2) 3 (CH2 CH=CH) 3 (CH2) 4COCH3
The conclusive evidence of gamma-linolenic acid formicester:
One, infrared absorption spectrum (1R)
Instrument model: Perkin-Elmer 983 type infrared spectrometers
Test condition: liquid-film method is measured Resolution 3.0 Ordinate mode%T
ORD?High?100.00，ORD?LOW?10.00
RANCE?4000-800.0?Abso.scale?0.50
Infrared spectrogram such as accompanying drawing 1, accompanying drawing 2 accompanying drawings 1 are linolenic acid formicester standard infrared spectrogram, and accompanying drawing 2 is for mycelium separates, the infrared spectrogram of the product of esterification product.
Mycelium separates, the esterification product the data absorption peak cm-1 oscillatory type group sucting strength 3009 stretching vibration C=C 64.002926 stretching vibrations-CH of mensuration
2-35.172855 stretching vibration CH
3-46.981742 stretching vibrations-C-O-CH
339.701459 scissoring vibration-CH
2-64.001434 stretching vibration C-O 63.971362 stretching vibration C-O-C 71.941172 in-plane bending vibration C=C 60.901018 out-of-plane deformation vibration C=C 76.03 722 flexural vibration-CH
2-70.30
Analysis result: by mycelium of the present invention through aforesaid method separate, after the esterification product in contain CH
3CH
2Groups such as CH=CHC-O, and conform to the main absorption peak 3040,1,735 720 of the gamma-linolenic acid formicester of document (J.Chem Soc2779 (1961)) report, identical with the linolenic acid standard diagram.
Conclusion: mycelium of the present invention after aforesaid method branch, esterification product be the gamma-linolenic acid formicester
Aforesaid mycelium separates, the ultraviolet absorption spectrum (UV) of esterification product:
Instrument model: Varion EMS-200 type ultraviolet spectrophotometer
Test condition: Ord Max/Min 1.000 0.000
WL?Max/Min?nm?300.0?200.0
Speed (nm/min) 200 etoh solvents
Ultraviolet spectrogram: as accompanying drawing 3
Accompanying drawing 3 for the mycelium that is rich in lipid acid of the present invention through aforesaid method separate, after the esterification the product ultraviolet spectrogram.
Test result: λ max=203.4nm is-charateristic avsorption band of C-OCH3
Resolve: therefore the gamma-linolenic acid formicester does not have the UV collection of illustrative plates of standard because of no conjugated system, and its ester group absorption peak has been measured in this evaluation.
The aforesaid mycelium that is rich in lipid acid separates, the esterification product
1H nuclear magnetic resonance spectrum (1HNMR):
Instrument model: Varion VXR-300MHz nuclear magnetic resonance analyser
Mark solvent C DCl3 in condition determination: the TMS
1H nuclear magnetic resonance map such as accompanying drawing 4,5;
Accompanying drawing 4 is linolenic acid formicester standard 1H nucleus magnetic resonance figure;
Accompanying drawing 5 is that the mycelium of lipid acid separates, the esterification product
1H nucleus magnetic resonance figure.
The corresponding proton a 0.894 3 three-CH3b 0.904-1.612 C more than 10 of determination data sequence number chemical shift ppm proton number multiplicity
3,4,15,16,17
-CH
2-c 1.637-2.342 C more than 6
2,5,14
0
-CH
2-C-,=CH-CH
2-d 2.805-2.820 C more than 4
8,11=CH-CH
2-CH=e 3.679 3 list-OCH3f 5.337-5.373 more than 6-CH=
Resolve---of the present invention be rich in the mycelium of lipid acid through the separation of aforesaid method, esterification after the product of gained contain CH
3-,-CH
2-, CH=CH ,-CH=CH-CH
2-CH=, C-OCH3;
Conclusion: the mycelium that is rich in lipid acid of the present invention through aforesaid method separate, after the esterification product and gamma-linolenic acid methyl esters bibliographical information data (herbal medicine .13 (9) 385 (1982)) identical (14), close with linolenic acid formicester standard diagram.
Aforesaid mycelium separates, the mass spectrum (MS) of esterification product:
Instrument model: KyKy-ZHP-5 velocitron
Test condition: resolving power 7000 acceleration voltage 6KV AEI MS 50
Mass spectrum: as accompanying drawing 6,7,8;
Accompanying drawing 6 is a gamma-linolenic acid formicester standard mass spectrum;
Accompanying drawing 7 is tested product mass spectrum;
Accompanying drawing 8 is high resolution mass spectrometry figure.
Determination data: mass-to-charge ratio (m/e) relative abundance mass-to-charge ratio (m/e) relative abundance 292 (M) 2.85 79 2.64 194 2.11 67 61.11 15 0 5.58 55 67.66 121 7.06 41 61.76 107 8.55 28 (base peak) 100.00 93 14.45---------------------91 9.80 81 46.82
Resolve: 292 M.
41 CH
3-C＝CH
2
29 (base peak) CH
3-CH
2
194 41+(CH
2)
n-1?n＝11
81 41+-CH
2-CH＝CH-
67 41+-CH＝CH-
55 41+-CH
2-
Conclusion: the mycelium that is rich in lipid acid of the present invention through aforesaid method separate, after the esterification this compound of product have and the identical peak of gamma-linolenic acid formicester standard diagram, identify through high resolution MS, 292.2400 peak value is wherein arranged, identical with the calculated value 292.2402 of C19 H32 O2.
Through above-mentioned parsing can prove conclusively that the mycelium that is rich in lipid acid of the present invention separates, product after the esterification has gamma-linolenic acid formicester structure, after separation, the esterification of surveying product contain gamma-linolenic acid.
Separate, after the esterification product in except that containing gamma-linolenic acid, also have unsaturated fatty acids, amino acid, VITAMIN etc. such as linolic acid.
In order further to determine mycelium composition of the present invention, be title and the content that routine means obtain other compositions below by one.
The mensuration of lipid acid (pressing GB9695.2-88) in the mycelium of the present invention:
Instrument: gas chromatograph, totalizing instrument
Test method:fatty acid composition is extracted grease with the Soxhlet extraction process, in the presence of boron trichloride, carry out the saponification of glyceride and the esterification of free fatty acids, air inlet chromatography (is the collection of illustrative plates of sample 2 as accompanying drawing 9) is surveyed its composition with area normalization method then; The below is the testing result (%) of various aliphatic acid in total grease of 4 samples: 414: 0 (myristic acids) 1.7 1.8 1.9 1.816: 0 (palmitic acid) of title sample 1 (%) sample 2 samples 3 samples, 26.9 26.1 26 19.916: 1 (palmitoleic acids) 3.4 3.1 3.0 2.118: 0 (stearic acid) 1.9 2.0 2.1 118: 1 (oleic acid) 49 49.4 49.3 43.518: 2 (linoleic acid), 12.4 12.1 12.3 12.118: 3 (gamma-Linolenic acids) 4.2 4.9 5 19.0
The mensuration (pressing GB12388-90) of vitamin-E in the mycelium that is rich in lipid acid of the present invention
Instrument: high pressure liquid chromatograph band ultraviolet spectrometry detector
Test method: the VE in the sample after saponification extract to be handled, with its not the sponifiable extracting section to organic solvent, VE is separated with high performance liquid chromatography C18 reversed-phase column, through the UV-detector detection, and measure with inner mark method ration.
Accompanying drawing 10 is the liquid chromatogram of the sample 2 of separated product, is respectively 4 test result of samples below.Detected result sample 5 samples 6 samples 7 sample 8 (mg/100g) content of vitamin E 69.23 68.02 68.0 67.78
Amino acid whose mensuration in the fatty acid composition of the present invention
Instrument: Beckman 6300 amino acidanalysers
Test method: according to China standard GB/T14965-94 about amino acid whose measuring method.
Testing result (mg/100g): title sample 9 samples 10 samples 11 samples 12 asparatates 1,350 1,320 1,330 1300 threonines 500 520 510 550 serines 400 500 440 430 glutamic acid 2,300 2,100 2,200 2300 proline 2,450 2,450 2,500 2550 glycine 4,100 4,200 4,000 4300 alanine 1,800 1,900 1,800 1900 valines 850 830 820 800 methionine 310 300 280 340 isoleucines 770 760 710 750 leucines 1,300 1,200 1,100 1000 tyrosine 370 350 310 380 phenylalanines 700 650 660 700 histidines 220 250 270 300 lysines 1,000 1,000 950 1050 arginine 1,750 1,700 1,600 1600
Experimental study by aforesaid method, confirm to contain in the mycelium of the present invention lipid acid such as gamma-linolenic acid, linolic acid, vitamin-E, A, 16 seed amino acids, trace element, multiple composition to the human body beneficial such as protein, above result is by Beijing Medical University and Chinese Academy Of Preventive Medicine Research Institute Of Nutrition And Food Hygiene's test approval.
As from the foregoing, the mycelium that is rich in lipid acid of fermentative Production provided by the invention is by saponification, methanolizing pre-treatment, identifies through ultraviolet, infrared, nucleus magnetic resonance and mass spectroscopy, contain gamma-linolenic acid in the proof oil, its structure is suitable-6,9,12-punicic acid, molecular formula are C
18H
30O
2, gamma-linolenic acid content is about 14.5% in the oil.According to China's national standard about protein, lipid acid, amino acid, VITAMIN and determination of trace elements method, record in the mycelium except that containing polyunsaturated fatty acids such as gamma-linolenic acid, linolic acid and 16 seed amino acids, also contain following minor component, described minor component comprises vitamin-E, A, iron, zinc, calcium, magnesium, selenium and other trace elements.
The distribution of other composition is as follows in the described stem body fatty acid composition:
Experimental study by aforesaid method, confirm to contain gamma-linolenic acid, linolic acid in the mycelium, vitamin-E, A, 16 seed amino acids, trace element, multiple composition to the human body beneficial such as protein, above result is by Beijing Medical University and Chinese Academy Of Preventive Medicine Research Institute Of Nutrition And Food Hygiene's test approval.
As from the foregoing, the mycelium of fermentative Production provided by the invention is by saponification, methanolizing pre-treatment, identify through ultraviolet, infrared, nucleus magnetic resonance and mass spectroscopy, contain gamma-linolenic acid in the proof oil, its structure is suitable-6,9, the 12-punicic acid, molecular formula is C18H30O2, and gamma-linolenic acid content is 14.5% in the oil.According to China's national standard about protein, lipid acid, amino acid, VITAMIN and determination of trace elements method, record in the mycelium except that containing polyunsaturated fatty acids such as gamma-linolenic acid, linolic acid, also contain following component: vitamin-E, A, iron, zinc, calcium, magnesium, selenium and other trace elements and 16 seed amino acids.
The mycelium of method for preparing has purposes widely, also can be used as the into raw material of purification single fat acid, the example that is rich in a mycelial success of lipid acid of the present invention is exactly at the healthcare products that cooperated other additive to have good result---and three ring health kings, these healthcare products and this case have proposed patent application simultaneously.
Claims (6)
1, a kind of mycelium that is rich in lipid acid is characterized in that containing in the described mycelium lipid acid more than or equal to about 30-50%.
2, the mycelium that is rich in lipid acid according to claim 1 is characterized in that described lipid acid comprises that 1.7～1.9% tetradecanoic acid is arranged approximately; 19.9～26.9% palmitinic acid; 2.1～3.4% physetoleic acid; 1～2.1% stearic acid; 43.5～49.4 oleic acid; 12.1～12.4 linolic acid; 4.2～19 gamma-linolenic acid.
3, the mycelium that is rich in lipid acid according to claim 1 and 2 is characterized in that described lipid acid comprises that 1.7～1.9% tetradecanoic acid is arranged approximately; 19.9～26.9% palmitinic acid; 2.1～3.4% physetoleic acid; 1～2.1% stearic acid; 43.5～49.4 oleic acid; 12.1～12.4 linolic acid; 6～19 gamma-linolenic acid.
4, the mycelium that is rich in lipid acid according to claim 1, the acid number that it is characterized in that described lipid acid is smaller or equal to 4.
5, the described mycelial method that is rich in lipid acid of a kind of fermentative Production claim 1, it is characterized in that bacterial classification by the mutagenic obtained product gamma-linolenic acid of relevant bacterial screening under the mucorales, cultivate through potato substratum (doing the inclined-plane), to in the potato substratum, forward seed bottle (interior dress seed culture medium) to from the inclined-plane by the bacterial classification through slant culture, last shaking table, 25-35 ℃, 24hr, the shaking table frequency is 45-50HZ; When treating in the seed bottle that growth is vigorous, receive 50L first class seed pot (interior dress seed culture medium) with 10% inoculum size, 25-35 ℃, stir and ventilate, cultivate 24-48hr, after forward 500L secondary seed jar (interior dress seed culture fluid) to, stir and ventilate, cultivate 24-48hr; Forward 5 tons of fermentor tanks (interior dress fermention medium) then to, 25-35 ℃, ventilate and stir, cultivate 72-96hr, nutrient solution keeps treating that the fermented liquid pol drops to about 1% between the pH value 3.7-5.1 during last seeding tank, the fermentor tank, when mycelium weight in wet base 30% is above, put jar, and carry out centrifugation, promptly make the mycelium that is rich in lipid acid of the present invention.
6, by the described fermentative Production method for compositions of claim 5, it is characterized in that the mycelium that makes cleans with distilled water, chopping, adopt the oven dry of vacuum dehydrating at lower temperature device, vacuum tightness 0.1 normal atmosphere, 45 ℃-65 ℃ of temperature, baking 4hr obtains water content at the mycelium of the present invention below 5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95119312 CN1157851A (en) | 1995-11-30 | 1995-11-30 | A kind of mycelium rich in aliphatic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95119312 CN1157851A (en) | 1995-11-30 | 1995-11-30 | A kind of mycelium rich in aliphatic acid |
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| Publication Number | Publication Date |
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| CN1157851A true CN1157851A (en) | 1997-08-27 |
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ID=5081943
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 95119312 Pending CN1157851A (en) | 1995-11-30 | 1995-11-30 | A kind of mycelium rich in aliphatic acid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114384105A (en) * | 2020-10-16 | 2022-04-22 | 仙乐健康科技股份有限公司 | Construction method and application method of probiotic tablet stability test prediction model |
-
1995
- 1995-11-30 CN CN 95119312 patent/CN1157851A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114384105A (en) * | 2020-10-16 | 2022-04-22 | 仙乐健康科技股份有限公司 | Construction method and application method of probiotic tablet stability test prediction model |
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