CN1157850A - Culture medium for producing fatty acid rich mycelium by fermentation method - Google Patents

Abstract

A culture medium for preparing fatty acid composition by fermentation process includes potato culture medium, seed culture medium and fermenting culture medium. By means of said culture medium and invented process, a composition rich in fatty acid, especially the fatty acid rich in gamma-linolenic acid, which can be used as additive for food, beverage, medicine and health-care product without by-effect, can be stably obtained. Said culture medium can be used in culture of Mortierellaceac strain, fermentation and production.

Description

Be used for the mycelial substratum that fermentative Production is rich in lipid acid

What the present invention relates to is a kind of mycelial substratum that fermentative Production is rich in lipid acid that is used for, and particularly a kind ofly is used for the mycelial substratum that fermentative Production is rich in gamma-linolenic acid.

The various culture medium prescriptions that are used to ferment are a lot of in the prior art, the raw material that is adopted also is similar, but in some specific occasions, adopt a kind of substratum with unique formula, be specifically designed to a kind of specific technology, will play and make technology performance maximum efficiency, and make the component content of technology products therefrom stable, effective constituent significantly increases, and a kind of just suitable fermentative Production of substratum of the present invention is rich in the mycelial substratum of lipid acid, is rich in gamma-linolenic acid in the mycelium particularly of the present invention.

Above-mentioned gamma-linolenic acid is that lipid acid is closed in a kind of senior many insatiable hungers, it is essential fatty acid, it is the structured material of forming each tissue biological's film of human body, it also is the precursor of a series of prostaglandin(PG)s of synthesized human, have reducing blood-fat, anticoagulant, blood are fastened the synthetic lipotropism matter oxidation of plain A2, suppress ulcer and gastrorrhagia, strengthen Regular Insulin and antiobesity action.

Utilize Production by Microorganism Fermentation to be rich in the mycelium of lipid acid, especially being rich in the linolenic mycelium of r-is research and that opened up both at home and abroad in recent years new way.

Gamma-linolenic acid is present in the cell of some bacterium, fungi and microalgae, and its content accounts for the 6%-24% of total fat.

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, in this mycelium, be rich in the r-linolenic acid through fermentative preparation.This bacterial classification is a kind of of zygomycetes (Zygomycetes) mucorales (Mucorales) Mortierellaceaes (Mortierellaceae) genus mortierellas (MortierillaCoemans), 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 and lipid coefficient.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, gamma-linolenic acid content height by suitable culture condition and substratum of the present invention, can make resulting mycelial lipid content greater than 30%, and r-linolenic acid content in lipid surpasses 4-21%;

Of the present inventionly be exclusively used in fermentative Production to be rich in the mycelial culture medium prescription of fatty acid composition of gamma-linolenic acid as follows:

1, 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)2SO4 0.5-1％

Wort 0.1%

Yeast extract paste 0.05-0.1%

KH2PO4 0.1-0.2％

MgSO4 0.04-0.08mg/L

Trisodium citrate 0.4-0.6% 3, fermention medium:

Glucose 8-10%

KH2PO4 0.2-0.3％

NH4NO3 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/L

Mn(MnCL2) 1-3mg/L

Trisodium citrate 1.0-1.2%

Citric acid 0.3-0.6%

Brief description adopts the mycelial technology of medium preparation of the present invention, and its schema is seen Fig. 5,

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 r-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 that r-linolenic acid mycelium separates, the infrared spectrogram of esterification product;

Accompanying drawing 3 is that r-linolenic acid mycelium separates, the gas chromatogram of esterification product;

Accompanying drawing 4 is that r-linolenic acid mycelium separates, the liquid chromatography of esterification product

Accompanying drawing 5 is the mycelial technical process of medium preparation

Prepared mycelium is with its formicester isolated in form, and collecting single Rf value part after the esterification can be for the sample of identifying usefulness.

Accompanying drawing 1 is a linolenic acid formicester standard infrared spectrogram,

Accompanying drawing 2 is for being rich in the mycelial infrared spectrogram of lipid acid.

Be the object lesson of substratum of the present invention below, by these examples and in conjunction with the content and the parameter of other paragraph of the present invention, can confirm further that substratum of the present invention just just makes the stable content of various compositions in the resulting mycelium that is rich in lipid acid, the content of effective height, and effective too in suitability for industrialized production.

Embodiment potato substratum, (doing the inclined-plane) 1234 potato % 20 25 30 24 agar % 1.6 1.5 1.8 2 glucose % 2.5 23 2.7 seed culture mediums 1234 glucose % 8675 urea % 0.5 0.6 0.55 0.7, (NH ₄) ₂SO ₄% 1 0.8 0.9 0.5 wort % 0.1 0.1 0.1 0.1 yeast extract paste % 0.05 0.06 0.08 0.1KH ₂PO ₄% 0.2 0.17 0.14 0.1MgSO ₄Mg/L 0.04 0.06 0.07 0.08 trisodium citrate % 0.6 0.55 0.5 0.4 fermention mediums 1234 glucose % 898 10KH ₂PO ₄% 0.2 0.3 0.2 0.3NH ₄NO ₃% 0.2 0.2 0.3 0.3MgSO ₄% 0.05 0.04 0.03 0.05 yeast powder % 0.03 0.05 0.05 0.03Fe ₂+ (FeSO ₄) mg/L 10 18 30 25Ca (CaCl ₂) mg/L 30 17 10 24Cu (CuSO ₄) % 0.4 0.5 0.6 0.8Zn (ZnSO ₄) mg/L 321 3Mn (MnCl ₂) mg/L 1123 trisodium citrate % 1.0 1.2 1.1 1.0 citric acid % 0.6 0.5 0.4 0.3

Adopt the mensuration (pressing GB9695.2-88) that is used for the fatty acid composition lipid acid that mycelial substratum that fermentative Production is rich in lipid acid produces 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 3) 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

Adopt the mensuration (pressing GB12388-90) that is used for the fatty acid composition vitamin-E that mycelial substratum that fermentative Production is rich in lipid acid produces 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 4 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

Adopt the amino acid whose mensuration of fatty acid composition that fermentative Production is rich in the mycelial substratum production of lipid acid that is used for 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 adopt and of the present inventionly to be used for the fatty acid composition that fermentative Production is rich in the mycelial substratum production of lipid acid and to contain 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 fatty acid composition 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.

Be the solid particulate of brown after the fatty acid composition vacuum-drying described in the present invention, wherein, the distribution of other composition is as follows in the fatty acid composition:

Wherein, the about 14.0g/100g of protein that contains in the described stem body fatty acid composition.

The about 0.079mg/100g. of VA that in described stem body fatty acid composition, contains

The trace element that contains in described stem body fatty acid composition comprises: the about 55.5mg/100g of iron; Zinc about 0.14; Copper about 0.28; Calcium about 375.4; Magnesium about 87.6; Potassium about 55.6; Sodium about 244; Manganese about 3.04; Phosphorus about 2125.8; Selenium about 1.65.

The distribution of other composition is as follows in the described stem body fatty acid composition:

Standard model 13 samples 14 samples 15 samples 16 total fatty %＞30 44.4 46.1 47.9 48.3 linolenic acid %＞6 4.2 9.7 19 13.7 acid numbers＜4 1.6 1.50 1.4--

Being exclusively used in of the present invention relates to substratum that fermentation method prepares fatty acid composition can stably obtain being rich in lipid acid in conjunction with technology of the present invention, especially is rich in the linolenic lipid acid of r-; Described substratum is used for mortierella sp spawn culture, fermentation and production; The fatty acid composition of being produced or be rich in that r-linolenic acid fatty acid composition composition is basicly stable, content of effective is high, and do not have toxic side effect.

Claims (4)

1, a kind ofly is exclusively used in the substratum that fermentative Production is rich in r-linolenic acid fatty acid composition, it is characterized in that described substratum comprises potato substratum, seed culture medium and fermention medium.

2, substratum according to claim 1 is characterized in that described potato substratum (doing the inclined-plane) comprising:

Potato 20-30%

Agar 1.5-2%

Glucose 2-3%

3, substratum according to claim 1 is characterized in that described seed culture medium comprises:

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%

4, substratum according to claim 1 is characterized in that described fermention medium comprises:

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/L

Mn(MnCL2) 1-3mg/L

Trisodium citrate 1.0-1.2%

Citric acid 0.3-0.6%

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