CN1329519C - Gamma-linolenic acid dry microbial powder enriched with organic celenium and its producing process - Google Patents
Gamma-linolenic acid dry microbial powder enriched with organic celenium and its producing process Download PDFInfo
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Abstract
The present invention relates to organic selenium enriched gamma-linolenic acid dry bacteria powder, a production process thereof and an induced mutagenesis strain used for the biosynthesis of the bacteria powder. Mutagenic cunninghamella echinulata CGMCC NO. 1652 is adopted to be fermented in the mode of selenium enriching on a medium which comprises an organic carbon source, an organic nitrogen source, inorganic salts and sodium selenite, and an organic selenium enriched gamma-linolenic acid bacteria body is obtained; then the gamma-linolenic acid dry bacteria powder is obtained through the adoption of microwave vacuum freezing sublimation drying technique, the content of grease is from 35 to 39%, the content of gamma-linolenic acid is from 15 to 18%, and the content of selenium is from 139 to 330 mu g/g. Through multiple production experiments, the present invention has the characteristics of mature technique, simple process, green, environmental protection adaption ot large-scale commercial process, extensive source of raw materials, low cost, good quality, etc. The product which is the organic selenium enriched gamma-linolenic acid dry bacteria powder can be extensively applied to fields of medicine, food, feed additives, etc.
Description
Technical field
The present invention relates to a kind of gamma-linolenic acid dry bacterial powder and production technique thereof, specifically, relate to gamma-linolenic acid dry bacterial powder product and technology thereof that a kind of mutagenic strain fermentative production of utilizing cunninghamella echinulata is rich in organoselenium.
Background technology
Gamma-linolenic acid (Gamma-Linolenic Acid) is called for short GLA, molecular formula: C
18H
30O
2, be a kind of punicic acid, belong to polyenoid class active substance.GLA is the unsaturated fatty acids of needed by human, is the material of big class mediator somatocyte self vigor.It has treatment hypertension, hyperlipidemia, hyperglycemia, diabetes, correction lipid metabolism disorders, treatment liver cirrhosis and nephrotic syndrome, anticancer breeding, strengthens the effect of immune function of human body.
Be from plants such as root of Redsepal Eveningprimrose, to extract gamma-linolenic acid always in the prior art, but, do not satisfy the demand of domestic and international market far away because the shortage of raw material causes the output of gamma-linolenic acid to be restricted.Develop a kind of method of biosynthesizing gamma-linolenic acid in recent years, adopt microbial strains fermentative production gamma-linolenic acid bacterium powder such as mortierella, little Ke Yinhan be mould.Biosynthetic gamma-linolenic acid main component, except that containing polyenoid linolic acid (main component of Maitong) and linolenic acid, also contain anti-mutation, radioprotective, antifatigue, antidotal superoxide-dismutase (SOD), strengthen the macromolecule polysaccharide of body immunity, and protein, multiple amino acids, VITAMIN and trace element.The comprehensive utilization of its plurality of active ingredients, a large amount of clinical pathologies of warp experiment showed, evident in efficacy, without any side effects, and do not contain any hormone.
The selenium element is the indispensable trace element of humans and animals vital movement.Scientific research shows that more than 40 kind of disease is relevant with the selenium element, and scarce selenium can cause anaemia, cardiovascular and cerebrovascular diseases, and hepatopathy, diabetes, cancer, childhood development is bad, human body premature aging etc.The selenium element is the active centre of multiple antioxidase, and its resistance of oxidation is 400 times of Ve.It is the division and the propagation of anticancer effectively, strengthens body immunity.Replenishing of selenium can purify the blood, strengthen myocardium power.There is the people that pure product of linolenic acid and selenium element are adopted the synthetic selenizing linolenic acid of chemical process at present, obtained the tangible product of antitumous effect.But have chemical lyase residual, harmful in the product that this employing chemosynthesis obtains, this problem is still unresolved at present.
Summary of the invention
One of purpose of the present invention provides a kind of novel process of utilizing microbe fermentation method (industrialization) production to be rich in the gamma-linolenic acid dry bacterial powder of organoselenium; Two of purpose has provided a kind of without any the residual gamma-linolenic acid dry bacterial powder product that is rich in organoselenium of chemical lyase; Three of purpose has provided the bacterial strain of anti-selenium of the high yield GLA that a strain obtains through mutagenesis.
The present invention adopts following technical proposals to solve the foregoing invention purpose:
1. bacterial strain mutagenesis: cunninghamella echinulata (Cunninghamella echinulata) the CGMCC NO.1652 that the present invention uses, be to be original strain, through mutagenic obtained with the AS3.2473 cunninghamella echinulata that China Microbial Culture Preservation Commission common micro-organisms center provides.Concrete mutagenic processes is: children mycelia in age is handled the acquisition protoplastis, through microwave, nitrosoguanidine Combined Processing, bacteria suspension after handling is coated on the PDA flat board that closes Sodium Selenite 10mg/L-200mg/L cultivates, choose the bacterial strain of form notable difference, after obtain through shaking bottle final election.This bacterial strain is compared with original strain has fast growth, and spore quantity is many, characteristics such as mycelia is sturdy, the back stabilization characteristics of genetics that goes down to posterity.
This bacterial strain on the PDA substratum, 28 ℃ of cultivations of temperature.2 days colony diameters are 2-5cm, and mycelia is a canescence, have the part conidium to form; Colony diameter was 4-10cm in 3-4 days, and conidium forms in a large number, and bacterium colony is a Dark grey.
The mycelia initial stage does not have separation, forms tabula later on.The sturdy multi-branched of mycelia, the about 11-19 micron of diameter.Sporangiophore is upright, branch is umbrella.Sporocyst Dan Sheng, spherical in shape.Conidium is spherical or oval, on lunge is arranged, and can come off.Spore diameter 9.5-13.2 micron.The conidium sympodium, plum blossom shape or irregular.
2. planting liquid cultivates: the spore suspension of making is inoculated in the seeding tank cultivates.The composition of substratum comprises: sucrose (or glucose) 3-5%, peptone 0.05-0.1%, potassium primary phosphate 0.01-0.03%, sal epsom 0.01-0.03%, lime carbonate 0.03-0.05%, Sodium Selenite 0.0005-0.001%, PH5.5-6, temperature 22-30 ℃, incubation time 24-36 hour.
3. fermentor cultivation: well-grown seed liquor is inserted in the fermentor tank.The composition of fermention medium comprises: sucrose (or starch or molasses) 8-15%, groundnut meal 1-5%, peptone 1-5%, potassium primary phosphate 0.01-0.03%, sal epsom 0.01-0.03%, lime carbonate 0.03-0.05%, Sodium Selenite 0.0005-0.001%, the somatomedin trace, PH5.5-6, temperature 22-30 ℃, air quantity 1:0.05-0.5v/v, stirring velocity 150-200rpm, tank pressure 0.02-0.08mpa, incubation time 96-100 hour.
4. microorganism collection: adopt microwave vacuum freeze sublimation drying method to carry out drying the gamma-linolenic acid fresh thalli that obtains.Reaction parameter is as follows:
Vacuum chamber: diameter 1.6m, long 13m, vacuum tightness 30mbar
Input microwave frequency: 2450MHz ± 50MHz
Input microwave power: 48KW ± 2KW
Use glass reinforced TFE transport tape to transmit.
To be placed on the travelling belt at the gamma-linolenic acid fresh thalli of-5 ℃ of following prefreezings, import microwave energy under the low pressure of 80-100T, heated 40 minutes, and keep the hot air convection of temperature 15-20%, evaporate moisture content from inside to outside.4-6 hour altogether time of drying.
Technical target of the product of the present invention is as follows: in every 100g dry bacterial powder,
Fat content: 35-39%
Gamma-linolenic acid content: 15-18%
Selenium content: 139-330 μ g/g
Aminoacids complex: 3.5-6.8g
Protein-polysaccharide: 4-7g
Superoxide-dismutase (SOD): 543.7 μ g/g
Adopt above-mentioned technical scheme to implement the present invention, its main beneficial effect is as follows:
1. use the strain fermentation of mutagenesis to produce gamma-linolenic acid, in screening high yield GLA bacterial strain, with the substratum that contains high density selenium bacterial strain is cultivated, filter out the bacterial strain of anti-selenium (the not ability selenium growth of common cunninghamella echinulata) that contains selenium, and in fermentation culture, taked corresponding condition, obtained the gamma-linolenic acid dry bacterial powder product that not only contains GLA but also be rich in organoselenium, wherein the content of gamma-linolenic acid reaches as high as 18% at 12-15%.Simultaneously, contain abundant selenium, selenium content is up to 139-330 μ g/g.Experiment showed, that multiple synergy with physiological function material can obviously improve the effect of product.
2. in the preparation process, main raw material (carbon source, nitrogenous source) all adopts natural organic matter, does not use inorganic raw material, has improved the level of safety of product, environmental protection.
3. at characteristics such as product fat content height, easy oxidations, adopt microwave vacuum freeze sublimation drying method that product is carried out drying.This method is that vacuum microwave drying and vacuum freeze-drying are combined together, and microwave mainly plays the effect of heating herein.Past adds devices such as thermal recovery electrically heated or heat pump set in the vacuum freezing sublimating technologe, behind the employing microwave, can cancel said apparatus.Cryodesiccated ultimate principle, the three-state that is based on water changes, and three kinds of phases not only can be changed mutually but also can be coexisted.When water when triple point (0.01 ℃ of temperature, water vapor pressure 610.5Pa), water, ice, water vapour three can coexist and balance mutually.Under high vacuum state, utilize the distillation principle, make moisture content in the gamma-linolenic acid thalline that freezes in advance without the dissolving of ice, be that water vapour is removed directly, thereby reach cryodesiccated purpose with the distillation of ice attitude.Freeze dried gamma-linolenic acid thalline becomes spongy, no drying shrinkage, fabulous, the moisture branch of rehydration few, can long-time at normal temperatures storage and transport behind the corresponding package.With respect to other drying means, the advantage of microwave vacuum freeze sublimation drying method is as follows:
Sex change or inactivation can not take place in many heat-sensitive substances;
When dry at low temperatures, some the volatile component losses in the gamma-linolenic acid thalline are very little;
Under the low temperature, the effect of microbial growth and enzyme can't be carried out, so product can keep original state;
Owing under frozen state, carry out drying, so small product size is almost constant, has kept the original structure of gamma-linolenic acid thalline, concentration phenomena can not take place;
Because the form with ice crystal after pre-freeze of the moisture in the gamma-linolenic acid thalline exists, inorganic salts material originally soluble in water is distributed in the thalline of gamma-linolenic acid uniformly.During distillation, dissolved substance soluble in water is just separated out, and has avoided in the general drying means gamma-linolenic acid thalline internal moisture to separate out on the surface to the entrained inorganic salt of surface transport and causes case-hardened phenomenon;
Dried gamma-linolenic acid thalline is loose porous, is spongy, adds that dissolving almost recovers original state rapidly and fully immediately behind the water;
Because drying is carried out under vacuum, oxygen is few, so some readily oxidizable substances have obtained protection;
The dry moisture that can get rid of more than the 95-99% makes dried gamma-linolenic acid thalline energy prolonged preservation and unlikely going bad.
Because the existence of above-mentioned advantage, adopt this method to carry out drying and can improve every technical indicator 1-3 percentage point of gamma-linolenic acid dry bacterial powder, effectively improve the added value of gamma-linolenic acid dry bacterial powder.
4. the gamma-linolenic acid dry bacterial powder that is rich in organoselenium that adopts biological fermentation process to obtain is compared with the selenizing linolenic acid of chemosynthesis, residual without any chemical lyase, without any side effects to human body, and multiple synergy with physiological function material can obviously improve the effect of product.
5. produce the fermentation whole process, set up aseptic area and adopt totally-enclosed operation, the computer complete monitoring can guarantee the quality of gamma-linolenic acid dry bacterial powder to reduce industrial production cost.
The cunninghamella echinulata (Cunninghamellaechinulata) that the present invention uses is preserved in China Microbial Culture Preservation Commission common micro-organisms center on March 16th, 2006, and preserving number is CGMCC NO.1652.
Embodiment
The following example will further specify the present invention.
Embodiment 1:1 ton fermentor tank is produced thalline
1. slant strains CGMCC NO.1652 is inoculated in enlarged culturing in the Kolle flask, temperature 22-30 ℃, cultivated 3-5 days.
2. bacterial classification is made bacterial suspension inoculation in seeding tank, the seeding tank volume is 100 liters, substratum: sucrose 4kg, peptone 0.4kg, potassium primary phosphate 20g, sal epsom 20g, lime carbonate 35g, Sodium Selenite 2.1g, PH5.5-6, temperature 25-30 ℃, air quantity 1: 0.5v/v, stirring velocity 150-200rpm, incubation time are 32 hours.
3. seed liquor is transferred in the fermentor tank 1000 liters of fermentor tank volumes.Substratum: sucrose 72kg, groundnut meal 4kg, potassium primary phosphate 200g, sal epsom 200g, lime carbonate 350g, Sodium Selenite 21g, somatomedin 2.3g, PH5.5-6, temperature 22-30 ℃, air quantity 1: 0.25-0.55v/V, stirring velocity 150-200rpm, tank pressure 0.02-0.08mpa cultivated 96 hours.
4. microorganism collection adopts the microwave vacuum freeze sublimating technologe, input microwave frequency 2400MHz, and input microwave power 46KW heated 40 minutes, and kept the hot air convection of temperature 15-20%, dry 4-6 hour.Get thalline 31.5kg, fat content 35.5% in wherein every 100g dry bacterial powder, GLA content 12.5%, selenium content 139.2 μ g/g.
Embodiment 2:5 ton fermentor tank is produced thalline
1. slant strains CGMCC NO.1652 is inoculated in enlarged culturing in the Kolle flask, temperature 22-30 ℃, cultivated 3 days.
2. bacterial classification is made bacterial suspension inoculation in seeding tank, the seeding tank volume is 500 liters, substratum: sucrose 15kg, peptone 3.5kg, potassium primary phosphate 90g, sal epsom 90g, lime carbonate 150g, Sodium Selenite 12g, PH5.5-6, temperature 22-30 ℃, air quantity 1: 0.3v/v, stirring velocity 150-200rpm, tank pressure 0.02-0.08mpa, incubation time are 28 hours.
3. seed liquor is transferred in the fermentor tank 5000 liters of fermentor tank volumes.Substratum: sucrose 400kg, groundnut meal 35kg, potassium primary phosphate 900g, sal epsom 900g, lime carbonate 1500g, Sodium Selenite 120g, somatomedin 10.5g, PH5.5-6, temperature 22-30 ℃, fermentation is during to sugared content 4%, add the molasses after 100kg sterilizes, air quantity 1: 0.1-0.3v/v, stirring velocity 150-200rpm, tank pressure 0.02-0.08mpa cultivated 100 hours.
4. microorganism collection is with embodiment 1.Get thalline 128kg, in wherein every 100g dry bacterial powder, fat content 32.3%, GLA content 18.1%, selenium content 142.4 μ g/g.
Embodiment 3:5 ton fermentor tank is produced thalline
1. slant strains CGMCC NO.1652 is inoculated in enlarged culturing in the Kolle flask, temperature 22-30 ℃, cultivated 3-4 days.
2. bacterial classification is made bacterial suspension inoculation in seeding tank, the seeding tank volume is 500 liters, substratum: sucrose 15kg, peptone 3.5kg, potassium primary phosphate 90g, sal epsom 90g, lime carbonate 150g, Sodium Selenite 12g, PH5.5-6, temperature 25-30 ℃, air quantity 1: 0.3v/v, stirring velocity 150-200rpm, tank pressure 0.02-0.08mpa, incubation time are 30 hours.
3. seed liquor is transferred in the fermentor tank 5000 liters of fermentor tank volumes.Substratum: sucrose 400kg, groundnut meal 35kg, potassium primary phosphate 900g, sal epsom 900g, lime carbonate 1500g, Sodium Selenite 120g, somatomedin 10.5g, PH5.5-6, temperature 25-30 ℃, fermentation is during to sugared content 10%, add the molasses after 80kg sterilizes, air quantity 1: 0.1-0.3v/v, stirring velocity 150-200rpm, tank pressure 0.02-0.08mpa cultivated 100 hours.
4. microorganism collection is with embodiment 1.Get thalline 120kg, fat content 37.3% in wherein every 100g dry bacterial powder, GLA content 16.2%, selenium content 168.5 μ g/g.
Embodiment 4:5 ton fermentor tank is produced thalline
1. slant strains CGMCC NO.1652 is inoculated in enlarged culturing in the Kolle flask, temperature 22-30 ℃, cultivated 3-4 days.
2. bacterial classification is made bacterial suspension inoculation in seeding tank, wherein temperature is 22 ℃, tank pressure 0.05mpa, and all the other are with embodiment 3.
3. seed liquor is transferred in the fermentor tank, wherein temperature is 30 ℃, tank pressure 0.08mpa, and stirring velocity 180rpm, all the other are with embodiment 3.
4. microorganism collection is with embodiment 1.Get thalline 118kg, fat content 35.2% in wherein every 100g dry bacterial powder, GLA content 17.0%, selenium content 156.3 μ g/g.
Comparative example 1:1 ton fermentor tank is produced thalline
1. with embodiment 1.2. with embodiment 1.3. with embodiment 1.
4. microorganism collection adopts sulfuration bed desiccating method.6-8 hour time of drying, temperature 50-70 ℃.
The result relatively
| Sulfuration bed desiccating method | Microwave vacuum freeze sublimation drying method | |
| Drying mode | Open, easily microbiological contamination | Vacuum is difficult for microbiological contamination |
| Heat-sensitive materials | Easily oxidation | Be difficult for oxidation |
| Fat content | 33.0％ | 35.5％ |
| GLA content | 10.2％ | 12.5％ |
| Selenium content | 110.4μg/g | 139.2μg/g |
| The compounded amino acid content | 3.0g | 5.8g |
| Proteoglycan content | 3.2g | 6.9g |
| SOD content | 540.1μg/g | 543.7μg/g |
| Preserving type | Cryopreservation | Normal temperature is preserved |
Comparative example 2:5 ton fermentor tank is produced thalline
1. with embodiment 3.
2. with embodiment 3.
3. with embodiment 3.
4. microorganism collection adopts sulfuration bed desiccating method.6-8 hour time of drying, temperature 50-70 ℃.
The result relatively
| Sulfuration bed desiccating method | Microwave vacuum freeze sublimation drying method | |
| Fat content | 32.7％ | 37.3％ |
| GLA content | 9.8％ | 16.2％ |
| Selenium content | 140.0μg/g | 168.5μg/g |
| The compounded amino acid content | 4.5g | 6.8g |
| Proteoglycan content | 3.6g. | 7.0g |
| SOD content | 538.2μg/g | 543.7μg/g |
What deserves to be explained is that the present invention can summarize with other the specific form without prejudice to spirit of the present invention or principal character.Therefore, no matter from which point, above-mentioned embodiment of the present invention all can only be thought can not limit the present invention to explanation of the present invention.
Claims (6)
1. production method that is rich in the gamma-linolenic acid of organoselenium is characterized in that: adopting cunninghamella echinulata (Cunninghamella echinulata) CGMCC NO.1652 is that bacterial strain carries out rich selenium fermentation, may further comprise the steps:
(1) spawn culture: slant strains inserted carry out enlarged culturing in the Kolle flask;
(2) planting liquid cultivates: spore suspension is inoculated in the seeding tank cultivates, substratum is a carbon source with glucose and/or sucrose, is nitrogenous source with the peptone, and adds Sodium Selenite, PH5.5-6, and temperature 22-30 ℃, incubation time is 24-36 hour;
(3) fermentation culture: will cultivate in well-grown seed liquor access fermentor tank, substratum is so that any one or a few is a carbon source in sucrose, molasses, the starch, with groundnut meal and/or peptone is nitrogenous source, and the interpolation Sodium Selenite, PH5.5-6, temperature 22-30 ℃, tank pressure 0.02-0.08mpa, air quantity 1:0.05-0.5v/v, stirring velocity 150-200rpm, incubation time 96-100 hour;
(4) microorganism collection: adopt microwave vacuum freeze sublimation drying method drying, wherein the condition of microwave vacuum freeze sublimation drying is: input microwave frequency 2450MHz ± 50MHz, input microwave power 48KW ± 2KW, microwave heating 40 minutes, and the hot air convection of maintenance temperature 15-20%, be 4-6 hour time of drying, must be rich in the gamma-linolenic acid dry bacterial powder of organoselenium.
2. production method according to claim 1, the condition of wherein said spawn culture is: the PDA substratum, temperature 22-30 ℃, cultivated 3-5 days.
3. production method according to claim 1, wherein seed culture medium comprises: sucrose 3-5%, peptone 0.05-0.1%, potassium primary phosphate 0.01-0.03%, sal epsom 0.01-0.03%, lime carbonate 0.03-0.05%, Sodium Selenite 0.0005-0.001%.
4. production method according to claim 1, wherein fermention medium comprises: sucrose 8-15%, groundnut meal 1-5%, peptone 1-5%, potassium primary phosphate 0.01-0.03%, sal epsom 0.01-0.03%, lime carbonate 0.03-0.05%, Sodium Selenite 0.0005-0.001%, somatomedin trace, incubation time 96 hours.
5. according to any gamma-linolenic acid dry bacterial powder that described method is produced of claim 1-4.
6. cunninghamella echinulata (Cunninghamella echinulata) CGMCC NO.1652.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4564634A (en) * | 1982-05-28 | 1986-01-14 | F.C.N.S.R.L. | Selenium compounds having antineoplastic activity, process for the preparation thereof and pharmaceutical compositions therefrom |
| CN1069070A (en) * | 1991-07-30 | 1993-02-17 | 中国科学院沈阳应用生态研究所 | Method for preparing gamma-linolenic acid and biological preparation mainly containing gamma-linolenic acid |
| CN1255540A (en) * | 1999-11-12 | 2000-06-07 | 沈阳大有生物工程有限责任公司 | Process for preparing biologic preparation containing linolenic acid |
-
2006
- 2006-03-29 CN CNB2006100658897A patent/CN1329519C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4564634A (en) * | 1982-05-28 | 1986-01-14 | F.C.N.S.R.L. | Selenium compounds having antineoplastic activity, process for the preparation thereof and pharmaceutical compositions therefrom |
| CN1069070A (en) * | 1991-07-30 | 1993-02-17 | 中国科学院沈阳应用生态研究所 | Method for preparing gamma-linolenic acid and biological preparation mainly containing gamma-linolenic acid |
| CN1255540A (en) * | 1999-11-12 | 2000-06-07 | 沈阳大有生物工程有限责任公司 | Process for preparing biologic preparation containing linolenic acid |
Non-Patent Citations (1)
| Title |
|---|
| 硒化亚麻酸的抗癌作用研究 薛少安 等，营养学报，第14卷第4期 1992 * |
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