CN103789358A - Method for preparing 2-phenethyl alcohol by adopting antrodia cinnamomea submerged fermentation technology - Google Patents
Method for preparing 2-phenethyl alcohol by adopting antrodia cinnamomea submerged fermentation technology Download PDFInfo
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- CN103789358A CN103789358A CN201410041209.2A CN201410041209A CN103789358A CN 103789358 A CN103789358 A CN 103789358A CN 201410041209 A CN201410041209 A CN 201410041209A CN 103789358 A CN103789358 A CN 103789358A
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Abstract
The invention relates to a method for preparing 2-phenethyl alcohol by adopting an antrodia cinnamomea submerged fermentation technology. The method comprises the following steps: (1) strain activation; (2) seed liquid preparation; 3) submerged fermentation cultivation of liquid; (4) solid-liquid separation and extraction of the fermentation liquor. The method is simple and convenient in production technology, and has a broad industrial development prospect; a fermentation culture solution is separated into mycelium and the fermentation liquor after being filtered, 2-phenethyl alcohol is obtained after the fermentation liquor is extracted, and the fermented mycelium can be applied to other biological researches; the method has important significance on effective utilization of agricultural by-products and wastes, improvement of additional value of edible and medicinal fungi industries, and comprehensive utilization of agriculture and forestry resources.
Description
Technical field
The invention belongs to the preparation field of 2 phenylethyl alcohol, particularly a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol.
Background technology
2 phenylethyl alcohol is quite popular to people because of its soft sweet fragrance, and in daily production and life, 2 phenylethyl alcohol is widely used at industrial circles such as cosmetics of everyday use, food, tobaccos.But also there is the effect that strengthens fragrance, can be for allocating multiple different odor type.In production of flavor field, 2 phenylethyl alcohol is also applied wide model, is second largest fragrance component, can allocate the essence of multiple odor type and use as foodstuff additive, increases the fragrance in food.At home and abroad, be applied at present in the industry such as medicine, makeup, and multiple derivative using value take it as raw material production is also very huge, as its ester derivative-Phenylethyl ethanoate etc., these derivatives also, because its fragrance is like rose and be with sweetly, can be used for daily use chemicals and food flavour equally.
By the end of at present, 2 phenylethyl alcohol is all cheap material chemosynthesis substantially, only has little directly extracting from rose.And the 2 phenylethyl alcohol market value of chemosynthesis is about 3.5 $/kg, natural 2 phenylethyl alcohol price is but up to 1000 $/kg.Along with scientific and technological development, the significantly improving of people's living standard, the enhancing of health perception, the consequent is the problem of food safety.For people's health, countries in the world also can be clearer and more definite for the restriction of food-processing aspect, and " green " this wording also more and more appears in the middle of our visual field.Current, the production of natural 2-benzyl carbinol in the world, mainly concentrates on the developed country such as France, Germany, and China is relatively short of in this respect, needs strengthening research.
Microorganism volume is little, breeding is fast, absorbs soon, and raw material sources are wide and low, therefore adopt the 2 phenylethyl alcohol cycle of biology-microbe transformation method production " green " short, efficiency is high, there is no the interference of other compounds etc., advantage is remarkable, therefore the quality of product also meets international standards than being easier to.Chinese patent CN1403581A discloses a kind of using tobacco as raw material, yeast fermentation method is produced the technique of perfume phenylethanol, but the method is used tobacco to do raw material, after organic solvent extraction, ion-exchange resin purification obtains phenylethyl alcohol, it is a kind of method of microbial method production crude benzene ethanol of novelty, but cost is higher, and tobacco material potential toxin situation during the fermentation does not relate to, and cheap, the safe agricultural byproducts of use, obtain the method for natural 2 phenylethyl alcohol by microbial method, become a kind of active demand.
Camphor tree sesame (Antrodia camphorata) claim again Antrodia camphorata, Cinnamomum kanahirai hay mushroom etc., belongs to Basidiomycota, Basidiomycotina, and Basidiomycetes, aptychus Zoopagales, polyporaceae, platform sesame belongs to, and is the distinctive medicinal fungi in Taiwan, is mainly distributed in TaiWan, China mountain area.Its nutritive ingredient is abundant, contains the multiple physiologically active ingredients such as polysaccharide, triterpenes, steroid and antioxidant, have protect the liver, the multiple efficacies such as antitumor, anti-oxidant, immunomodulatory, removing toxic substances, anti-inflammatory, become in recent years the focus of research and development.Because Cinnamomum kanahirai hay tree is listed in rare and protected trees, and the solid culture cycle of camphor tree sesame is also long, therefore the submerged fermentation technology of camphor tree sesame is more and more by people are paid attention to.The sporophore of wild camphor tree sesame is few, and resource is deficient all the more, and large-scale artificial growth also fails really to realize, therefore liquid submerged fermentation technology, can obtain at short notice a large amount of mycelium with it, and without advantages such as the restrictions in season, become gradually the main method that camphor tree sesame is cultivated.The nearly more than ten years, the research of associated each side has all been carried out in TaiWan, China, continent, but most research mainly concentrates on mycelium and mycelia active composition aspect.The general fermentation filtered solution as waste in technological process, all topple over, but research finds wherein to contain fragrance matter 2 phenylethyl alcohol, therefore by Optimal Medium condition and fermentation condition, improve the content of 2 phenylethyl alcohol in camphor tree sesame fermented liquid, can effectively utilize fermented liquid, carry out the production of natural 2-benzyl carbinol, there is very large value of exploiting and utilizing at aspects such as medicine, food, perfume industries.Due to material safety, with low cost, can join in chewing gum, candy, refreshment, soft drink, sport-drink and milk preparation, also can be for the production of the production of shampoo, soap, body lotion and toothpaste in makeup and perfume and reodorant.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol, the method production technique is easy, there is industrialization development prospect widely, fermentation culture is divided into mycelium and fermented liquid after filtering, fermented liquid is extracted to rear acquisition 2 phenylethyl alcohol, fermentation mycelium can be used for other biological study, to effectively utilizing agricultural-food by product, waste, have great importance to improving food, the added value of medicinal fungus industry and the comprehensive utilization of agricultural resource.
A kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol of the present invention, comprising:
(1) under aseptic condition, camphor tree sesame bacterial strain (ATCC200183) is transferred on PDA inclined-plane, 26～30 ℃ of constant temperature lucifuges are cultivated 12～20d, for subsequent use after mycelia is covered with inclined-plane, transfer 2-3 time during this time;
(2) above-mentioned cultured slant strains is cut into bacterium piece (0.2～0.5cm × 0.2～0.5cm) access PD liquid nutrient medium and (take 200g potato, clean peeling section, the 1000mL that adds water boils 15min, after filtered through gauze, add deionized water and complement to 1000mL, add again 12g glucose, boil to solid and fully dissolve rear packing sterilizing, obtain PD liquid nutrient medium) in, 150～200rpm, 26～30 ℃ of shaking table constant temperature culture 6～10d; Under aseptic condition, after homogenate, access in fresh PD liquid nutrient medium, inoculum size is 10%～20%, and 150～200rpm, 26～30 ℃ of shaking table constant temperature culture 5～8d, obtain liquid seeds liquid;
(3) by after the homogenate of aforesaid liquid seed liquor, in access fermention medium, inoculum size is 10%～20%, and 100～200rpm, 23 ℃～28 ℃ ferment at constant temperature 5～6d obtain camphor tree sesame fermented liquid; Wherein, consisting of of fermention medium: 3%～8% glucide, 0.3%～0.8% nitrogenous source, 0.08%～0.2% K
2hPO
4, 0.03%～0.08% MgSO
4, 0.01% V
b1, all the other are deionized water, sterilizing 20～30min at 121 ℃;
(4) will after above-mentioned camphor tree sesame filtering fermentation liquor, obtain supernatant liquor, supernatant liquor concentrating under reduced pressure, obtains Fermented Condensed liquid; By Fermented Condensed liquid and the ultrasonic extraction of organic solvent equal-volume, combining extraction liquid, concentrating under reduced pressure, obtains the concentrated solution (HPLC detection level exceedes 50%) of 2 phenylethyl alcohol.
Glucide in described step (3) is the one in glucose, fructose, sucrose, N.F,USP MANNITOL, Zulkovsky starch, maltose, wheat bran.Wherein wheat bran needs 100 ℃ of boiling water 1 hour, the centrifugal 10min of 5000g.
Nitrogenous source in described step (3) is the one in yeast powder, peptone, extractum carnis, analysis for soybean powder, ammonium sulfate.Wherein analysis for soybean powder granularity is for by 100～200 sieve apertures, 100 ℃ of boiling water baths 1 hour, the centrifugal 10min of 5000g.
Consisting of of fermention medium in described step (3): 5% glucose, 0.5% yeast powder, 0.05%MgSO
4, 0.1%K
2hPO
4and 0.01%V
b1, all the other are deionized water, sterilizing 30min at 121 ℃.
When described step (3) amplification culture, pass into sterile air, every liter of substratum air flow is 1L/min.
In described step (3), inoculum size is 20%, 150rpm, 26 ℃～28 ℃ ferment at constant temperature.
10 times of supernatant liquor concentrating under reduced pressure in described step (4); 40 times of extraction liquid concentrating under reduced pressure.
Organic solvent in described step (4) is chloroform, ethyl acetate, ether, sherwood oil, Skellysolve A, Skellysolve A: methylene dichloride=2:1(V/V), Skellysolve A: ether=1:1(V/V), one in methylene dichloride.Take Skellysolve A: methylene dichloride=2:1(V/V) as best.
Extraction temperature in described step (4) is 10～40 ℃, and the pH of Fermented Condensed liquid is 3～5.
beneficial effect
Production technique of the present invention is easy, there is industrialization development prospect widely, fermentation culture is divided into mycelium and fermented liquid after filtering, fermented liquid is extracted to rear acquisition 2 phenylethyl alcohol, fermentation mycelium can be used for other biological study, to effectively utilizing agricultural-food by product, waste, have great importance to improving food, the added value of medicinal fungus industry and the comprehensive utilization of agricultural resource.
Accompanying drawing explanation
Fig. 1 is that inoculum size is on producing the impact of 2 phenylethyl alcohol;
Fig. 2 is that rotating speed is on producing the impact of 2 phenylethyl alcohol;
Fig. 3 is that culture temperature is on producing the impact of 2 phenylethyl alcohol.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
The technique of camphor tree sesame submerged fermentation 2 phenylethyl alcohol, concrete zymotechnique is as follows:
First order seed is cultivated: the bacterium piece that will cultured fresh slant strains cuts into 0.2～0.5cm × 0.2～0.5cm accesses in PD liquid nutrient medium 4～5,150rpm, and 26 ℃ of shaking table constant temperature culture 6d, little mycelium pellet is covered with after shaking flask for subsequent use.
Secondary seed is cultivated: primary seed solution, under aseptic condition, after the homogenate of homogeneous instrument, access in fresh PD liquid nutrient medium, and inoculum size is 10%～20%, 150rpm, after 26 ℃ of constant-temperature shaking culture 5d, little mycelium pellet is covered with shaking flask and is liquid seeds liquid.Take out, after the homogenate of homogeneous instrument, in access fermention medium.
Shake flask fermentation: the culture medium prescription of shake flask fermentation is: 50g/L glucose (or the one in other glucides), 5g/L yeast powder, 0.5g/L magnesium sulfate, the dipotassium hydrogen phosphate of 1g/L, V
b1100mg/L, natural pH, inoculum size is 5-30%, and liquid amount is 120mL/250mL triangular flask, and rotating speed is 150r/min, 26 ℃ of constant temperature culture 5d, after fermentation ends, sampling is measured the 2 phenylethyl alcohol content in camphor tree sesame fermented liquid by HPLC method, the results are shown in Figure shown in 1.
As seen from Figure 1, preferred fermentation condition is: inoculum size is with 20%(V/V) best, inoculum size directly affects the length of fermentation period, in substratum, access a large amount of mycelium, can shorten fermentation period, thereby save the power consumption in fermenting process, but, inoculum size is too much, can cause the deficiency of nutriment and oxygen supply in substratum, thereby affects the metabolism and growth of thalline.Leavening temperature 26-28 ℃, in final fermented liquid, the content of 2 phenylethyl alcohol reaches 1.13g/L.
Embodiment 2
Except shaking speed in shake flask fermentation step is 90-170rpm, all the other processing condition are all with embodiment 1.
After fermentation ends, sampling is measured the 2 phenylethyl alcohol content in camphor tree sesame fermented liquid by HPLC method, the results are shown in Figure shown in 2.
As seen from Figure 2, suitable fermentation condition is:
Inoculum size 20%(V/V) be best, shaking speed is that 150rpm is that (shaking speed is passed through the metabolism growth that affects dissolved oxygen and then affect microorganism to the best, but in the time that rotating speed is too fast, will affect thalline, particularly the mycelia of macro fungi can be cut off).Now in fermented liquid, the content of 2 phenylethyl alcohol reaches 1.18g/L.
Embodiment 3
Except culture temperature is 23-38 ℃, all the other processing condition are all with embodiment 1.
After fermentation ends, sampling is measured the 2 phenylethyl alcohol content in camphor tree sesame fermented liquid by HPLC method, the results are shown in Figure shown in 3.
As seen from Figure 3, suitable fermentation condition is:
Inoculum size 20%(V/V) be best, shaking speed is that 150rpm is best, culture temperature is 26-28 ℃, and best (temperature can affect in organism enzymic activity and then have influence on microbial growth metabolism, and the needed optimum temperuture of different microorganisms and meta-bolites is neither identical), the content of the man of wild origin's 2 phenylethyl alcohol that now ferments reaches 1.19g/L.
From embodiment 1～3, under top condition, camphor tree sesame deep liquid is cultivated and is produced 2 phenylethyl alcohol, has obtained good effect, and for this reason, amplification culture below is further set forth content of the present invention.
Embodiment 4
Scale-up: in the fermentor tank of a 30L, take camphor tree sesame bacterial strain as bacterial classification, play the cultural method of seed, the proportioning of fermention medium, and the sterilising conditions of fermention medium etc. is all with embodiment 1, putting tank volume is 20L, fermentation condition is inoculum size 20%(V/V), pass into sterile air, air flow is 20L/min, mixing speed is 100-200rpm, 26 ℃～28 ℃ ferment at constant temperature 5 days, put after tank, fermented liquid 10-30 ℃, the centrifugal 10min of 5000g, can obtain Antrodia Camphorata mycelium and fermented supernatant fluid, the wherein about 20L of supernatant liquor, the content of 2 phenylethyl alcohol is 1.25g/L, total amount is 25g.After fermented supernatant fluid concentrating under reduced pressure, obtain the Fermented Condensed liquid of 2L, stand-by.
Fermented supernatant fluid organic solvent extractionprocess extracts 2 phenylethyl alcohol, and extractant condition is:
Skellysolve A: methylene dichloride=2:1(V/V) be organic phase, Fermented Condensed liquid is water, water/organic phase=1(V/V), extraction temperature is 10-40 ℃, pH=3～5 of water, ultrasonic extraction three times, combining extraction liquid, after extraction liquid concentrating under reduced pressure, can obtain 2 phenylethyl alcohol product 50mL, 2 phenylethyl alcohol content is 458g/L, and total yield is 91.6%(take the total content of 2 phenylethyl alcohol in fermented liquid as standard), reclaim solvent, recycle.
Embodiment 5
Except organic extraction is chloroform mutually, other condition is with embodiment 4, after extraction liquid concentrating under reduced pressure, can obtain 2 phenylethyl alcohol product 49mL, 2 phenylethyl alcohol content is 437g/L, and total yield is 85.7%(take the total content of 2 phenylethyl alcohol in fermented liquid as standard), the chloroform of recovery recycles.
From embodiment 4～5, organic solvent extraction, several conventional organic solvents can reach reasonable extraction yield, are all the good methods of separation and purification 2 phenylethyl alcohol from fermented liquid; Can meet the needs that suitability for industrialized productionization is produced.
Claims (10)
1. adopt camphor tree sesame submerged fermentation technology to prepare a method for 2 phenylethyl alcohol, comprising:
(1) under aseptic condition, camphor tree sesame bacterial strain is transferred on PDA inclined-plane, 26～30 ℃ of constant temperature lucifuges are cultivated 12～20d, for subsequent use after mycelia is covered with inclined-plane, transfer 2-3 time during this time;
(2) above-mentioned cultured slant strains is cut in bacterium piece access PD liquid nutrient medium to 150～200rpm, 26～30 ℃ of shaking table constant temperature culture 6～10d; Under aseptic condition, after homogenate, access in fresh PD liquid nutrient medium, inoculum size is 10%～20%, and 150～200rpm, 26～30 ℃ of shaking table constant temperature culture 5～8d, obtain liquid seeds liquid;
(3) by after the homogenate of aforesaid liquid seed liquor, in access fermention medium, inoculum size is 10%～20%, and 100～200rpm, 23 ℃～28 ℃ ferment at constant temperature 5～6d obtain camphor tree sesame fermented liquid; Wherein, consisting of of fermention medium: 3%～8% glucide, 0.3%～0.8% nitrogenous source, 0.08%～0.2% K
2hPO
4, 0.03%～0.08% MgSO
4, 0.01% V
b1, all the other are deionized water, sterilizing 20～30min at 121 ℃;
(4) will after above-mentioned camphor tree sesame filtering fermentation liquor, obtain supernatant liquor, supernatant liquor concentrating under reduced pressure, obtains Fermented Condensed liquid; By Fermented Condensed liquid and the ultrasonic extraction of organic solvent equal-volume, combining extraction liquid, concentrating under reduced pressure, obtains the concentrated solution of 2 phenylethyl alcohol.
2. a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol according to claim 1, is characterized in that: the glucide in described step (3) is the one in glucose, fructose, sucrose, N.F,USP MANNITOL, Zulkovsky starch, maltose, wheat bran.
3. a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol according to claim 1, is characterized in that: the nitrogenous source in described step (3) is the one in yeast powder, peptone, extractum carnis, analysis for soybean powder, ammonium sulfate.
4. a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol according to claim 1, is characterized in that: the consisting of of the fermention medium in described step (3): 5% glucose, 0.5% yeast powder, 0.05%MgSO
4, 0.1%K
2hPO
4and 0.01%V
b1, all the other are deionized water, sterilizing 30min at 121 ℃.
5. a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol according to claim 1, is characterized in that: when described step (3) amplification culture, pass into sterile air, every liter of substratum air flow is 1L/min.
6. a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol according to claim 1, is characterized in that: in described step (3), inoculum size is 20%, 150rpm, 26 ℃～28 ℃ ferment at constant temperature.
7. a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol according to claim 1, is characterized in that: 10 times of supernatant liquor concentrating under reduced pressure in described step (4); 40 times of extraction liquid concentrating under reduced pressure.
8. a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol according to claim 1, is characterized in that: the organic solvent in described step (4) is one or more in chloroform, ethyl acetate, ether, sherwood oil, Skellysolve A, methylene dichloride.
9. a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol according to claim 8, is characterized in that: described organic solvent is that volume ratio is Skellysolve A and the methylene dichloride of 2:1.
10. a kind of method that adopts camphor tree sesame submerged fermentation technology to prepare 2 phenylethyl alcohol according to claim 1, is characterized in that: the extraction temperature in described step (4) is 10～40 ℃, and the pH of Fermented Condensed liquid is 3～5.
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Cited By (3)
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| CN104127366A (en) * | 2014-07-31 | 2014-11-05 | 深圳市仁泰生物科技有限公司 | Antrodia cinnamomea Chinese herbal toothpaste and preparation method thereof |
| CN104324057A (en) * | 2014-10-20 | 2015-02-04 | 三生源生物科技（天津）有限公司 | Method for extracting triterpenoids |
| WO2016015283A1 (en) * | 2014-07-31 | 2016-02-04 | 深圳市仁泰生物科技有限公司 | Antrodia cinnamomea chinese herbal toothpaste and preparation method therefor |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104127366A (en) * | 2014-07-31 | 2014-11-05 | 深圳市仁泰生物科技有限公司 | Antrodia cinnamomea Chinese herbal toothpaste and preparation method thereof |
| WO2016015283A1 (en) * | 2014-07-31 | 2016-02-04 | 深圳市仁泰生物科技有限公司 | Antrodia cinnamomea chinese herbal toothpaste and preparation method therefor |
| CN104127366B (en) * | 2014-07-31 | 2016-08-24 | 深圳市仁泰生物科技有限公司 | A kind of camphor tree sesame Chinese medicinal toothpaste and preparation method thereof |
| CN104324057A (en) * | 2014-10-20 | 2015-02-04 | 三生源生物科技（天津）有限公司 | Method for extracting triterpenoids |
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Application publication date: 20140514 |