CN109370920A - A kind of cultural method and a kind of purple sesame liquid fermentation method of purple sesame bacterial strain - Google Patents

A kind of cultural method and a kind of purple sesame liquid fermentation method of purple sesame bacterial strain Download PDF

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CN109370920A
CN109370920A CN201811511381.4A CN201811511381A CN109370920A CN 109370920 A CN109370920 A CN 109370920A CN 201811511381 A CN201811511381 A CN 201811511381A CN 109370920 A CN109370920 A CN 109370920A
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maple
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water
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CN109370920B (en
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张雷
杨淑琴
郭秀茹
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Beijing belilles Biotechnology Co., Ltd
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Qian'an Belle Biotechnology Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract

The present invention provides a kind of cultural methods of purple sesame bacterial strain, wherein in the Tube propagation stage, Tube propagation base includes: that nitrogen source, carbon source, phosphate, sulfate, agar and maple water mention solution.The present invention also provides a kind of cultural methods of purple sesame bacterial strain and a kind of purple sesame liquid fermentation method, wherein shake-flask seed culture solution and/or fermentation culture include that nitrogen source, carbon source, phosphate, sulfate and maple water mention solution.Present inventor has been surprisingly found that the purple sesame bacterial strain for wild breeding on maple, in artificial incubation, if certain density maple water is added in the medium mentions solution as nutritional supplement, the growth of purple sesame is greatly facilitated and promotes the secretion of purple sesame effective component, such as with the fermentor of identical inoculum concentration and identical capacity, the dry mycelial weight of acquisition can improve at least 1.5 times；The purple sesame polysaccharide obtained in fermentation liquid at least improves 1.6 times.

Description

A kind of cultural method and a kind of purple sesame liquid fermentation method of purple sesame bacterial strain
Technical field
The present invention relates to a kind of cultural method of purple sesame bacterial strain and a kind of purple sesame liquid fermentation methods.
Background technique
Purple sesame is the fine work of the ganoderma lucidum of Chinese tradition, and the effective component of purple sesame mainly has purple sesame polysaccharide, triterpenes (ganoderma lucidum Acid) etc..The medical value of purple sesame polysaccharide mainly has anticancer, hypoglycemic, immunological regulation, promotes protein and the synthesis of nucleic acid etc.； The medical value of ganoderic acid mainly has protect liver and toxin expelling, the release for reducing cholesterol, inhibiting histamine, inhibition angiotensin to turn Change etc..
Purple sesame manually more difficult domestication culture, thus purple sesame product can be seldom seen on the market.The study found that purple sesame mycelia Equally contain the effective component of a large amount of purple sesame, such as purple sesame polysaccharide in body.Therefore, people begin trying the mycelia of culture purple sesame Body uses purple sesame liquid fermentation to obtain the effective component of purple sesame.However, cultivating the method for purple sesame at present or using purple The effect is unsatisfactory for sesame liquid fermentation, and the concentration of obtainable purple sesame polysaccharide is not generally high in fermentation liquid, usually in 4-5 weight ‰ Left and right.
Summary of the invention
The present invention in order to overcome in the prior art by culture G.japonicum mycelium to obtain the low defect of purple sesame polysaccharide yield, Provide a kind of method for cultivating G.japonicum mycelium and a kind of purple sesame liquid fermentation method.
Therefore, on the one hand, the present invention provides a kind of cultural methods of purple sesame bacterial strain, which is characterized in that in Tube propagation Stage, Tube propagation base include: that nitrogen source, carbon source, phosphate, sulfate, agar and maple water mention solution；The nitrogen source is preferred It is preferably glucose, potato for potato, the carbon source；Wherein, the phosphate is preferably the sylvite of phosphoric acid, more preferably KH2PO4；The sulfate is preferably MgSO4。
The present invention also provides a kind of cultural methods of purple sesame bacterial strain, which is characterized in that in shaking flask cultivation stage, shaking flask kind Sub- culture solution includes that nitrogen source, carbon source, phosphate, sulfate and maple water mention solution；The nitrogen source is preferably potato or ferment The combination of female powder and potato, the carbon source are preferably glucose, potato；Wherein, the phosphate is preferably the potassium of phosphoric acid Salt and ammonium salt, more preferably KH2PO4And NH4H2PO4；The sulfate is preferably MgSO4。
The present invention also provides a kind of purple sesame liquid fermentation methods, which is characterized in that this method includes fermentation culture packet It includes nitrogen source, carbon source, phosphate, sulfate and maple water and mentions solution；The nitrogen source is preferably potato or yeast powder and Ma Ling The combination of potato, the carbon source are preferably glucose, potato；Wherein, the phosphate is preferably the sylvite and ammonium salt of phosphoric acid, more Preferably KH2PO4And NH4H2PO4；The sulfate is preferably MgSO4。
Applicant has been surprisingly found that the purple sesame bacterial strain for wild breeding on maple, in artificial incubation, if Certain density maple water is added in culture medium and mentions solution as nutritional supplement, the growth and promotion of purple sesame is greatly facilitated The secretion of purple sesame effective component, for example, by using cultural method of the invention, in the test tube stage, culture can cover with test tube in 4 days, And test tube can just be covered with by using conventional culture medium culture at least to cultivate 5 days；In the shaking flask stage, in other same culture item Under part, using the mycelium that cultural method culture of the invention is collected into be using conventional method culture collect it is mycelial extremely It is 1.5 times few；With the fermentor of identical inoculum concentration and identical capacity, the dry mycelial weight of acquisition can improve at least 1.5 (traditional jade Rice & peanut milk culture medium zymotechnique, the dry mycelial weight content in fermentation liquid are 1.1% or so) times, it can preferably improve about 2 times；Fermentation The purple sesame polysaccharide obtained in liquid at least improves 1.6 times of (traditional corn pulp culture medium zymotechnique, purple sesame polyoses contents 5.0 ‰ Left and right), it can preferably improve 3 times.In addition, present inventor also found, used in shaking flask cultivation stage and in fermentation stage The culture medium of identical component is not only saved using the inconvenient of different culture medium, and effective tunning of bacterial strain is still than existing There is effective tunning in technology to increase；In situations where it is preferred, the content that maple water mentions solution in fermentation medium is higher than Maple water in shaking flask cultivation stage in culture medium proposes the content of solution.
Specific embodiment
The present invention provides a kind of cultural methods of purple sesame bacterial strain, which is characterized in that in the Tube propagation stage, Tube propagation Base includes: that nitrogen source, carbon source, phosphate, sulfate, agar and maple water mention solution.
In the Tube propagation stage, the nitrogen source can for this field use any nitrogen source, such as yeast powder, peanut powder, Dehydrated potato powder, ammonium hydroxide, urea etc.；But preferably potato and preferably potato it is to boil water be used as nitrogen source.
In the Tube propagation stage, the carbon source can be the carbon source generallyd use in this field, such as glucose, corn form sediment Powder, dehydrated potato powder, cane molasses etc.；But preferably glucose, potato, potato are preferably that potato is to boil water as carbon source.
The phosphate can be phosphate commonly employed in the art, but the sylvite of preferably phosphoric acid, more preferably KH2PO4。
The sulfate can be sulfate commonly employed in the art, but preferably MgSO4, more preferably MgSO4· 7H2O。
The maple water mentions solution and can be obtained using the various pieces of maple to be contacted to a period of time with water, according to contact Temperature it is different, time of contact is accordingly adjusted, such as can be contacted several days in room temperature, and dozens of minutes can also be boiled.
It in order to further improve the culture effect of purple sesame bacterial strain, is preferably carried out in mode in one kind, the maple water mentions Solution is by including the following steps preparation: maple and boiling are boiled 10 minutes to 3 hours, more preferably 10 minutes small to 1 When, most preferably 15-40 minutes；Wherein, the maple (in terms of dry weight) and water weight ratio are 1:1-100, preferably 1:3- 50, more preferably 1:5-30, most preferably 1:10-20.
Although for being specifically partially not particularly limited for maple, it is preferable that the maple is the branch bar of maple.
It in order to preferably cultivate purple sesame bacterial strain, is preferably carried out in mode in one kind, with the gross weight of the Tube propagation base On the basis of amount, the dosage of the potato is 15-30 weight %, and the content of the glucose is 1-5 weight %, KH2PO4Contain Amount is 0.1-0.5 weight %, MgSO4Content be 0.05-0.2 weight %, VB1Content be 0.0005-0.003 weight %, fine jade The content of rouge is 0.5-3 weight %, and surplus is that maple water mentions solution, and potato is to be added in the following manner: by the use The potato of amount is to boil water, and filtered liquid is added for filtering；The maple water mentions solution through the following steps that preparation: institute Stating maple (in terms of dry weight) and water weight ratio is 1:10-20, is boiled 15-40 minutes.
It in order to preferably cultivate purple sesame bacterial strain, is preferably carried out in mode in one kind, wherein the Tube propagation base includes VB1。
It is preferably carried out in mode in one kind, wherein maple is added in shaking flask cultivation stage, shake-flask seed culture solution Water mentions solution.
The present invention also provides a kind of cultural methods of purple sesame bacterial strain, which is characterized in that in shaking flask cultivation stage, shaking flask kind Sub- culture solution includes that nitrogen source, carbon source, phosphate, sulfate and maple water mention solution.
Any nitrogen source that the nitrogen source can use for this field, such as corn pulp, beancake powder, yeast powder, peanut powder, horse Bell sweet potato starch, ammonium hydroxide, urea etc.；But preferably potato and preferably dehydrated potato powder；Or preferably yeast powder and potato The combination of (preferably dehydrated potato powder).
The carbon source can be the carbon source that generallys use in this field, for example, glucose, cornstarch, potato starch, Cane molasses etc.；But preferably glucose, potato, potato are preferably dehydrated potato powder.
The phosphate can be phosphate commonly employed in the art, but the sylvite and ammonium salt of preferably phosphoric acid is more excellent It is selected as KH2PO4And NH4H2PO4。
The sulfate can be sulfate commonly employed in the art, but preferably MgSO4, more preferably MgSO4· 7H2O。
The maple water mentions solution and can be obtained using the various pieces of maple to be contacted to a period of time with water, according to contact Temperature it is different, time of contact is accordingly adjusted, such as can be contacted several days in room temperature, and dozens of minutes can also be boiled.
In order to further improve the culture effect of purple sesame bacterial strain, in shaking flask cultivation stage, mode is preferably carried out in one kind In, wherein it is by including the following steps preparation that the maple water, which mentions solution: 10 minutes to 3 hours are boiled in maple and boiling, More preferably 10 minutes to 1 hour, most preferably 15-40 minutes；Wherein, the maple (in terms of dry weight) is with water weight ratio 1:1-100, preferably 1:3-50, more preferably 1:5-30, most preferably 1:10-20.
Although for being specifically partially not particularly limited for maple, it is preferable that the maple is the branch bar of maple.
It in order to further improve the culture effect of purple sesame bacterial strain, is preferably carried out in mode in one kind, with shaking flask training On the basis of the total weight of nutrient solution, the content of the potato is 0.4-0.8 weight %, and the content of the glucose is 0.8-3 weight % is measured, the content of yeast powder is 0.1-0.5 weight %, KH2PO4Content be 0.08-0.3 weight %, MgSO4·7H2O's contains Amount is 0.01-0.4 weight %, NH4H2PO4Content be 0.005-0.02 weight %, VB1Content be 0.0005-0.003 weight Measure %, surplus is that maple water mentions solution, and the maple water mentions solution through the following steps that preparation: the maple (in terms of dry weight) It is 1:10-20 with water weight ratio, boils 15-40 minutes；Wherein, the pH value of the culture medium is preferably 5.5-7, more preferably 6-6.5。
It in order to further improve the culture effect of purple sesame bacterial strain, is preferably carried out in mode in one kind, wherein the shaking flask Seed culture fluid further includes VB1。
It in order to further improve the culture effect of purple sesame bacterial strain, is preferably carried out in mode in one kind, wherein trained in shaking flask The stage of supporting mentions solution added with maple water in Shake flask medium.
The present invention provides a kind of purple sesame liquid fermentation methods, which is characterized in that this method includes that fermentation medium includes Nitrogen source, carbon source, phosphate, sulfate and maple water mention solution.
Any nitrogen source that the nitrogen source can use for this field, such as corn pulp, beancake powder, yeast powder, peanut powder, horse Bell sweet potato starch, ammonium hydroxide, urea etc.；But preferably potato and preferably dehydrated potato powder；Or preferably yeast powder and potato The combination of (preferably dehydrated potato powder).
The carbon source can be the carbon source that generallys use in this field, for example, glucose, cornstarch, potato starch, Cane molasses etc.；But preferably glucose, potato, potato are preferably dehydrated potato powder.
The phosphate can be phosphate commonly employed in the art, but the sylvite and ammonium salt of preferably phosphoric acid is more excellent It is selected as KH2PO4And NH4H2PO4。
The sulfate can be sulfate commonly employed in the art, but preferably MgSO4, more preferably MgSO4· 7H2O。
In order to further obtain more G.japonicum myceliums and improve the amount of purple sesame polysaccharide in fermentation liquid, it is a kind of preferably In embodiment, wherein it is by including the following steps preparation that the maple water, which mentions solution: maple is boiled 10 minutes with boiling to arrive 3 hours, more preferably 10 minutes to 1 hour, most preferably 15-40 minutes；Wherein, the maple (in terms of dry weight) and water weight Amount ratio is 1:1-100, preferably 1:3-50, more preferably 1:5-30, most preferably 1:8-15.
Although for being specifically partially not particularly limited for maple, it is preferable that the maple is the branch bar of maple.
In order to further obtain more G.japonicum myceliums and improve the amount of purple sesame polysaccharide in fermentation liquid, it is a kind of preferably In embodiment, on the basis of the total weight of the fermentation culture, the content of the potato is 0.4-0.8 weight %, institute The content for stating glucose is 0.8-3 weight %, and the content of yeast powder is 0.1-0.5 weight %, KH2PO4Content be 0.08-0.3 Weight %, MgSO4Content be 0.01-0.4 weight %, NH4H2PO4Content be 0.005-0.02 weight %, VB1Content be 0.0005-0.003 weight %, surplus are that maple water mentions solution, and the maple water mentions solution through the following steps that preparation: described Maple (in terms of dry weight) and water weight ratio are 1:8-15, are boiled 15-40 minutes；Wherein, the pH value of the culture medium is preferably 5.5-7 more preferably 6-6.5.
In order to further obtain more G.japonicum myceliums and improve the amount of purple sesame polysaccharide in fermentation liquid, it is a kind of preferably In embodiment, wherein the fermentation culture further includes VB1。
Preferably, the method also includes the step of after fermentation separating fermentation liquid and mycelium；The separation Method can be separated by solid-liquid separation using simple, for example, filtering, centrifugation.Preferably, the content of purple sesame polysaccharide is 8 ‰ in fermentation liquid More than, preferably 10 ‰ or more, more preferably 12 ‰ or more, most preferably 15 ‰ or more；Wherein mycelium dry weight be 1.5% with On, preferably 2% or more.
Separated fermentation liquid can be utilized directly, be utilized after can also being concentrated and dried, and can also come out separation of polysaccharides After utilize；The fermentation liquid can be used for food, health care product, cosmetics etc.；For example, fermentation liquid can be used as beverage after sterilization, Nutritional supplement as food and health care product；Fermentation liquid can also be used in cosmetic field as stoste, or as battalion Support replenishers.
Separated mycelium can be used for food, health care product etc. after being pulverized.
In order to further obtain more G.japonicum myceliums and improve the amount of purple sesame polysaccharide in fermentation liquid, it is a kind of preferably In embodiment, wherein in purple sesame strain tube cultivation stage, mention solution added with maple water in Tube propagation base；With/ Or, mentioning solution added with maple water in shake-flask seed culture solution in purple sesame bacterial strain shaking flask cultivation stage；Preferably, it is fermenting The maple water that the concentration that maple water in the culture medium in stage mentions solution is higher than in the culture medium of shaking flask cultivation stage mentions solution Concentration and the culture medium in Tube propagation stage in maple water propose the concentration of solution, in shaking flask cultivation stage and fermentation stage Other ingredients of culture medium are identical, while being conducive to simplify step；Preferably, the maple water in the culture medium of fermentation stage mentions The concentration of solution is higher than the concentration that the maple water in the culture medium of shaking flask cultivation stage mentions solution by least 1% or more, more preferably Higher than 2% or more, most preferably higher than 3% or more.
Preferably, in shake-flask seed cultivation stage and fermentation stage, culture solution is with the total weight benchmark of culture solution, institute The content for stating potato is 0.4-0.8 weight %, and the content of the glucose is 0.8-3 weight %, and the content of yeast powder is 0.1-0.5 weight %, KH2PO4Content be 0.08-0.3 weight %, MgSO4Content be 0.01-0.4 weight %, NH4H2PO4 Content be 0.005-0.02 weight %, VB1Content be 0.0005-0.003 weight %, surplus be maple water mention solution, and The maple water mentions solution through the following steps that preparation: the maple (in terms of dry weight) is 1:8-15 with water weight ratio, is boiled 15-40 minutes；Wherein, the pH value of the culture medium is preferably 5.5-7, more preferably 6-6.5；Preferably, in fermentation stage The concentration that maple water in culture medium mentions solution is higher than the concentration that the maple water in the culture medium of shaking flask cultivation stage mentions solution At least 1% or more, more preferably higher than 2% or more, most preferably higher than 3% or more.
Embodiment 1
1, purple sesame strain inclined plane Tube propagation:
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 7:100,20min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
PDA culture medium (g/L): potato 250g is added 500ml deionized water and boils 30min, and filtered through gauze takes filtrate； Glucose 30g, KH is added2PO42g, MgSO4·7H2O 0.8g, VB10.01g, agar 15g, in addition the maple water for stating preparation mentions Solution is settled to 1L.
The Spawn incubation stage: from purple sesame strain (Chinese agriculture Microbiological Culture Collection administrative center, deposit number: ACCC50045) take 1 piece of (1*1cm) lawn in storage test tube and be inoculated in solid PDA medium, activated, rejuvenation, be inoculated with After strain at 24 DEG C insulating box culture 4 days, obtain growing vigorous (covering with test tube), the purple sesame activated strains of no miscellaneous bacteria.
2, purple sesame shake-flask seed liquid culture:
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 9:100,20min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
Shake-flask seed liquid culture medium (g/L): glucose 15g, dehydrated potato powder 6g, yeast powder 2g, KH2PO41.5g MgSO4·7H2O0.7g, NH4H2PO40.3g, VB10.01g, the maple water that above-mentioned preparation is added mention solution and are settled to 1L, pH tune Section is 6.
Shaking flask cultivation stage: using connect bacterium shovel take activated, 4 pieces of eugonic purple sesame lawn (1*1cm), be inoculated in In 90mL shake-flask seed liquid culture medium, 25 DEG C of cultivation temperature, shaking table culture 4 days, purple sesame fermentation seed liquid is obtained.Collect wherein one The mycelium of bottle, dry weighing, obtains 0.009g mycelium/ml fermentation liquid.
3, purple sesame liquid fermentation and culture
Fermentation broth (g/L): glucose 450g, dehydrated potato powder 180g, yeast powder 60g, KH2PO445g, MgSO4·7H2O 21g, NH4H2PO49g, VB10.3g, is added the maple water for preparing in step 2 and mentions solution and be settled to 30L, pH It is adjusted to 6.
Fermentation culture stage: it is inoculated into fermentation broth using cultured purple sesame seed liquor, inoculum concentration is 9%, fermentor total capacity is 30L, and fermentation liquid hold-up is the 70% of fermentor total volume.Adjusting fermentation temperature is 25 DEG C, is passed through Filtrated air.When content of reducing sugar is lower than 3 ‰ in fermentation medium, fermentation is terminated.By centrifuge separation, purple sesame mycelia is obtained Body and fermentation liquid.By the dry weighing of mycelia, 0.018g mycelium/ml fermentation liquid is obtained, fermentation liquid is detected by phend-sulphuric acid The concentration of middle purple sesame polysaccharide is 9.5 ‰.
Embodiment 2
1, purple sesame strain inclined plane Tube propagation:
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 5:100,50min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
PDA culture medium (g/L): potato 150g is added 500ml deionized water and boils 30min, and filtered through gauze takes filtrate； Glucose 50g, KH is added2PO45g, MgSO4·7H2O 0.5g, VB10.03g, agar 30g, in addition the maple water for stating preparation mentions Solution is settled to 1L.
The Spawn incubation stage: from purple sesame strain (Chinese agriculture Microbiological Culture Collection administrative center, deposit number: ACCC50045) take 1 piece of (1*1cm) lawn in storage test tube and be inoculated in solid PDA medium, activated, rejuvenation, be inoculated with After strain at 24 DEG C insulating box culture 4 days, obtain growing vigorous (covering with test tube), the purple sesame activated strains of no miscellaneous bacteria.
2, purple sesame shake-flask seed liquid culture:
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 7:100,40min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
Shake-flask seed liquid culture medium (g/L): glucose 30g, mealy potato 4g, yeast powder 5g, KH2PO43g, MgSO4· 7H2O 0.2g, NH4H2PO40.8g, VB10.008g, the maple water that above-mentioned preparation is added mention solution and are settled to 1L, and pH is adjusted to 6.5。
Shaking flask cultivation stage: using connect bacterium shovel take activated, 4 pieces of eugonic purple sesame lawn (1*1cm), be inoculated in In 90mL shake-flask seed liquid culture medium, 25 DEG C of cultivation temperature, shaking table culture 4 days, purple sesame fermentation seed liquid is obtained.Collect wherein one The mycelium of bottle, dry weighing, obtains 0.011g mycelium/ml fermentation liquid.
3, purple sesame liquid fermentation and culture
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 9:100,40min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
Fermentation broth (g/L): glucose 900g, mealy potato 120g, yeast powder 150g, KH2PO490g, MgSO4·7H2O 6g, NH4H2PO42.4g, VB12.4g, the maple water that above-mentioned preparation is added mention solution and are settled to 30L, pH tune Section is 6.5.
Fermentation culture stage: it is inoculated into fermentation broth using cultured purple sesame seed liquor, inoculum concentration is 9%, fermentor total capacity is 30L, and fermentation liquid hold-up is the 70% of fermentor total volume.Adjusting fermentation temperature is 25 DEG C, is passed through Filtrated air.When content of reducing sugar is lower than 3 ‰ in fermentation medium, fermentation is terminated.By centrifuge separation, purple sesame mycelia is obtained Body and fermentation liquid.By the dry weighing of mycelia, 0.021g mycelium/ml fermentation liquid is obtained, fermentation liquid is detected by phend-sulphuric acid The concentration of middle purple sesame polysaccharide is 10.3 ‰.
Embodiment 3
1, purple sesame strain inclined plane Tube propagation:
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 7:100,40min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
PDA culture medium (g/L): potato 300g is added 500ml deionized water and boils 30min, and filtered through gauze takes filtrate； Glucose 10g, KH is added2PO43g, MgSO4·7H2O 1.5g, VB10.01g, agar 5g, in addition the maple water for stating preparation mentions Solution is settled to 1L.
The Spawn incubation stage: from purple sesame strain (Chinese agriculture Microbiological Culture Collection administrative center, deposit number: ACCC50045) take 1 piece of (1*1cm) lawn in storage test tube and be inoculated in solid PDA medium, activated, rejuvenation, be inoculated with After strain at 24 DEG C insulating box culture 4 days, obtain growing vigorous (covering with test tube), the purple sesame activated strains of no miscellaneous bacteria.
2, purple sesame shake-flask seed liquid culture:
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 6:100,50min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
Shake-flask seed liquid culture medium (g/L): glucose 15g, mealy potato 8g, yeast powder 3g, KH2PO42g, MgSO4· 7H2O 1.5g, NH4H2PO40.6g, VB10.03g, the maple water that above-mentioned preparation is added mention solution and are settled to 1L, and pH is adjusted to 6.5。
Shaking flask cultivation stage: using connect bacterium shovel take activated, 4 pieces of eugonic purple sesame lawn (1*1cm), be inoculated in In 90mL shake-flask seed liquid culture medium, 25 DEG C of cultivation temperature, shaking table culture 4 days, purple sesame fermentation seed liquid is obtained.Collect wherein one The mycelium of bottle, dry weighing, obtains 0.013g mycelium/ml fermentation liquid.
3, purple sesame liquid fermentation and culture
Maple water proposes the preparation of solution: maple branch (dry weight) mixed with deionized water with 12:100,20min is boiled, Filtered through gauze obtains maple water and mentions solution.
Fermentation broth (g/L): glucose 450g, mealy potato 240g, yeast powder 90g, KH2PO460g, MgSO4· 7H2O 45g, NH4H2PO418g, VB10.9g, the maple water that above-mentioned preparation is added mention solution and are settled to 30L, and pH is adjusted to 6.5.
Fermentation culture stage: it is inoculated into fermentation broth using cultured purple sesame seed liquor, inoculum concentration is 9%, fermentor total capacity is 30L, and fermentation liquid hold-up is the 70% of fermentor total volume.Adjusting fermentation temperature is 25 DEG C, is passed through Filtrated air.When content of reducing sugar is lower than 3 ‰ in fermentation medium, fermentation is terminated.By centrifuge separation, purple sesame mycelia is obtained Body and fermentation liquid.By the dry weighing of mycelia, 0.026g mycelium/ml fermentation liquid is obtained, fermentation liquid is detected by phend-sulphuric acid The concentration of middle purple sesame polysaccharide is 16.38 ‰.
Embodiment 4
1, purple sesame strain inclined plane Tube propagation:
PDA culture medium (g/L): potato 250g is added 500ml deionized water and boils 30min, and filtered through gauze takes filtrate； Glucose 30g, KH is added2PO42g, MgSO4·7H2O 0.8g, VB10.01g, agar 15g add such as deionized water, are settled to 1L。
The Spawn incubation stage: from purple sesame strain (Chinese agriculture Microbiological Culture Collection administrative center, deposit number: ACCC50045) take 1 piece of (1*1cm) lawn in storage test tube and be inoculated in solid PDA medium, activated, rejuvenation, be inoculated with After strain at 24 DEG C insulating box culture 4 days, obtain growing vigorous (covering with test tube), the purple sesame activated strains of no miscellaneous bacteria.
2, purple sesame shake-flask seed liquid culture:
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 9:100,20min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
Shake-flask seed liquid culture medium (g/L): glucose 10g, dehydrated potato powder 6g, yeast powder 2g, KH2PO41.5g MgSO4·7H2O0.7g, NH4H2PO40.3g, VB10.01g, the maple water that above-mentioned preparation is added mention solution and are settled to 1L, pH tune Section is 6.
Shaking flask cultivation stage: using connect bacterium shovel take activated, 4 pieces of eugonic purple sesame lawn (1*1cm), be inoculated in In 90mL shake-flask seed liquid culture medium, 25 DEG C of cultivation temperature, shaking table culture 4 days, purple sesame fermentation seed liquid is obtained.Collect wherein one The mycelium of bottle, dry weighing, obtains 0.008g mycelium/ml fermentation liquid.
3, purple sesame liquid fermentation and culture
Fermentation broth (g/L): glucose 300g, dehydrated potato powder 180g, yeast powder 60g, KH2PO445g, MgSO4·7H2O 21g, NH4H2PO49g, VB10.3g, is added the maple water for preparing in step 2 and mentions solution and be settled to 30L, pH It is adjusted to 6.
Fermentation culture stage: it is inoculated into fermentation broth using cultured purple sesame seed liquor, inoculum concentration is 9%, fermentor total capacity is 30L, and fermentation liquid hold-up is the 70% of fermentor total volume.Adjusting fermentation temperature is 25 DEG C, is passed through Filtrated air.When content of reducing sugar is lower than 3 ‰ in fermentation medium, fermentation is terminated.By centrifuge separation, purple sesame mycelia is obtained Body and fermentation liquid.By the dry weighing of mycelia, 0.017g mycelium/ml fermentation liquid is obtained, fermentation liquid is detected by phend-sulphuric acid The concentration of middle purple sesame polysaccharide is 9.3 ‰.
Embodiment 5
1, purple sesame strain inclined plane Tube propagation:
PDA culture medium (g/L): potato 250g is added 500ml deionized water and boils 30min, and filtered through gauze takes filtrate； Glucose 30g, KH is added2PO42g, MgSO4·7H2O 0.8g, VB10.01g, agar 15g add such as deionized water, are settled to 1L。
The Spawn incubation stage: from purple sesame strain (Chinese agriculture Microbiological Culture Collection administrative center, deposit number: ACCC50045) take 1 piece of (1*1cm) lawn in storage test tube and be inoculated in solid PDA medium, activated, rejuvenation, be inoculated with After strain at 24 DEG C insulating box culture 5 days, obtain growing vigorous (covering with test tube), the purple sesame activated strains of no miscellaneous bacteria.
2, purple sesame shake-flask seed liquid culture:
Shake-flask seed liquid culture medium (g/L): glucose 15g, dehydrated potato powder 6g, yeast powder 2g, KH2PO41.5g MgSO4·7H2O0.7g, NH4H2PO40.3g, VB10.01g is added deionized water, is settled to 1L, pH is adjusted to 6.
Shaking flask cultivation stage: using connect bacterium shovel take activated, 4 pieces of eugonic purple sesame lawn (1*1cm), be inoculated in In 90mL shake-flask seed liquid culture medium, 25 DEG C of cultivation temperature, shaking table culture 4 days, purple sesame fermentation seed liquid is obtained.Collect wherein one The mycelium of bottle, dry weighing, obtains 0.007g mycelium/ml fermentation liquid.
3, purple sesame liquid fermentation and culture
Fermentation broth (g/L): glucose 450g, dehydrated potato powder 180g, yeast powder 60g, KH2PO445g, MgSO4·7H2O 21g, NH4H2PO49g, VB10.3g, is added the maple water for preparing in step 2 and mentions solution and be settled to 30L, pH It is adjusted to 6.
Fermentation culture stage: it is inoculated into fermentation broth using cultured purple sesame seed liquor, inoculum concentration is 9%, fermentor total capacity is 30L, and fermentation liquid hold-up is the 70% of fermentor total volume.Adjusting fermentation temperature is 25 DEG C, is passed through Filtrated air.When content of reducing sugar is lower than 3 ‰ in fermentation medium, fermentation is terminated.By centrifuge separation, purple sesame mycelia is obtained Body and fermentation liquid.By the dry weighing of mycelia, 0.014g mycelium/ml fermentation liquid is obtained, fermentation liquid is detected by phend-sulphuric acid The concentration of middle purple sesame polysaccharide is 8.3 ‰.
Embodiment 6
1, purple sesame strain inclined plane Tube propagation:
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 7:100,40min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
PDA culture medium (g/L): potato 300g is added 500ml deionized water and boils 30min, and filtered through gauze takes filtrate； Glucose 10g, KH is added2PO43g, MgSO4·7H2O 1.5g, VB10.01g, agar 5g, in addition the maple water for stating preparation mentions Solution is settled to 1L.
The Spawn incubation stage: from purple sesame strain (Chinese agriculture Microbiological Culture Collection administrative center, deposit number: ACCC50045) take 1 piece of (1*1cm) lawn in storage test tube and be inoculated in solid PDA medium, activated, rejuvenation, be inoculated with After strain at 24 DEG C insulating box culture 4 days, obtain growing vigorous (covering with test tube), the purple sesame activated strains of no miscellaneous bacteria.
2, purple sesame shake-flask seed liquid culture:
Maple water proposes the preparation of solution: maple branch (dry weight) being mixed with deionized water with 6:100,50min, yarn are boiled Cloth is obtained by filtration maple water and mentions solution.
Shake-flask seed liquid culture medium (g/L): glucose 15g, mealy potato 8g, yeast powder 3g, KH2PO42g, MgSO4· 7H2O 2g, NH4H2PO40.6g, the maple water that above-mentioned preparation is added mention solution and are settled to 1L, and pH is adjusted to 6.5.
Shaking flask cultivation stage: using connect bacterium shovel take activated, 4 pieces of eugonic purple sesame lawn (1*1cm), be inoculated in In 90mL shake-flask seed liquid culture medium, 25 DEG C of cultivation temperature, shaking table culture 4 days, purple sesame fermentation seed liquid is obtained.Collect wherein one The mycelium of bottle, dry weighing, obtains 0.012g mycelium/ml fermentation liquid.
3, purple sesame liquid fermentation and culture
Maple water proposes the preparation of solution: maple branch (dry weight) mixed with deionized water with 12:100,20min is boiled, Filtered through gauze obtains maple water and mentions solution.
Fermentation broth (g/L): glucose 450g, mealy potato 240g, yeast powder 90g, KH2PO460g, MgSO4· 7H2O 60g, NH4H2PO418g, the maple water that above-mentioned preparation is added mention solution and are settled to 30L, and pH is adjusted to 6.5.
Fermentation culture stage: it is inoculated into fermentation broth using cultured purple sesame seed liquor, inoculum concentration is 9%, fermentor total capacity is 30L, and fermentation liquid hold-up is the 70% of fermentor total volume.Adjusting fermentation temperature is 25 DEG C, is passed through Filtrated air.When content of reducing sugar is lower than 3 ‰ in fermentation medium, fermentation is terminated.By centrifuge separation, purple sesame mycelia is obtained Body and fermentation liquid.By the dry weighing of mycelia, 0.024g mycelium/ml fermentation liquid is obtained, fermentation liquid is detected by phend-sulphuric acid The concentration of middle purple sesame polysaccharide is 14.43 ‰.
Comparative example 1
1, purple sesame strain inclined plane Tube propagation:
PDA culture medium (g/L): potato 250g is added 500ml deionized water and boils 30min, and filtered through gauze takes filtrate； Glucose 20g, KH is added2PO41.5g, MgSO4·7H2O 1g, VB10.01g, agar 15g, adds deionized water, is settled to 1L.
The Spawn incubation stage: from purple sesame strain (Chinese agriculture Microbiological Culture Collection administrative center, deposit number: ACCC50045) take 1 piece of (1*1cm) lawn in storage test tube and be inoculated in solid PDA medium, activated, rejuvenation, be inoculated with After strain at 24 DEG C insulating box culture 5 days, obtain growing vigorous (covering with test tube), the purple sesame activated strains of no miscellaneous bacteria.
2, purple sesame shake-flask seed liquid culture:
Shake-flask seed liquid culture medium (g/L): glucose 15g, corn pulp 20g, yeast powder 3g, KH2PO42g, MgSO4· 7H2O 1.5g, NH4H2PO40.6g, adds deionized water to be settled to 1L, and pH is adjusted to 6.5.
Shaking flask cultivation stage: using connect bacterium shovel take activated, 4 pieces of eugonic purple sesame lawn (1*1cm), be inoculated in In 90mL shake-flask seed liquid culture medium, 25 DEG C of cultivation temperature, shaking table culture 5 days, purple sesame fermentation seed liquid is obtained.Collect wherein one The mycelium of bottle, dry weighing, obtains 0.004g mycelium/ml fermentation liquid.
3, purple sesame liquid fermentation and culture
Fermentation broth (g/L): glucose 450g, corn pulp 600g, yeast powder 90g, KH2PO460g, MgSO4· 7H2O 45g, NH4H2PO418g, adds deionized water to be settled to 30L, and pH is adjusted to 6.5.
Fermentation culture stage: it is inoculated into fermentation broth using cultured purple sesame seed liquor, inoculum concentration is 9%, fermentor total capacity is 30L, and fermentation liquid hold-up is the 70% of fermentor total volume.Adjusting fermentation temperature is 25 DEG C, is passed through Filtrated air.When content of reducing sugar is lower than 3 ‰ in fermentation medium, fermentation is terminated.By centrifuge separation, purple sesame mycelia is obtained Body and fermentation liquid.By the dry weighing of mycelia, 0.016g mycelium/ml fermentation liquid is obtained, fermentation liquid is detected by phend-sulphuric acid The concentration of middle purple sesame polysaccharide is 5.2 ‰.

Claims (15)

1. a kind of cultural method of purple sesame bacterial strain, which is characterized in that in the Tube propagation stage, Tube propagation base includes: nitrogen source, carbon Source, phosphate, sulfate, agar and maple water mention solution；It is preferably grape that the nitrogen source, which is preferably potato, the carbon source, Sugar, potato；Wherein, the phosphate is preferably the sylvite of phosphoric acid, more preferably KH2PO4；The sulfate is preferably MgSO4。
2. the cultural method of purple sesame bacterial strain according to claim 1, wherein the maple water mention solution be by include with Lower step preparation: 10 minutes to 3 hours, more preferably 10 minutes to 1 hour, most preferably 15-40 are boiled in maple and boiling Minute；Wherein, the maple (in terms of dry weight) and water weight ratio are 1:1-100, preferably 1:3-50, more preferably 1:5- 30, most preferably 1:10-20；Preferably, the maple is the branch bar of maple.
3. the cultural method of purple sesame bacterial strain according to claim 1 or 2, wherein with the total weight of the Tube propagation base On the basis of, the dosage of the potato is 15-30 weight %, and the content of the glucose is 1-5 weight %, KH2PO4Content For 0.1-0.5 weight %, MgSO4Content be 0.05-0.2 weight %, VB1Content be 0.0005-0.003 weight %, agar Content be 0.5-3 weight %, surplus is that maple water mentions solution, and potato is to be added in the following manner: by the dosage Potato it is to boil water, filtering, filtered liquid is added；The maple water mentions solution through the following steps that preparation: described Maple (in terms of dry weight) and water weight ratio are 1:10-20, are boiled 15-40 minutes.
4. the cultural method of purple sesame bacterial strain according to claim 1 or 2, wherein the Tube propagation base includes VB1。
5. the cultural method of purple sesame bacterial strain according to claim 1 or 2, wherein in shaking flask cultivation stage, shake-flask seed training Solution is mentioned added with maple water in nutrient solution.
6. a kind of cultural method of purple sesame bacterial strain, which is characterized in that in shaking flask cultivation stage, shake-flask seed culture solution includes nitrogen Source, carbon source, phosphate, sulfate and maple water mention solution；The nitrogen source is preferably potato or yeast powder and potato Combination, the carbon source are preferably glucose, potato；Wherein, the phosphate is preferably the sylvite and ammonium salt of phosphoric acid, more preferably For KH2PO4And NH4H2PO4；The sulfate is preferably MgSO4。
7. cultural method according to claim 6, wherein it is by including the following steps preparation that the maple water, which mentions solution, : maple and boiling are boiled 10 minutes to 3 hours, and more preferably 10 minutes to 1 hour, most preferably 15-40 minutes；Wherein, The maple (in terms of dry weight) and water weight ratio are 1:1-100, preferably 1:3-50, more preferably 1:5-30, most preferably 1:10-20；Preferably, the maple is the branch bar of maple.
8. cultural method according to claim 6 or 7, wherein described on the basis of the total weight of the shake flask culture The content of potato is 0.4-0.8 weight %, and the content of the glucose is 0.8-3 weight %, and the content of yeast powder is 0.1- 0.5 weight %, KH2PO4Content be 0.08-0.3 weight %, MgSO4Content be 0.01-0.4 weight %, NH4H2PO4Contain Amount is 0.005-0.02 weight %, VB1Content be 0.0005-0.003 weight %, surplus is that maple water mentions solution, and described Maple water mentions solution through the following steps that preparation: the maple (in terms of dry weight) is 1:10-20 with water weight ratio, is boiled 15-40 minutes；Wherein, the pH value of the culture medium is preferably 5.5-7, more preferably 6-6.5.
9. cultural method according to claim 6 or 7, wherein the shake-flask seed culture solution further includes VB1。
10. cultural method according to claim 6 or 7, wherein in the Tube propagation stage, added in Tube propagation base There is maple water to mention solution.
11. a kind of purple sesame liquid fermentation method, which is characterized in that this method includes that fermentation culture includes nitrogen source, carbon source, phosphoric acid Salt, sulfate and maple water mention solution；The nitrogen source is preferably the combination of potato or yeast powder and potato, the carbon Source is preferably glucose, potato；Wherein, the phosphate is preferably the sylvite and ammonium salt of phosphoric acid, more preferably KH2PO4With NH4H2PO4；The sulfate is preferably MgSO4。
12. fermentation process according to claim 11, wherein it is by including the following steps system that the maple water, which mentions solution, Standby: maple and boiling are boiled 10 minutes to 3 hours, and more preferably 10 minutes to 1 hour, most preferably 15-40 minutes；Its In, the maple (in terms of dry weight) and water weight ratio are 1:1-100, preferably 1:3-50, more preferably 1:5-30, most preferably For 1:8-15；Preferably, the maple is the branch bar of maple.
13. fermentation process according to claim 11 or 12, wherein on the basis of the total weight of the fermentation culture, The content of the potato is 0.4-0.8 weight %, and the content of the glucose is 0.8-3 weight %, and the content of yeast powder is 0.1-0.5 weight %, KH2PO4Content be 0.08-0.3 weight %, MgSO4Content be 0.01-0.4 weight %, NH4H2PO4 Content be 0.005-0.02 weight %, VB1Content be 0.0005-0.003 weight %, surplus be maple water mention solution, and The maple water mentions solution through the following steps that preparation: the maple (in terms of dry weight) is 1:8-15 with water weight ratio, is boiled 15-40 minutes；Wherein, the pH value of the culture medium is preferably 5.5-7, more preferably 6-6.5.
14. fermentation process according to claim 11 or 12, wherein the fermentation culture further includes VB1；Preferably, institute The method of stating further includes the steps that after fermentation separating fermentation liquid and mycelium；Wherein in fermentation liquid purple sesame polysaccharide content It is 8 ‰ or more, preferably 10 ‰ or more, more preferably 12 ‰ or more, most preferably 15 ‰ or more；Wherein mycelium dry weight is 1.5% or more, preferably 2% or more.
15. fermentation process according to claim 11 or 12, wherein in purple sesame strain tube cultivation stage, trained in test tube It supports in base and mentions solution added with maple water；And/or it in purple sesame bacterial strain shaking flask cultivation stage, is added in shake-flask seed culture solution There is maple water to mention solution；Preferably, the concentration that the maple water in the culture medium of fermentation stage mentions solution is higher than in shaking flask culture Maple water in the culture medium in stage mentions the maple water in the concentration and the culture medium in Tube propagation stage of solution and mentions the dense of solution Degree is identical with other ingredients of the culture medium of fermentation stage in shaking flask cultivation stage；Preferably, in the culture medium of fermentation stage Maple water mention the concentration higher than the concentration that the maple water in the culture medium of shaking flask cultivation stage mentions solution at least 1% of solution with On, more preferably higher than 2% or more, most preferably higher than 3% or more.
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