CN105400833A - A method of increasing a fermentation yield of natural abscisic acid - Google Patents

Abstract

The invention relates to method of increasing a fermentation yield of natural abscisic acid and belongs to the technical field of microorganism fermentation. The method includes inoculating botrytis cinerea capable of producing abscisic acid to a potato dextrose agar (PDA) solid plate, activating, culturing, subjecting the botrytis cinerea activated on the plate to seed culture, inoculating a cultured seed solution to a liquid fermentation culture medium, performing fermentation culture, and adding one or more than two of a plant leach liquor, sodium succinate and ammonium molybdate into the liquid fermentation culture medium, thus increasing the yield of the natural active abscisic acid produced by fungus fermentation. The method increases the oxygen utilization rate of a strain, accelerates thallus growth, and allows the strain to produce the abscisic acid in a high product synthesis rate. Materials used by the method are wide in source. The production cost is reduced. Processes are simplified. Subsequent industrial application is facilitated.

Description

A kind of method improving PBI 58 fermentation yield

Technical field

The invention belongs to technical field of microbial fermentation, relate to a kind of novel method improving PBI 58 fermentation yield.

Background technology

Dormin is one of large spontaneous growth conditioning agent of plant five.It plays many-sided effect in the growing of plant, and is agriculturally having broad application prospects, can improve drought resisting and the Salt-endurance of plant, has high value to exploitation low-yield land and afforestation, afforestation desert etc.; Dormin still suppresses effective inhibitor of seed germination, therefore may be used for the storage of seeds, ensures the storage quality of seed, fruit; In addition, dormin can also cause the rapid closedown of Stoma of Leaves, and can be used for the fresh-keeping of flower, florescence alternation, hestening rooting etc., flower gardening has larger using value.But due to ABA content in plant materials extremely low, therefrom extract infeasible; The racemic form dormin of synthetic is the mixture of natural type and non-natural type dormin, needs to split, not only expensive, and physiologically active is low, significantly limit its research application.

In order to solve and meet the needs that dormin is applied to agriculture production, in recent years, utilize fermentable to produce dormin to receive publicity, current fermentable bacterial classification used is in the majority with Botrytis cinerea, such as, nineteen eighty-two, Japanese MarumoS etc. find a kind of ABA-Producing Strains-Botrytis cinerea, and this bacterium is fermented and within 7 days, can accumulate the dormin of 52 μ g/g; 1993, the report such as Wu Shaobai produced dormin output with Botrytis cinerea solid fermentation method and is only 29ng/mL; Nineteen ninety-five, just the clear report that waits does not screen the fungi-Botrytis cinerea producing dormin from soil, and output is 18 μ g/mL.In order to improve the output of Botrytis cinerea fermentative production dormin, people have carried out optimization and the improvement of different aspect to fermention medium and zymotechnique.Such as, 2007, Zhang Hui etc. utilized the fermentation condition of orthogonal test to ABA-Producing Strains to be optimized, and define the best of breed making dormin output increased, but the amplitude that its output promotes is not the highest; 2002, the ratio etc. that the report such as Zheng Hang adds nutritive substance Semen Maydis powder, precursor substance trisodium citrate in the fermentation medium and changes glucose and sucrose can improve Botrytis cinerea and ferment and produce the output of dormin, but to add the concentration of precursor substance higher, usage quantity is large, causes the waste of resource; Publication number is that a kind of stream during the fermentation that application discloses of CN101041837B adds the technique of inositol releasing glucose repression to prepare the method for PBI 58, can make the output increased of dormin; Publication number is that the patent of CN1067724C passes through the existing ABA-Producing Strains of transformation, and change culture medium prescription, changing liquid Batch fermentation technique is continuous flow feeding discharging, and the method for adding key substrate improves the output of dormin; Publication number is that a kind of stream during the fermentation that application discloses of CN102399827B adds the precursor substance of acetyl-CoA as bacterial strain synthesis dormin, simultaneously, add bio-surfactant tween 20 and improve the dissolved oxygen situation of fermented liquid to prepare the method for PBI 58 to improve the output of dormin, but inositol and acetyl-CoA price relatively costly, be unfavorable for cost-saving.Publication number is the method that application discloses a kind of fungi solid state fermentation production dormin of CN103409474A, and compared with liquid fermenting, dormin output significantly improves.Although above prior art improves the fermentation yield of dormin to a certain extent, zymotechnique is loaded down with trivial details, and the by product that fermentation produces is more, insufficient to utilizing of oxygen; In addition, the promotion material cost of interpolation is higher, is unfavorable for the follow-up popularization of enterprise; Meanwhile, dormin solid state fermentation complicated operation, is unsuitable for enterprise and produces on a large scale.

Summary of the invention

The invention provides a kind of method improving PBI 58 fermentation yield, bacterial strain can be made to produce dormin with higher Product formation speed, be beneficial to follow-up suitability for industrialized production.

Technical problem to be solved by this invention is that thalline is insufficient to utilizing of oxygen, and the fermentation accelerant cost of interpolation is higher, the easy thickness of fermented liquid.

In order to solve the problems of the technologies described above, the present invention adopts following technical scheme:

A kind of method improving PBI 58 fermentation yield is provided, comprise the following steps: the Botrytis cinerea that first can produce dormin is seeded in activation culture 4-5 days on potato agar glucose (PDA) solid plate, culture temperature is 25-28 DEG C, then the Botrytis cinerea activated on flat board is cultivated 48-72h in seed culture medium, by seed liquor with 5% ~ 10% inoculum size access liquid fermentation medium, fermentation culture 9 ~ 10 days produces dormin; Described liquid fermentation medium needs to add the one in plant vat liquor, Soduxin, ammonium molybdate or two or more combination.

Of the present inventionly provide a kind of method improving PBI 58 fermentation yield, it is characterized in that, in liquid fermentation medium, add plant vat liquor, Soduxin, the one of ammonium molybdate or two or more combination.Thus improve the output that fungi fermentation produces PBI 58.In the present invention, fungi is the Botrytis cinerea producing PBI 58, is that we obtain from separation screening the sick fruit of tomato.

Of the present inventionly provide a kind of method improving PBI 58 fermentation yield, it is characterized in that, composition and the content of liquid seed culture medium used are as follows, glucose 20g/L, MgSO ₄7H ₂o0.2g/L, KCl0.5g/L, KH ₂pO ₄0.8g/L, VB11 mg/L, monosodium glutamate 3.0g/L, FeSO ₄7H ₂o0.5mg/L, ZnSO ₄.7H ₂o2.5mg/L, CuSO ₄.7H ₂o4mg/L, supplies 1L with distilled water.

Of the present inventionly provide a kind of method improving PBI 58 fermentation yield, it is characterized in that, liquid fermentation medium used is PD liquid nutrient medium or dedicated liquid fermention medium; Composition and the ratio of PD liquid nutrient medium are as follows, glucose 20g/L, potato 200g, clean peeling, get its filtrate, supply 1L with distilled water after stripping and slicing boiling; Composition and the content of dedicated liquid fermention medium are as follows, glucose 20g/L, MgSO ₄.7H ₂o0.2g/L, KCl0.5g/L, CaCO ₃5g/L, KH ₂pO ₄0.8g/L, VB11 mg/L, monosodium glutamate 3.0g/L, FeSO ₄7H ₂o0.5mg/L, ZnSO ₄7H ₂o2.5mg/L, CuSO ₄7H ₂o4mg/L, Semen Maydis powder 4g/L, supplies 1L with distilled water.

Of the present inventionly provide a kind of method improving PBI 58 fermentation yield, it is characterized in that, plant vat liquor used is Folium Capsici, the vat liquor of tomato leaf, during lixiviate, the phosphate buffered saline buffer of employing 0.1M and the plant leaf tissue of pulverizing are according to weight ratio 1:(0.02-0.2) lixiviate 12-24h at 4 DEG C, after lixiviate, its supernatant liquor of centrifuging and taking is as vat liquor, and plant vat liquor aperture is add in liquid fermentation medium after the film filter of 0.22-0.45 μm filters.

In liquid fermentation medium, the add-on of plant vat liquor is 2mL-10mL/L.

Of the present inventionly provide a kind of method improving PBI 58 fermentation yield, it is characterized in that, in liquid fermentation medium used, the concentration of Soduxin is 1-15mM/L.

Of the present inventionly provide a kind of method improving PBI 58 fermentation yield, it is characterized in that, in liquid fermentation medium used, the concentration of ammonium molybdate is 0.05-5mM/L.

Of the present inventionly provide a kind of method improving PBI 58 fermentation yield, it is characterized in that, in liquid fermentation medium used, the mass ratio of Soduxin and ammonium molybdate is 1:(0.2-12).

Of the present inventionly provide a kind of method improving PBI 58 fermentation yield, have obvious promoter action to Botrytis cinerea fermentative production PBI 58, the material added is nontoxic, environmentally safe.Wherein, the effective constituent in plant vat liquor can accelerate the growth of Botrytis cinerea, improves the utilization ratio to oxygen, makes bacterial strain produce dormin with higher Product formation speed; Soduxin is as prerequisite material, and the output of Botrytis cinerea fermentation being produced to PBI 58 has obvious castering action; Ammonium molybdate can make yeasting stablize, and fermentation broth viscosity is reduced, and promotes the synthesis of dormin; Meanwhile, this zymotechnique institute substance wide material sources, cost is lower, and technique is simple, is beneficial to the production application of follow-up industry.

Embodiment

The following example further illustrates of the present invention, instead of limitation of the present invention.

The experimental technique used in following embodiment if no special instructions, is ordinary method.

The bacterial classification used in following embodiment is Botrytis cinerea, is us from separation and purification the sick fruit of tomato.

embodiment 1

Bacterial classification activation culture on PDA flat board of first will preserve, is down flat plate by the PDA substratum prepared sterilizing in 121 DEG C of high-pressure sterilizing pots after 30 minutes for subsequent use.Then bacterial classification is inoculated on solid plate in Bechtop, activation culture 4-5 days at 26-28 DEG C.By the thalline aseptic inoculation ring picking that activated in liquid seed culture medium, break up and shake up, shake-flask culture about 72h at 28 DEG C.

By bacterial classification cultured in seed culture medium with 5% inoculum size access shaking flask that PD liquid nutrient medium is housed carry out fermentation culture, the composition of PD liquid nutrient medium and content are: glucose 20g/L, potato 200g, clean the rear diced shape of peeling, put on the inherent electromagnetic oven of pot and boil, timing 30 minutes, then with four layers of gauze, well-done potato is filtered, by its filtrate with distilled water diluting to 1L; Pepper Leaves is fully pulverized, according to the weight ratio 1:0.2 lixiviate of the phosphate buffered saline buffer of 0.1M and the Pepper Leaves tissue of pulverizing, extraction temperature 4 DEG C, extraction time is 24h, is for subsequent use after the film filter of 0.45 μm filters after centrifugal by its supernatant liquor aperture; Then be divided into two groups by after the PD liquid nutrient medium sterilizing prepared, one group adds Folium Capsici vat liquor by the consumption of 10mL/L, and another group does not add Folium Capsici vat liquor in contrast; With 150 revs/min of oscillation and fermentation cultivation 9 days at 28 DEG C.

Recording control group dormin output by high performance liquid chromatography is 4.65mg/L, and adding Folium Capsici vat liquor group dormin output is 29.81mg/L, improves 541.08% than control group.

embodiment 2

Actication of culture and seed culture method are with embodiment 1.By the bacterial classification activated with 10% inoculum size access shaking flask that dedicated liquid fermention medium is housed carry out fermentation culture, the composition of dedicated liquid fermention medium and content are: glucose 20g/L, MgSO ₄7H ₂o0.2g/L, KCl0.5g/L, CaCO ₃5g/L, KH ₂pO ₄0.8g/L, VB11 mg/L, monosodium glutamate 3.0g/L, FeSO ₄7H ₂o0.5mg/L, ZnSO ₄7H ₂o2.5mg/L, CuSO ₄7H ₂o4mg/L, Semen Maydis powder 4g/L, supplies 1L with distilled water.Pepper Leaves is fully pulverized, according to the weight ratio 1:0.1 lixiviate of the phosphate buffered saline buffer of 0.1M and the Pepper Leaves tissue of pulverizing, extraction temperature 4 DEG C, extraction time is 12h, is for subsequent use after the film filter of 0.45 μm filters after centrifugal by its supernatant liquor aperture; Be divided into two groups by after the dedicated liquid fermention medium sterilizing prepared, one group adds Folium Capsici vat liquor by the consumption of 5mL/L, and another group does not add Folium Capsici vat liquor in contrast; With 150 revs/min of oscillation and fermentation cultivation 9 days at 28 DEG C.

Recording control group dormin output by high performance liquid chromatography is 7.07mg/L, and adding Folium Capsici vat liquor group dormin output is 23.89mg/L, improves 237.91% than control group.

embodiment 3

Actication of culture and seed culture method are with embodiment 1.By the bacterial classification activated with 10% inoculum size access shaking flask that dedicated liquid fermention medium is housed carry out fermentation culture, the composition of dedicated liquid fermention medium and content are: glucose 20g/L, MgSO ₄7H ₂o0.2g/L, KCl0.5g/L, CaCO ₃5g/L, KH ₂pO ₄0.8g/L, VB11 mg/L, monosodium glutamate 3.0g/L, FeSO ₄7H ₂o0.5mg/L, ZnSO ₄7H ₂o2.5mg/L, CuSO ₄7H ₂o4mg/L, Semen Maydis powder 4g/L, supplies 1L with distilled water.The dedicated liquid fermention medium prepared is divided into two groups, one group adds Soduxin and ammonium molybdate, make Soduxin and the concentration of ammonium molybdate in fermenting substratum be respectively 5mM/L and 1mM/L, another group does not then add in contrast, accesses bacterial classification after sterilizing; With 150 revs/min of oscillation and fermentation cultivation 9 days at 28 DEG C.

Recording control group dormin output by high performance liquid chromatography is 8.18mg/L, adds Soduxin and ammonium molybdate group dormin output is 26.29mg/L, improves 221.39% than control group.

embodiment 4

Actication of culture and seed culture method are with embodiment 1.By the bacterial classification activated with 5% inoculum size access shaking flask that PD liquid nutrient medium is housed carry out fermentation culture, the composition of PD liquid nutrient medium and content are: glucose 20g/L, potato 200g, clean the rear diced shape of peeling, put on the inherent electromagnetic oven of pot and boil, timing 30 minutes, then with four layers of gauze, well-done potato is filtered, by its filtrate with distilled water diluting to 1L; The PD liquid nutrient medium prepared is divided into two groups, and one group adds Soduxin and ammonium molybdate, makes Soduxin and the concentration of ammonium molybdate in PD liquid nutrient medium be respectively 5mM/L and 5mM/L, and another group does not then add in contrast, accesses bacterial classification after sterilizing; With 150 revs/min of oscillation and fermentation cultivation 9 days at 28 DEG C.

Recording control group dormin output by high performance liquid chromatography is 4.92mg/L, adds Soduxin and ammonium molybdate group dormin output is 17.76mg/L, improves 260.98% than control group.

embodiment 5

Actication of culture and seed culture method are with embodiment 1.By the bacterial classification activated with 5% inoculum size access shaking flask that dedicated liquid fermention medium is housed carry out fermentation culture, the composition of dedicated liquid fermention medium and content are: glucose 20g/L, MgSO ₄7H ₂o0.2g/L, KCl0.5g/L, CaCO ₃5g/L, KH ₂pO ₄0.8g/L, VB11 mg/L, monosodium glutamate 3.0g/L, FeSO ₄7H ₂o0.5mg/L, ZnSO ₄7H ₂o2.5mg/L, CuSO ₄7H ₂o4mg/L, Semen Maydis powder 4g/L, supplies 1L with distilled water.The dedicated liquid fermention medium prepared is divided into two groups, and one group adds Soduxin, makes the concentration of Soduxin in dedicated liquid fermention medium be 15mM/L, and another group does not then add in contrast, accesses bacterial classification after sterilizing; With 150 revs/min of oscillation and fermentation cultivation 10 days at 28 DEG C.

Recording control group dormin output by high performance liquid chromatography is 8.18mg/L, and adding Soduxin group dormin output is 35.18mg/L, improves 330.07% than control group.

embodiment 6

Actication of culture and seed culture method are with embodiment 1.By the bacterial classification activated with 8% inoculum size access shaking flask that PD liquid nutrient medium is housed carry out fermentation culture, the composition of PD liquid nutrient medium and content are: glucose 20g/L, potato 200g, clean the rear diced shape of peeling, put on the inherent electromagnetic oven of pot and boil, timing 30 minutes, then with four layers of gauze, well-done potato is filtered, by its filtrate with distilled water diluting to 1L; Tomato blade is pulverized, according to the weight ratio 1:0.02 lixiviate of the phosphate buffered saline buffer of 0.1M and the tomato leaf tissue of pulverizing, extraction temperature 4 DEG C, extraction time is 12h, and after centrifugal, its supernatant liquor aperture is that the film filter of 0.22 μm filters for subsequent use afterwards; The PD liquid nutrient medium prepared is divided into two groups, one group adds Soduxin, the concentration of Soduxin in PD liquid nutrient medium is made to be 5mM/L, another group does not then add in contrast, after sterilizing, tomato leaf vat liquor is added in the PD liquid nutrient medium of Soduxin group by the consumption of 2mL/L, then access bacterial classification; With 150 revs/min of oscillation and fermentation cultivation 9 days at 28 DEG C.

Recording control group dormin output by high performance liquid chromatography is 4.92mg/L, adds Soduxin and tomato leaf vat liquor group dormin output is 20.57mg/L, improves 318.09% than control group.

Claims (10)

1. one kind is improved the method for PBI 58 fermentation yield, comprise the following steps: the Botrytis cinerea that first can produce dormin to be seeded on potato agar glucose (PDA) flat board activation culture 4 ~ 5 days, culture temperature is 25 ~ 28 DEG C, then the Botrytis cinerea activated is cultivated 48 ~ 72h in seed culture medium, by seed liquor with 5% ~ 10% inoculum size access liquid fermentation medium, fermentation culture 9 ~ 10 days produces dormin; Described liquid fermentation medium needs to add the one in plant vat liquor, Soduxin, ammonium molybdate or two or more combination.

2. the method for raising PBI 58 fermentation yield according to claim 1, is characterized in that, composition and the content of seed culture medium used are as follows, glucose 20g/L, MgSO ₄ .7H ₂o0.2g/L, KCl0.5g/L, KH ₂pO ₄0.8g/L, VB11 mg/L, monosodium glutamate 3.0g/L, FeSO ₄ .7H ₂o0.5mg/L, ZnSO ₄ .7H ₂o2.5mg/L, CuSO ₄ .7H ₂o4mg/L, supplies 1L with distilled water.

3. the method for raising PBI 58 fermentation yield according to claim 1, is characterized in that, liquid fermentation medium used is PD liquid nutrient medium or dedicated liquid fermention medium.

4. the method for raising PBI 58 fermentation yield according to claim 3, is characterized in that, composition and the content of dedicated liquid fermention medium are as follows, glucose 20g/L, MgSO ₄ .7H ₂o0.2g/L, KCl0.5g/L, CaCO ₃5g/L, KH ₂pO ₄0.8g/L, VB11 mg/L, monosodium glutamate 3.0g/L, FeSO ₄ .7H ₂o0.5mg/L, ZnSO ₄ .7H ₂o2.5mg/L, CuSO ₄ .7H ₂o4mg/L, Semen Maydis powder 4g/L, supplies 1L with distilled water.

5. the method for raising PBI 58 fermentation yield according to claim 1, is characterized in that, plant vat liquor used is the vat liquor of Folium Capsici, tomato leaf, and vat liquor aperture is use after the film filter of 0.22 ~ 0.45 μm filters.

6. the method for raising PBI 58 fermentation yield according to claim 5, it is characterized in that, adopt the plant leaf tissue of the phosphate buffered saline buffer of 0.1M and pulverizing according to weight ratio 1:(0.02 ~ 0.2) carry out lixiviate, extraction temperature 4 DEG C, time is 12 ~ 24h, and after lixiviate, its supernatant liquor of centrifuging and taking is as vat liquor.

7. improve the method for PBI 58 fermentation yield according to claim 5, it is characterized in that, in liquid fermentation medium used, the add-on of plant vat liquor is 2mL ~ 10mL/L.

8. improve the method for PBI 58 fermentation yield according to claim 1, it is characterized in that, in liquid fermentation medium used, the concentration of Soduxin is 1 ~ 15mM/L.

9. improve the method for PBI 58 fermentation yield according to claim 1, it is characterized in that, in liquid fermentation medium used, the concentration of ammonium molybdate is 0.05 ~ 5mM/L.

10. improve the method for PBI 58 fermentation yield according to claim 1, it is characterized in that, in liquid fermentation medium used, the mass ratio of Soduxin and ammonium molybdate is 1:(0.2 ~ 12).