CN106967769B - Fermentation medium and culture method for improving leucotrichin production of lilac purpurea - Google Patents
Fermentation medium and culture method for improving leucotrichin production of lilac purpurea Download PDFInfo
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Abstract
The invention relates to a fermentation culture medium for improving the yield of leucotrichin from lilac spore fungus, which comprises the following raw materials in parts by weight: 45-55 parts of glucose, 4.5-5.5 parts of peptone, 4.5-5.5 parts of yeast extract and K2HPO40.5 to 2.0 parts of Na2CO38.5 to 12.5 parts of MgSO4·7H20.1-0.5 part of O, 1.5-10 parts of corn flour and FeSO4·7H20.005-0.05 part of O and 1.5-10.5 parts of urea. The culture method for improving the leucotrichin production of the violaxanthin by using the culture medium comprises the following steps: the method comprises the steps of carrying out fermentation culture on the purple rhodosporium shake flask culture liquid, carrying out seeding tank culture by using the fermentation medium, and inoculating the seeding tank culture liquid into a fermentation tank for fermentation. The method for culturing the lilac purple spore bacteria by adopting the fermentation culture medium has the characteristics of low cost, short fermentation period, high yield of white ash prepared bacterin and the like.
Description
Technical Field
The invention belongs to the field of microbial fermentation, and particularly relates to a fermentation culture medium and a culture method for improving the yield of leucotrichin from lilac purpurea.
Background
Root knot nematode of cucumber (A)Meloidogyne spp.) Meloidogyne cucumerina belonging to the class of the lateral coccales (Secernencea), order of Staphylotrichales (Tylenchida), Heterodermoideae (Heterodioidea), Meloidogyne cucumeriae (Meloidogyne cucumeride) ((MeloidogyneGoeldi), an important group of phytopathogenic nematodes that severely endanger commercial crops and are widely distributed around the world. The cucumber root-knot nematode has a very wide host range, and can infect not only economic grain crops, vegetables and ornamental flowers and trees, but also even weeds. The root-knot nematode disease of cucumber can reduce the yield of economic crops by 10-20%, and even cause the loss of crop by over 75% when the crop is serious. In China, due to the high-speed development of agricultural economy, the continuous reform and adjustment of an industrial structure, the continuous reasonable optimization of a planting system, the rapid development of greenhouse and greenhouse cultivation areas and the slight management consciousness of farmers, the disease degree of the cucumber root-knot nematode disease tends to increase year by year, for example, the annual yield reduction of host crops caused by the cucumber root-knot nematode in southern China is 15-20% and is more than 70% in severe cases. The cucumber root-knot nematode not only causes the yield reduction of crops, but also induces other diseases such as root rot, bacterial wilt, blight and tobacco black shank of hosts. At present, diseases caused by cucumber root-knot nematodes severely restrict the development and stable yield of crops in protected areas of China, and influence the sustainable, rapid and healthy development of national agricultural economy.
Purple lilac spore fungus (purple lilac spore fungus)Purpureocillium lilacinum) As a nematode biocontrol factor, a large number of researches show that the nematode has a wide host range, can adapt to different climatic conditions and can parasitize various nematodes such as meloidogyne incognita, cyst nematode, heterodera, golden nematode, nematode of a puncture nematode, pearl nematode and the like. The viola lilacina has the characteristics of epidemic contact killing, safety, no pollution and conformity with the development of an environment-friendly society, can be used as a new effective biocontrol factor, overcomes a plurality of problems brought by the traditional chemical pesticide, and is an application biocontrol bacterium and a functional bacterium with great popularization potential.
Park et al detected the presence of leucotrichin the fermentation broth of viola glaucescens and used as an indicator of nematode activity, which has been widely used to control plant nematodes at present. Leupeptin is a nonacycline substance produced by non-ribosomal metabolic pathways and widely present in plants, bacteria and fungi. Griseofulvin consists of many components, of which griseofulvin a and B are widely and abundantly present, and others, such as griseofulvin D, E, F, H, K and L, of which griseofulvin a functions to inhibit mitochondrial ATP synthesis and participate in different phosphate metabolic pathways.
However, in the prior art, the rapid propagation of the purple lilac spore and the improvement of the yield of the griseofulvin still have the technical problems to be solved urgently.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a fermentation culture medium for improving the yield of leucovorin produced by lilac violet spore, and the culture of the lilac violet spore by adopting the fermentation culture medium has the characteristics of low cost, short fermentation period, high yield of the leucovorin produced and the like.
In order to solve the problems, the invention is realized by the following technical scheme:
designing a fermentation culture medium PPDB for improving the yield of leucoderma preparation of the violonyeum, which comprises the following raw materials in parts by weight: 45-55 parts of glucose, 4.5-5.5 parts of peptone, 4.5-5.5 parts of yeast extract and K2HPO40.5 to 2.0 parts of Na2CO38.5 to 12.5 parts of MgSO4·7H20.1-0.5 part of O, 1.5-10 parts of corn flour and FeSO4·7H20.005-0.05 part of O and 1.5-10.5 parts of urea.
Preferably, the fermentation medium comprises the following raw materials in parts by weight: 50 parts of glucose, 5 parts of peptone, 5 parts of yeast extract and K2HPO41.0 part of Na2CO310 parts of MgSO (MgSO)4·7H20.2 part of O, 3 parts of corn flour and FeSO4·7H20.047 part of O and 5 parts of urea.
More preferably, the fermentation medium contains the following concentrations of the starting materials: glucose 50.0g/L, peptone 5.0g/L, yeast extract5g/L、K2HPO4 1.0g/L、Na2CO3 10.0g/L、MgSO4·7H2O0.2 g/L, corn flour 3g/L, FeSO4·7H2O0.047 g/L and urea 5 g/L.
The pH value of the fermentation medium is 7.5-8.5, preferably 7.8-8.0.
The glucose is industrial glucose for cultivating bacteria, and the urea is agricultural urea.
The invention also designs a culture method for improving the leucotrichin production of the lilac purpurea, which comprises the following steps:
(1) shake flask fermentation culture
Inoculating the activated lilium purple spore bacteria liquid into a shake flask for culture to obtain a suspension, and then performing shake culture for 36-48 h under the conditions that the rotation speed is 200rpm and the temperature is 28 ℃ until OD is reached600= 0.8-1.2, obtaining shake flask culture liquid;
the fermentation medium in the shake flask is potato glucose liquid medium, and each liter of the culture medium is prepared by adding water into 200g of potatoes and 20g of glucose to a constant volume, uniformly mixing and sterilizing.
(2) Seeding tank culture
Arranging the fermentation culture medium for improving the limescin production by the lilac purple spore in a seeding tank, inoculating the shake flask culture bacterial liquid obtained in the step (1) into the seeding tank (the inoculation amount of the shake flask culture bacterial liquid is 1-3 wt% of the fermentation culture medium), and carrying out fermentation culture for 36-54 h under the conditions of the ventilation volume of 1.4L/h, the temperature of 28 ℃ and the rotation speed of 180rpm (pH 7.8) until the dissolved oxygen content is reduced to 20-30% (air saturation) to obtain the seeding tank culture bacterial liquid;
the ventilation and temperature control method comprises the following steps:
when fermentation is started, controlling the volume ratio of gas to liquid to be 0.4-0.7; in the middle stage of fermentation, when the dissolved oxygen is reduced to 50%, the gas-liquid volume ratio is adjusted to 1.4-1.6; and in the later stage of fermentation, when the dissolved oxygen is reduced to 50%, adjusting the gas-liquid volume ratio to 2.1-2.3, and keeping the ventilation until the fermentation is finished.
(3) Fermentation of
Inoculating the obtained seed tank culture bacterial liquid into a fermentation tank according to the inoculation amount of 5-10 wt% for fermentation, wherein in the middle stage of fermentation: when the dissolved oxygen content is reduced to 50%, adjusting the gas-liquid volume ratio to 1.0-1.8; and (3) in the later fermentation stage: when the dissolved oxygen content is reduced to 50%, the gas-liquid volume ratio is adjusted to 2.1-2.7, the ventilation volume is kept until the fermentation is finished, and the temperature is maintained at 25-30 ℃ in the process, and the rotation speed is 200 rpm.
The invention has the following positive and beneficial technical effects:
(1) the fermentation medium has balanced and reasonable component ratio, can provide balanced nutrient substances for mass propagation of the lilac purple spore and production of leuconystatin, promotes the lilac purple spore to grow and propagate quickly, and promotes the leuconystatin to produce a large amount in a short time.
(2) The raw materials of the fermentation medium are mainly from cheap and easily-obtained agricultural and sideline products, and in the process of fermentation of the lilac violet spore, the spore quantity of the lilac violet spore is increased, the fermentation period is shortened, and the yield of the white lime nystatin is increased.
Detailed Description
The following examples are given to illustrate specific embodiments of the present invention, but are not intended to limit the scope of the present invention in any way. The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the biochemical reagents and raw materials are all conventional products on the market if not specified.
Example 1: a fermentation culture medium PPDB for improving the production of leuconystatin by violet spore bacteria contains the following raw materials in concentration: glucose 50.0g/L, peptone 5.0g/L, and yeast extract 5g/L, K2HPO4 1.0g/L、Na2CO3 10.0g/L、MgSO4·7H2O0.2 g/L, corn flour 3g/L, FeSO4·7H2O0.047 g/L and urea 5 g/L.
Example 2: a fermentation culture medium PPDB for improving the yield of leuconystatin by using violet spore bacteria comprises the following raw materials in parts by weight: 55 g/L glucose, 4.5g/L peptone and 5.5 g/L, K yeast extract2HPO4 0.5 g/L、Na2CO3 12.5 g/L、MgSO4·7H2O0.1 g/L, corn flour 10 g/L, FeSO4·7H20.005 g/L of O and 10.5 g/L of urea.
Example 3: a fermentation culture medium PPDB for improving the yield of leuconystatin by using violet spore bacteria comprises the following raw materials in parts by weight: 45 g/L glucose, 5.5 g/L peptone and 4.5g/L, K yeast extract2HPO4 2.0 g/L、Na2CO38.5g/L、MgSO4·7H2O0.5 g/L, corn flour 1.5g/L, FeSO4·7H2O0.05 g/L and urea 1.5 g/L.
Example 4: a culture method for improving the leucoderma preparation produced by violet spore fungus comprises the following steps:
(1) shake flask fermentation culture
Inoculating the activated purple violet spore bacteria liquid into a shake flask for culturing to obtain suspension, and performing shake culture at the rotation speed of 200rpm and the temperature of 28 ℃ for 40h until OD is achieved600=1.0, obtaining shake flask culture liquid; the fermentation medium in the shake flask is potato glucose liquid medium, and each liter of the culture medium is prepared by adding water into 200g of potatoes and 20g of glucose to a constant volume, uniformly mixing and sterilizing.
(2) Seeding tank culture
Setting the PPDB of the fermentation culture medium in the example 1 in a seeding tank, inoculating the shake flask culture bacterial liquid obtained in the step (1) into the seeding tank (the inoculation amount of the shake flask culture bacterial liquid is 2wt% of the fermentation culture medium), and performing fermentation culture for 48 hours under the conditions of the ventilation volume of 1.4L/h, the temperature of 28 ℃ and the rotating speed of 180rpm (pH 7.8) until the dissolved oxygen is reduced to 25% (air saturation) to obtain the culture bacterial liquid of the seeding tank;
the ventilation and temperature control method comprises the following steps: when fermentation is started, controlling the volume ratio of gas to liquid to be 0.5-0.6; in the middle stage of fermentation, when the dissolved oxygen is reduced to 50%, the gas-liquid volume ratio is adjusted to 1.5-1.6; and in the later stage of fermentation, when the dissolved oxygen is reduced to 50%, adjusting the gas-liquid volume ratio to 2.1-2.2, and keeping the ventilation volume until the fermentation is finished.
(3) Fermentation of
Inoculating the obtained seed tank culture solution into a fermentation tank according to the inoculation amount of 8wt% for fermentation (the effect data are shown in table 1), wherein in the middle stage of fermentation: when the dissolved oxygen content is reduced to 50%, adjusting the gas-liquid volume ratio to 1.4-1.5; and (3) in the later fermentation stage: when the dissolved oxygen content is reduced to 50%, adjusting the gas-liquid volume ratio to 2.4-2.5, and keeping the ventilation volume until the fermentation is finished, wherein the temperature is maintained at 27-28 ℃ in the process, and the rotation speed is 200 rpm.
Comparative example
The comparative example was carried out substantially the same as example 4 except that the conventional control PDB medium was placed in the seed tank in step (2), and the test results of example 4 and the comparative example are shown in Table 1.
TABLE 1 comparison of fermentation effects of different treatments on lilac violet spore bacteria
Claims (2)
1. A method for increasing the content of violet sporePurpureocillium lilacinum) The culture method for producing leupeptin is characterized by comprising the following steps:
(1) shake flask fermentation culture
Inoculating the activated lilium purple spore bacteria liquid into a shake flask for culture to obtain a suspension, and then performing shake culture for 36-48 h under the conditions that the rotation speed is 200rpm and the temperature is 28 ℃ until OD is reached600= 0.8-1.2, obtaining shake flask culture liquid;
(2) seeding tank culture
A fermentation culture medium for improving the production of leucoderma preparation rhzomorph by violet spore bacteria is arranged in a seeding tank, and the fermentation culture medium contains the following raw materials in concentration: glucose 50.0g/L, peptone 5.0g/L, and yeast extract 5g/L, K2HPO4 1.0g/L、Na2CO310.0g/L、MgSO4·7H2O0.2 g/L, corn flour 3g/L, FeSO4·7H2O0.047 g/L and urea 5 g/L; the pH value of the fermentation medium is 7.8-8.0;
inoculating the shake flask culture bacterial liquid obtained in the step (1) into a seed tank, wherein the inoculation amount of the shake flask culture bacterial liquid is 1-3 wt% of a fermentation culture medium, and carrying out fermentation culture for 36-54 h under the conditions of the ventilation amount of 1.4L/h, the temperature of 28 ℃ and the rotation speed of 180rpm until the dissolved oxygen is reduced to 20-30%, so as to obtain a seed tank culture bacterial liquid;
(3) inoculating the obtained seed tank culture bacterial liquid into a fermentation tank according to the inoculation amount of 5-10 wt% for fermentation;
the parameters of the fermentation were controlled as follows: in the middle stage of fermentation, when the dissolved oxygen is reduced to 50%, the gas-liquid volume ratio is adjusted to 1.0-1.8; in the later stage of fermentation, when the dissolved oxygen is reduced to 50%, adjusting the gas-liquid volume ratio to 2.1-2.7, and keeping the ventilation volume until the fermentation is finished, wherein the temperature is kept at 25-30 ℃ in the process, and the rotation speed is 200 rpm;
the ventilation and temperature control method comprises the following steps: when fermentation is started, controlling the volume ratio of gas to liquid to be 0.4-0.7; in the middle stage of fermentation, when the dissolved oxygen is reduced to 50%, the gas-liquid volume ratio is adjusted to 1.4-1.6; and in the later stage of fermentation, when the dissolved oxygen is reduced to 50%, adjusting the gas-liquid volume ratio to 2.1-2.3, and keeping the ventilation until the fermentation is finished.
2. The culture method according to claim 1, wherein: in the step (1), the fermentation medium in the shake flask is potato glucose liquid medium, and each liter of the culture medium is prepared by adding water into 200g of potatoes and 20g of glucose to fix the volume, uniformly mixing and sterilizing.
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