CN103146601A - Bacillus spp culture medium with coconut water as main raw material - Google Patents

Abstract

The invention discloses a Bacillus spp culture medium with coconut water as a main raw material. The culture medium comprises: (1) a growth stimulation culture medium (/L); (2) a liquid amplification culture medium (/L); and (3) a colony counting culture medium (/L). According to the invention, economical, cheap and widely available coconut water is used as the main raw material for the Bacillus spp culture medium, and a scientific and reasonable application method of the Bacillus spp culture medium is constructed at the same time; with the application method, the culture medium can meet requirements of scale-up culture of Bacillus spp; a leftover of coconut processing in Hainan province, i.e., coconut water, is made full use of, the Bacillus spp culture medium with coconut water as the main raw material is created for the first time, a good cheap culture medium formula is provided for development of scale-up culture of Bacillus spp in the future, and the Bacillus spp culture medium has a good application prospect; an effective approach is provided for high-valued utilization of coconut water, an added value of coconut processing can be obviously increased, and pollution of the industry of coconut processing to the environment can be reduced at the same time.

Description

Bacillus culture medium take Sucus Cocois as main raw material

Technical field

The present invention relates to a kind of bacillus culture medium take Sucus Cocois as main raw material.

Background technology

Born of the same parents' exoamylases, proteolytic enzyme and the lipase that genus bacillus produces in breeding can be the biological residual body in aquaculture water, residual bait and other organic matter degradations small organic molecule, genus bacillus all has obvious Degradation to the ammonia nitrogen in aquaculture water, nitrite nitrogen, nitric nitrogen and COD simultaneously, aging and the eutrophication that this has reduced aquaculture water to a great extent, the purpose that reaches improvement and repair aquaculture water; In addition, genus bacillus can also suppress growing of various pathogens in aquaculture water by antagonistic action.Therefore, the microbial preparation of developing based on genus bacillus is applied to sea farming system, has broad application prospects.

In the genus bacillus mass-producing is cultivated; mainly take starch, wheat bran, soybean protein and yeast powder etc. as main raw material; after the substratum of setting up take these raw materials as main component is cultivated the genus bacillus mass-producing; viable bacteria concentration also can reach the requirement of suitability for industrialized production, but substratum (liquid) cost per ton be more than 200 yuan.In order further to reduce the genus bacillus industrial production cost; seeking a kind of culture medium raw material of relatively cheap and wide material sources and set up bacillus culture medium take this raw material as main component, is the only way of realizing that the genus bacillus mass-producing is cultivated, satisfied the wide popularization and application of said preparation.

Coconut palm is a kind of modal tropical food energy crop of various places, Hainan plantation.Sucus Cocois wherein contains abundant glucose, fructose, sucrose, protein, fat, multi mineral prime element and VITAMIN etc., is the important medium component of the microorganisms such as acetobacter xylinum.And in the traditional problem in Hainan, Sucus Cocois is only directly entered surrounding enviroment as a kind of tankage, and this not only causes serious environmental pollution, has also caused the great wasting of resources.

Summary of the invention

Having the purpose of this invention is to provide Sucus Cocois economic, cheap, wide material sources is the bacillus culture medium of main raw material, sets up simultaneously its scientific and rational using method; By this using method, these substratum can satisfy the requirement that the genus bacillus mass-producing is cultivated.

To achieve these goals, technical scheme of the present invention is: the bacillus culture medium take Sucus Cocois as main raw material is provided, comprises:

(1) the stimulating growth substratum (/L): Sucus Cocois 70 ~ 200ml, analysis for soybean powder 4 ~ 8g, K ₂HPO ₄1g, MnSO ₄H ₂O 0.02g, sterilization seawater 800 ~ 930ml, 7.2,121 ℃ of sterilization 20min of pH;

(2) the fluid enlargement culture base (/L): Sucus Cocois 100 ~ 250ml, analysis for soybean powder 4 ~ 8g, Semen Maydis powder 2g, K ₂HPO ₄2g, MnSO ₄H ₂O 0.05g, MgSO ₄7H ₂O 0.1g, sterilization seawater 750 ~ 900ml, 7.2,121 ℃ of sterilization 20min of pH;

(3) the enumeration substratum (/L): Sucus Cocois 100 ~ 250ml, analysis for soybean powder 4 ~ 8g, Semen Maydis powder 2g, K ₂HPO ₄2g, MnSO ₄H ₂O 0.05g, MgSO ₄7H ₂O 0.1g, agar 16g, sterilization seawater 750 ~ 900ml, 7.2,121 ℃ of sterilization 20min of pH.

The using method of the bacillus culture medium of the present invention take Sucus Cocois as main raw material:

(1) the single bacterium colony of picking genus bacillus is to 100ml stimulating growth substratum, cultivates 24h under 37 ℃, 180r/min condition;

(2) draw 1ml stimulating growth substratum bacterium liquid and be forwarded in fresh fluid enlargement culture base, cultivate 20h under 37 ℃, 180r/min condition.

Advantage of the present invention:

1, the present invention takes full advantage of the tankage----Sucus Cocois of Hainan coconut processing; set up first the bacillus culture medium take Sucus Cocois as main raw material; provide good Cheap Medium Compound for carrying out from now on genus bacillus mass-producing cultivation, had a good application prospect.

2, the present invention for the higher value application of Sucus Cocois provides an effective way, can obviously increase the added value of coconut processing, has reduced simultaneously the pollution of this processing industry to environment.

Description of drawings

Fig. 1 is the gemma of genus bacillus; In figure, arrow is depicted as gemma, is green, and ellipse or cylindricality, end life or middle life, size mostly are 0.6 ~ 0.7 μ m * 0.8 ~ 1.5 μ m; Nourishing body takes on a red color, and shaft-like, size mostly is 0.7 ~ 0.8 μ m * 3.0 ~ 4.0 μ m.

Fig. 2 is the genus bacillus colonial morphology on the enumeration substratum.In figure, the arrow indication is bacterium colony, and coarse, surface drying, canescence, edge are irregular, diffusion growth, opaque, d=2～5mm.

Embodiment

The invention will be further described with concrete case study on implementation for the below.

Case study on implementation one

Bacillus culture medium take Sucus Cocois as main raw material, the subtilis that follows these steps to preserve 1 year under p-80 ℃ of conditions carry out mass-producing and cultivate test, and be specific as follows:

1, will preserve the subtilis quick-thawing of 1 year under-80 ℃ of conditions with the warm water of 40 ℃, draw bacteria suspension that 1ml thaws to 100ml stimulating growth substratum, cultivate 24h under 37 ℃, 180r/min condition;

2, draw 1ml and cultivate stimulating growth substratum bacterium liquid after 24h to 100ml fluid enlargement culture base, cultivate 20h under 37 ℃, 180r/min condition;

3, draw 1ml fluid enlargement culture base bacterium liquid, gradient dilution is 10 ^-6, 10 ^-7, 10 ^-8Bacterium liquid; Drawing above-mentioned each gradient dilution bacterium liquid of 0.1ml is coated with on the enumeration substratum, counts; If 3 parallel group.Concrete outcome sees Table 1.

Table 1

*, bacterium colony covers with whole flat board, can't effectively count.

Result shows, the bacillus culture medium take Sucus Cocois as main raw material that the present invention sets up and carry out mass-producing and cultivate by the subtilis that preserved 1 year under the p-80 ℃ of conditions of its using method after, viable bacteria concentration is 8.4 * 10 ⁹CFU/ml.

Case study on implementation two

Bacillus culture medium take Sucus Cocois as main raw material follows these steps to that common on the market subtilis solid phase prod is carried out mass-producing and cultivates test, and is specific as follows:

1, aseptic technique takes 1g subtilis solid phase prod to 100ml stimulating growth substratum, cultivates 24h under 37 ℃, 200r/min condition;

2, draw 1ml and cultivate stimulating growth substratum bacterium liquid after 24h to 100ml fluid enlargement culture base, cultivate 20h under 37 ℃, 180r/min condition;

3, draw 1ml fluid enlargement culture base bacterium liquid, gradient dilution is 10 ^-6, 10 ^-7, 10 ^-8Bacterium liquid; Drawing above-mentioned each gradient dilution bacterium liquid of 0.1ml is coated with on the enumeration substratum, counts; If 3 parallel group.Concrete outcome sees Table 2.

Table 2

Result shows, the bacillus culture medium take Sucus Cocois as main raw material that the present invention sets up and by its using method, common on the market subtilis solid phase prod is carried out mass-producing and cultivates after, viable bacteria concentration is 5.2 * 10 ⁹CFU/ml.

Case study on implementation three

Bacillus culture medium take Sucus Cocois as main raw material follows these steps to that the lichens bacillus living is carried out mass-producing and cultivates test, and is specific as follows:

1, picking LB solid medium (/L: peptone 10g, yeast powder 5g, agar 15g, pH 7.2, sterilization seawater 1000ml, 121 ℃ of sterilization 20min) single bacterium colony of the Bacillus licheniformis of upper purifying is to 100ml stimulating growth substratum, cultivates 24h under 37 ℃, 180r/min condition;

2, draw 1ml and cultivate stimulating growth substratum bacterium liquid after 24h to 100ml fluid enlargement culture base, cultivate 20h under 37 ℃, 180r/min condition;

3, draw 1ml fluid enlargement culture base bacterium liquid, gradient dilution is 10 ^-6, 10 ^-7, 10 ^-8Bacterium liquid; Drawing above-mentioned each gradient dilution bacterium liquid of 0.1ml is coated with on the enumeration substratum, counts; If 3 parallel group.Concrete outcome sees Table 3.

Table 3

*, bacterium colony covers with whole flat board, can't effectively count.

Result shows, the bacillus culture medium take Sucus Cocois as main raw material that the present invention sets up and by its using method, lichens bacillus living list bacterium colony is carried out mass-producing and cultivates after, viable bacteria concentration is 2.1 * 10 ¹⁰CFU/ml.

Fig. 1 is the gemma of genus bacillus; In figure, arrow is depicted as gemma, is green, and ellipse or cylindricality, end life or middle life, size mostly are 0.6 ~ 0.7 μ m * 0.8 ~ 1.5 μ m; Nourishing body takes on a red color, and shaft-like, size mostly is 0.7 ~ 0.8 μ m * 3.0 ~ 4.0 μ m.

Fig. 2 is the genus bacillus colonial morphology on the enumeration substratum.In figure, the arrow indication is bacterium colony, and coarse, surface drying, canescence, edge are irregular, diffusion growth, opaque, d=2～5mm.

Above disclosed is only preferred embodiment of the present invention, certainly can not limit with this interest field of the present invention, and the equivalent variations of therefore doing according to claim of the present invention still belongs to the scope that the present invention is contained.

Claims (1)

1. the bacillus culture medium take Sucus Cocois as main raw material is characterized in that comprising:

(1) the stimulating growth substratum (/L): Sucus Cocois 70 ~ 200ml, analysis for soybean powder 4 ~ 8g, K ₂HPO ₄1g, MnSO ₄H ₂O 0.02g, sterilization seawater 800 ~ 930ml, 7.2,121 ℃ of sterilization 20min of pH;

(2) the fluid enlargement culture base (/L): Sucus Cocois 100 ~ 250ml, analysis for soybean powder 4 ~ 8g, Semen Maydis powder 2g, K ₂HPO ₄2g, MnSO ₄H ₂O 0.05g, MgSO ₄7H ₂O 0.1g, sterilization seawater 750 ~ 900ml, 7.2,121 ℃ of sterilization 20min of pH;

(3) the enumeration substratum (/L): Sucus Cocois 100 ~ 250ml, analysis for soybean powder 4 ~ 8g, Semen Maydis powder 2g, K ₂HPO ₄2g, MnSO ₄H ₂O 0.05g, MgSO ₄7H ₂O 0.1g, agar 16g, sterilization seawater 750 ~ 900ml, 7.2,121 ℃ of sterilization 20min of pH.

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