阅读说明：本技术 一种利用淀粉废水发酵生产多糖的方法 (Method for producing polysaccharide by fermenting starch wastewater ) 是由 周文文 高凌霄 王珏帆 汪旖清 钱超 于 2019-10-17 设计创作，主要内容包括：本发明公开了一种利用淀粉废水发酵生产多糖的方法,以淀粉废水为原料,利用多粘类芽孢杆菌进行发酵。本发明包括生产菌在淀粉废水中生长与多糖发酵适用性验证以及淀粉废水中芽孢杆菌生长与发酵曲线的测定,其中多粘类芽孢杆菌在淀粉废水中发酵多糖的生产方法,主要流程是在pH调节至中性的淀粉废水中接入芽孢杆菌在恒温下震荡培养。本发明公开的多糖生产方法不仅能够以较高的产糖效率生产得到具有多种用途的胞外多糖,而且以工业淀粉废水为原料,能够大大降低生产成本,同时发酵过程中能够明显降低废水中的淀粉和蛋白质等有机物含量,有效减少工业废水污染,具有极大的发展潜力。(The invention discloses a method for producing polysaccharide by fermenting starch wastewater, which takes the starch wastewater as a raw material and utilizes paenibacillus polymyxa to ferment. The invention comprises the verification of the growth of production bacteria in starch wastewater and the suitability of polysaccharide fermentation and the determination of growth and fermentation curves of bacillus in the starch wastewater, wherein the production method for fermenting polysaccharide by paenibacillus polymyxa in the starch wastewater mainly comprises the step of inoculating bacillus into the starch wastewater with the pH adjusted to be neutral and carrying out shake culture at a constant temperature. The polysaccharide production method disclosed by the invention can be used for producing extracellular polysaccharide with multiple purposes at high sugar production efficiency, and can be used for greatly reducing the production cost by taking industrial starch wastewater as a raw material, and meanwhile, the content of organic matters such as starch, protein and the like in the wastewater can be obviously reduced in the fermentation process, so that the industrial wastewater pollution is effectively reduced, and the polysaccharide production method has great development potential.)

1. A method for producing polysaccharide by fermenting starch wastewater is characterized by comprising the following steps:

(1) activating a paenibacillus polymyxa strain on a plate culture medium, inoculating the activated strain into a seed culture medium, and performing shake-flask culture at the temperature of 30-37 ℃ for 8-12h to obtain a paenibacillus polymyxa seed solution;

(2) adjusting the pH value of the starch wastewater to be neutral; inoculating the seed solution obtained in the step (1) to the starch wastewater by an inoculation amount of 1.5-2.5% in volume percentage, and carrying out shake culture in a shaking table at the temperature of 25-35 ℃ and the rotation speed of 150-220rpm for 48-72 h.

2. The method for producing polysaccharide by fermenting starch wastewater according to claim 1, wherein the mass volume fractions of the components of the plate culture medium are respectively: 1-2% of sucrose, 0.5-1% of peptone, 0.5% of yeast extract powder, 0.3% of disodium hydrogen phosphate and 2% of agar strips, sterilizing by high-pressure steam at 115 ℃, and pouring into a flat plate.

3. The method for producing polysaccharide by fermenting starch wastewater according to claim 1, wherein the mass volume fractions of the components of the seed culture medium are respectively: 5% of sucrose, 0.5% of tryptone, 0.1% of yeast extract powder and 0.3% of disodium hydrogen phosphate.

4. The method for producing polysaccharide by fermenting starch wastewater according to claim 1, wherein before the Paenibacillus polymyxa is inoculated into the starch wastewater, the starch wastewater is firstly verified to have microbial growth and polysaccharide fermentation applicability in the wastewater by the following steps:

a) verifying the growth applicability of microorganisms in wastewater: adjusting the pH value of the starch wastewater to be neutral, inoculating a paenibacillus polymyxa colony, performing shake culture at the temperature of 30-37 ℃ for 24-48h, coating and counting, and if the maximum number reaches 10⁸CFU/mL satisfies the applicability of microorganism growth;

b) polysaccharide fermentation applicability verification: adjusting the pH value of the starch wastewater to be neutral, adding agar strips to prepare a starch wastewater flat plate, taking a paenibacillus polymyxa bacterial liquid for streaking, carrying out shake culture at the temperature of 30-37 ℃ for 24-48h, observing the periphery of a bacterial colony on the wastewater flat plate, and if continuous pasty substances are generated on the periphery of the bacterial colony and filamentous adhesion is picked up, the applicability of polysaccharide fermentation is met, otherwise, the applicability is not met.

5. Use of a polysaccharide prepared by the method of any one of claims 1 to 4 for the preparation of a skin care product for repairing sun damage.

6. The use as claimed in claim 5, wherein: the addition amount of the polysaccharide in the skin care product is 100 mu g/mL-1000 mu g/mL.

Technical Field

The invention belongs to the technical field of microbial technology and sugar production, and particularly relates to a method for producing polysaccharide by fermenting paenibacillus polymyxa by taking starch wastewater as a raw material.

Background

The starch wastewater is waste liquid generated in the starch raw material processing process, is acidic wastewater containing high-concentration organic matters, and is one of several types of wastewater with higher pollution degree generated in the food industry at present. According to the technical specification of starch wastewater treatment engineering issued by the ministry of environmental protection of China, starch production factories can select a wastewater treatment method according to the biochemical characteristics of the finally obtained starch wastewater, and the mainstream treatment methods at present are flocculation precipitation and biological treatment or the combination of the two methods. However, starch wastewater often contains high concentrations of organic compounds such as starch, polysaccharide, crude protein, fat, crude fiber, etc., and has a very high value for recycling (Jin B, Huang LP, Lant P. Rhizopus precursors-a procedure for the utilization of starch and purification of static water materials Letters,2003,25(23): 1983-. Therefore, if the starch wastewater is reasonably treated and the recycling conditions are improved and then is recycled, the resource utilization rate can be effectively improved, and the effects of reducing toxicity and harm can be achieved.

The paenibacillus polymyxa exopolysaccharide is a fermentation product with high application value and can be used as a stabilizer for food and dye; plasma substitute for medical use or stable emulsion; the method is used for biological ore dressing to improve the hydrophilicity of the ore surface; ingredients for cosmetics; and an adsorbent, an immobilized enzyme, or a carrier for immobilized cells, which are used in biochemical, pharmaceutical industry, and laboratory (Liu Zheng, well Long, Zhou Chen Yan., research progress of polysaccharide of biocontrol bacterium Paenibacillus polymyxa [ J ]. Shanghai agricultural science 2015,31(4): 146-. Exopolysaccharide produced by Paenibacillus polymyxa has strong in vitro and in vivo antioxidant activity, and can be used as a natural antioxidant in food and pharmaceutical industries and the like. Therefore, the paenibacillus polymyxa is used for treating the starch wastewater, not only can extracellular polysaccharide products be obtained, but also the content of main organic pollutants in the starch wastewater can be reduced, and the method has good prospects in production of microbial polysaccharides, reduction of industrial pollution and environmental protection.

Disclosure of Invention

The invention aims to provide a method for producing polysaccharide by fermenting starch wastewater and simultaneously provide a method for verifying the applicability of the starch wastewater, so that extracellular polysaccharide with various application fields can be produced and obtained at high efficiency, the pollution of the starch wastewater to the environment can be reduced, and the method has great development potential.

The invention is realized by the following technical scheme.

The Paenibacillus polymyxa strain is preserved in China general microbiological culture collection center with the preservation number of CGMCC No.16444 and the preservation date of 2018, 9 months and 10 days, and has the preservation name of Paenibacillus polymyxa PYQ 1. Said paenibacillus polymyxa strain PYQ1 has been published in the invention with application number CN 201811216217.0.

A method for producing polysaccharide by fermenting starch wastewater comprises the following steps:

(1) activating a paenibacillus polymyxa strain on a plate culture medium, inoculating the activated strain into a seed culture medium, and performing shake-flask culture at the temperature of 30-37 ℃ for 8-12h to obtain a paenibacillus polymyxa seed solution;

(2) adjusting the pH value of the starch wastewater to be neutral; inoculating the seed solution obtained in the step (1) to the starch wastewater by an inoculation amount of 1.5-2.5% in volume percentage, and carrying out shake culture in a shaking table at the temperature of 25-35 ℃ and the rotation speed of 150-220rpm for 48-72 h.

Preferably, the mass volume fractions of the components of the plate culture medium are respectively as follows: 1-2% of sucrose, 0.5-1% of peptone, 0.5% of yeast extract powder, 0.3% of disodium hydrogen phosphate and 2% of agar strips, sterilizing by high-pressure steam at 115 ℃, and pouring into a flat plate.

Preferably, the mass volume fractions of the components of the seed culture medium are respectively as follows: 5% of sucrose, 0.5% of tryptone, 0.1% of yeast extract powder and 0.3% of disodium hydrogen phosphate.

Preferably, before the paenibacillus polymyxa strain is inoculated into the starch wastewater, the starch wastewater is firstly verified to be subjected to microbial growth and polysaccharide fermentation applicability in the wastewater, and the verification method comprises the following steps:

a) verifying the growth applicability of microorganisms in wastewater: adjusting the pH value of the starch wastewater to be neutral, inoculating a paenibacillus polymyxa colony, performing shake culture at the temperature of 30-37 ℃ for 24-48h, coating and counting, and if the maximum number reaches 10⁸CFU/mL satisfies the applicability of microorganism growth;

b) polysaccharide fermentation applicability verification: adjusting the pH value of the starch wastewater to be neutral, adding agar strips to prepare a starch wastewater flat plate, taking a paenibacillus polymyxa bacterial liquid for streaking, carrying out shake culture at the temperature of 30-37 ℃ for 24-48h, observing the periphery of a bacterial colony on the wastewater flat plate, and if continuous pasty substances are generated on the periphery of the bacterial colony and filamentous adhesion is picked up, the applicability of polysaccharide fermentation is met, otherwise, the applicability is not met.

The invention also provides application of the polysaccharide as a post-basking injury repairing agent. The polysaccharide is used as an additive of the skin care product for repairing the damage after the sun is damaged, and the skin care product containing the polysaccharide is smeared on the damaged skin within 0-4h after the skin is damaged by UV so as to play a role in repairing the damaged skin after the sun is damaged. The addition amount of polysaccharide in the skin care product is 100 μ g/mL-1000 μ g/mL.

The invention has the beneficial effects that:

according to the microbial growth and polysaccharide fermentation applicability verification experiment provided by the invention, the starch wastewater which can be used for producing sugar through the normal growth and fermentation of paenibacillus polymyxa can be preliminarily screened out, and the sugar production applicability and sugar production capacity conclusion of the starch wastewater can be obtained through drawing a fermentation curve and a growth curve within 60h, so that the system evaluation on the fermentation feasibility of the starch wastewater is achieved, and the possibility of fermentation failure is greatly avoided.

The polysaccharide production mode provided by the invention can efficiently produce sugar in a short time, and the starch wastewater is used as a raw material, and other nutrient substances are not required to be added, so that the production cost can be greatly reduced, organic pollutants such as starch, protein and the like in the starch wastewater can be effectively reduced, the effect of reducing environmental pollution is achieved, and the polysaccharide production mode has the advantages of cost saving, simplicity, high efficiency, pollution reduction and the like.

Drawings

FIG. 1 shows the results of the determination of polysaccharide fermentation conditions in different starch waste waters according to the method of the present invention; wherein A is wheat starch extraction waste liquid, B is potato starch extraction waste liquid, and C is corn starch extraction waste liquid;

FIG. 2 shows the growth curves of Paenibacillus polymyxa in three starch waste waters according to the method of the present invention;

FIG. 3 is a graph showing the fermentation and sugar production curves of Paenibacillus polymyxa in three types of starch wastewater according to the method of the present invention;

FIG. 4 DEAE elution curve of wheat starch wastewater fermentation polysaccharide.

Detailed Description

The following examples of the present invention are described in detail, but the following examples are not intended to limit the scope of the present invention.

Before the preparation of polysaccharide, a culture medium is prepared, and a seed solution is prepared. The culture medium used includes a plate culture medium, a seed culture medium, a wastewater fermentation medium and a sugar production medium, wherein the sugar production medium serves as a positive control group.

Plate culture medium: 2% of sucrose, 1% of peptone, 0.5% of yeast extract powder, 0.3% of disodium hydrogen phosphate and 2% of agar strips, sterilizing by high-pressure steam at 115 ℃ and pouring the mixture into a flat plate.

Seed culture medium: 5 percent of sucrose, 0.5 percent of tryptone, 0.1 percent of yeast extract powder and 0.3 percent of disodium hydrogen phosphate, and the shake flask culture is carried out under the condition of 30 ℃.

Wastewater fermentation medium: starch wastewater.

A sugar production culture medium: 5% of cane sugar, 0.5% of tryptone, 0.1% of yeast powder and Na₂HPO₄·12H₂0.3 percent of O and the balance of distilled water. The addition amount of all the components is equivalent to g/100ml in unit%.

Activating the strain of Paenibacillus polymyxa on a plate culture medium, inoculating the activated strain in a seed culture medium, and performing shake-flask culture at 30 ℃ for 12h to obtain a Paenibacillus polymyxa seed solution.