CN110885806A - Method for producing pectinase by using pickling brine and troxerutin brine - Google Patents
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Abstract
The invention discloses a method for producing pectinase by using pickling brine and kojic brine, belonging to the field of microorganisms. The method comprises the following steps: step 1: inoculating the bacillus subtilis into a solid activation culture medium, and performing activation culture at 37 ℃ for 48 hours to obtain activated bacillus subtilis; step 2: inoculating the activated bacillus subtilis obtained in the step 1 into a liquid activation culture medium, and performing shake cultivation for 12 hours at 35 ℃ to obtain a seed solution; and step 3: and (3) inoculating the seed liquid obtained in the step (2) into a fermentation culture medium of pickling saline water and kojic saline water for fermentation culture to obtain a crude pectinase liquid. The method for producing the pectinase by using the pickling brine and the qu-sulfonated brine can fully utilize the waste brine in the food industry, save the cost and provide a new method for the industrial fermentation production process of the pectinase.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a method for producing pectinase by using pickling brine and kombusu brine.
Background
Pectinase is a collective term for various enzymes that break down pectin, and galacturonic acid is the final product of its action. In nature, there are three main classes of pectin degrading enzymes: pectinesterase, polygalacturonase and pectin lyase. In nature, there are three main classes of pectin degrading enzymes: pectinesterase, polygalacturonase and pectin lyase. Pectinases have been widely used in various biological processes, such as food, feed, pectin waste water treatment, paper and textile industries. In industry, acid pectinases are mainly polygalacturonases from fungi used for the extraction and clarification of fruit juices and wines in the beverage industry. The alkalophilic pectinase is greatly utilized in biotechnology-based industries such as fiber degumming, paper industry, oil extraction and waste water treatment from fruit processing plants. In view of the wide application range of pectinase, optimizing pectinase production is the basis for reducing cost and improving production efficiency. In the Chinese patent CN107904195A, the pectinase is produced by using marine bacteria (Maribacterpp.) T28, the enzyme activity of the pectinase in the fermentation broth can reach 120-160U/mL, but most raw materials in the formula of the culture medium are industrial preparations, and the production cost is high. Therefore, a method for producing pectinase with high yield and low cost needs to be researched.
The fermented bean curd is also called fermented bean curd, is folk food which has been well-known in China, is deeply popular with common people in China and people in southeast Asia due to good taste and high nutrition, and is a durable delicious dish. In the food industry, the preparation process of the preserved beancurd comprises the following steps: firstly, putting the bean curd blocks in a food steamer flatly, controlling the temperature in the food steamer at 15-18 ℃, keeping the temperature at a certain humidity, and culturing for 3-5 days to ensure that hyphae grow vigorously until the bean curd grows mucor; and putting the bean curd blocks full of mucor in order by layers, adding salt layer by layer, pickling for about 8 days, and adding brine for storage. During the process of making preserved beancurd, a large amount of pickling brine is generated, and the pickling brine is usually directly discarded. However, the pickling saline water is rich in sufficient sodium chloride and contains a plurality of nutrient substances, and waste is caused by direct discarding, so that the method has environmental protection significance for finding available parts for the preserved bean curd pickling saline water. Similarly, in the process of preparing sauce foods such as soybean paste, the phenomenon that the kombucha brine is directly discarded also exists. Therefore, it is necessary to find a method for fully utilizing the pickling brine and the kojic brine to make the best use of the pickling brine and the kojic brine.
Disclosure of Invention
Aiming at the problem that the waste water of the pickling brine and the qu-sulfonated brine is unavailable in the prior art, the invention provides a method for producing pectinase by using the pickling brine and the qu-sulfonated brine.
The invention provides a method for producing pectinase by using pickling brine and kombusu brine, which comprises the following steps:
step 1: inoculating the bacillus subtilis into a solid activation culture medium, and performing activation culture at 37 ℃ for 48 hours to obtain activated bacillus subtilis;
wherein, the solid activation medium in the step 1 comprises: 2-5g/L beef extract, 5-10g/L peptone, 2-10g/L sodium chloride, 15g/L agar, pH7.0, and the balance of distilled water.
Step 2: inoculating the activated bacillus subtilis obtained in the step 1 into a liquid activation culture medium, and performing shake cultivation for 12 hours at 35 ℃ to obtain a seed solution;
wherein, the liquid activation culture medium in the step 2 comprises: 2-5g/L beef extract, 5-10g/L peptone, 2-10g/L sodium chloride, pH7.0, and the balance of distilled water.
And step 3: inoculating the seed liquid obtained in the step 2 into a fermentation culture medium of pickling saline water and kojic saline water for fermentation culture to obtain a crude pectinase liquid;
wherein the fermentation culture medium of the pickling brine and the kojic brine in the step 3 comprises: glucose, pectin, peptone, urea, pickling saline, kojic saline, potassium dihydrogen phosphate, ferric sulfate, mycotoxin and distilled water;
preferably, the fermentation medium comprises: 10-16g/L of glucose, 5-10g/L of pectin, 10-20g/L of peptone, 10-20g/L of urea, 0.5-1.5% (v/v) of preserved beancurd saline, 12.5-37.5% (v/v) of kombu saline, 2-8g/L of monopotassium phosphate, 2-8g/L of ferric sulfate, 0.5-2g/L of mycotoxin and the balance of distilled water;
further preferably, the volume ratio of the pickled fermented bean curd saline to the kombucha saline in the fermentation culture medium is 1: 25;
most preferably, the fermentation medium comprises: 12g/L of glucose, 6g/L of pectin, 15g/L of peptone, 15g/L of urea, 1% (v/v) of preserved fermented bean curd saline, 25% (v/v) of kojie saline, 5g/L of monopotassium phosphate, 5g/L of ferric sulfate, 1.0g/L of mycotoxin and the balance of distilled water.
Wherein the temperature of the fermentation culture in the step 3 is 40 ℃, and the ventilation volume is 8.0L/min.
Wherein, the time of fermentation culture in the step 3 is 20-30 hours; preferably, the fermentation culture time is 24 hours.
The invention has the beneficial effects that:
(1) the present invention creatively uses the pickling brine and the qu-huang brine, and recycles the original food industry wastewater to make the waste water become the raw material for producing pectinase.
(2) According to the invention, on the basis of the traditional fermentation culture medium, the pickling brine and the kojic brine are reasonably proportioned, and the carbon source, the nitrogen source, the inorganic ions and the nutrient substances are fully utilized to promote the fermentation of the bacillus subtilis and produce the high-activity pectinase, so that the highest activity of the finally obtained pectinase can reach 138U/mL.
Detailed Description
The present invention will be further explained with reference to specific embodiments in order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easy to understand, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments are possible. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.
The experimental materials, reagents and methods used in examples 1-4 and comparative examples 1-5 described in this specification are as follows:
1. test materials and reagents
Materials: bacillus subtilis.
Reagent:
(1) 1mg/mL pectin substrate buffer: 0.1g of pectin powder is added into 100mL of disodium hydrogen phosphate-citric acid buffer solution, and the mixture is placed on a magnetic stirrer to be stirred until the pectin powder is completely dissolved, so that 1mg/mL of substrate buffer solution is obtained.
(2) 3, 5-dinitrosalicylic acid (DNS) reagent is prepared by weighing 6.3g of DNS with an electronic balance, adding 500mL of distilled water, stirring for dissolving, placing in a 45 ℃ water bath kettle, and sequentially adding 21.0g of NaOH, 182.0g of sodium potassium tartrate, 5.0g of phenol and anhydrous Na2SO35.0g, stirred until completely dissolved, and then the volume is up to 1000 mL. DNS reagent requires brown riceThe color bottles are stored and can be used after being placed for 7 days.
Experimental methods
2.1 Process for the production of pectinase using pickling brine and kombusu brine comprising the steps of:
step 1: inoculating the bacillus subtilis into a solid activation culture medium, and performing activation culture at 37 ℃ for 48 hours to obtain activated bacillus subtilis; wherein the solid activation medium comprises: 3.5g/L beef extract, 7.5g/L peptone, 6g/L sodium chloride, 15g/L agar, pH7.0, and the balance of distilled water.
Step 2: inoculating the activated bacillus subtilis obtained in the step 1 into a liquid activation culture medium, and performing shake cultivation for 12 hours at 35 ℃ to obtain a seed solution; wherein the liquid activation medium comprises: 3.5g/L beef extract, 7.5g/L peptone, 6g/L sodium chloride, pH7.0, and the balance of distilled water.
And step 3: inoculating the seed liquid obtained in the step 2 into a fermentation culture medium of pickling saline water and kojic saline water for fermentation culture to obtain a crude pectinase liquid; wherein, the fermentation culture medium of the pickling brine and the quhuang brine comprises: glucose, pectin, peptone, urea, pickling saline, kojic saline, potassium dihydrogen phosphate, ferric sulfate, mycotoxin and distilled water;
wherein the fermentation culture temperature is 40 deg.C, and the ventilation amount is 8.0L/min; the fermentation culture time is 20-30 hours.
The raw pectinase solution prepared by the above method, the concentrations (or volume ratios) of the components of the fermentation medium of the pickling brine and the qu-sulfonated brine and the fermentation time are shown in Table 1.
2.2 detection of the enzyme Activity of pectinase
2.2.1 drawing a standard curve
Taking a standard solution of lactobionic acid, gradient-loading in an EP tube by using 200 mu L, then, supplementing to 2mL, and mixing uniformly. mu.L of DNS reagent was added to 500. mu.L of the mixture, and the mixture was boiled to develop color. After dilution, 0.5mL of each was taken, and the absorbance at 540nm was measured to prepare a calibration curve.
2.2.1 measurement of the enzyme Activity of pectinase
And centrifuging the fermentation liquor to obtain supernatant, namely crude enzyme liquid, and measuring the enzyme production activity of the strain by using the crude enzyme liquid. Sucking 1.5mL of 1mg/mL pectin substrate buffer solution into a 2mL EP tube, adding 0.5mL crude enzyme solution, mixing uniformly, reacting in a 15 ℃ water bath for 30min, taking out 500 μ L reaction solution, adding 800 μ L of LDNS to terminate the reaction, placing in boiling water, boiling for 5min, and then rapidly cooling with flowing water. The inactivated crude enzyme solution was used as a control. Definition of pectinase activity: the ability of 1mL of enzyme solution to hydrolyze a pectin substrate per minute to 1. mu. mol galacturonic acid under reaction conditions of 15 ℃ and pH5.0 is defined as 1 enzyme activity unit (U).
Examples and comparative examples
Comparative example 1
Compared with example 1, only the pickled fermented bean curd brine and the kombucha brine are lacked in the fermentation medium.
Comparative example 2
In contrast to example 1, only the pickled fermented bean curd brine was absent from the fermentation medium.
Comparative example 3
In contrast to example 1, only the fermentation medium was lacking in the kosmoothie brine.
Comparative example 4
Pectinase is produced by using a method provided in the literature, research on conditions for producing alkaline pectinase by solid fermentation of bacillus subtilis (food industry, vol 35, 12, 2014).
Comparative example 5
Pectinase was produced by the method provided in "optimization of fermentation conditions for producing pectinase by Bacillus subtilis FM 208849" (proceedings of university of Dalian Industrial science, vol. 29, 2, 2010).
Results of the experiment
In summary, the enzyme activity of the crude pectinase solution produced by the method provided by the invention is up to 138U/mL at most, which indicates that the fermentation medium containing the pickling brine and the qu-sulfonated brine provided by the invention can realize the technical effect of producing high-activity pectinase.
The foregoing is a preferred embodiment of the present invention, and is not intended to limit the invention in any way, so that any simple modification and equivalent changes made to the above embodiment without departing from the technical spirit of the present invention should be considered as the protection scope of the present invention.
Claims (9)
1. A method for producing pectinase by using pickling brine and kombusu brine is characterized by comprising the following steps of:
step 1: inoculating the bacillus subtilis into a solid activation culture medium, and performing activation culture at 37 ℃ for 48 hours to obtain activated bacillus subtilis;
step 2: inoculating the activated bacillus subtilis obtained in the step 1 into a liquid activation culture medium, and performing shake cultivation for 12 hours at 35 ℃ to obtain a seed solution;
and step 3: and (3) inoculating the seed liquid obtained in the step (2) into a fermentation culture medium of pickling saline water and kojic saline water for fermentation culture to obtain a crude pectinase liquid.
2. The method of claim 1, wherein the solid activation medium of step 1 comprises: 2-5g/L beef extract, 5-10g/L peptone, 2-10g/L sodium chloride, 15g/L agar, pH7.0, and the balance of distilled water.
3. The method of claim 1, wherein the liquid activation medium of step 2 comprises: 2-5g/L beef extract, 5-10g/L peptone, 2-10g/L sodium chloride, pH7.0, and the balance of distilled water.
4. The method as claimed in claim 1, wherein the said salting and kombursenig saline fermentation media in step 3 comprises: glucose, pectin, peptone, urea, pickling saline, kojic saline, potassium dihydrogen phosphate, ferric sulfate, mycotoxin and distilled water.
5. The method of claim 4, wherein said salting and kombursenizing saltwater fermentation media comprises: 10-16g/L of glucose, 5-10g/L of pectin, 10-20g/L of peptone, 10-20g/L of urea, 0.5-1.5% (v/v) of preserved beancurd saline, 12.5-37.5% (v/v) of kombu saline, 2-8g/L of monopotassium phosphate, 2-8g/L of ferric sulfate, 0.5-2g/L of mycotoxin and the balance of distilled water.
6. The method as claimed in claim 5, wherein the volume ratio of the pickled fermented bean curd brine to the komburse brine in the fermentation medium is 1: 25.
7. The method of claim 6, wherein: the fermentation culture medium of the pickling saline and the kombucha saline comprises: 12g/L of glucose, 6g/L of pectin, 15g/L of peptone, 15g/L of urea, 1% (v/v) of preserved fermented bean curd saline, 25% (v/v) of kojie saline, 5g/L of monopotassium phosphate, 5g/L of ferric sulfate, 1.0g/L of mycotoxin and the balance of distilled water.
8. The method of claim 1, wherein: the temperature of the fermentation culture in the step 3 is 40 ℃, and the ventilation volume is 8.0L/min.
9. The method of claim 1, wherein: the time of fermentation culture in the step 3 is 20-30 hours.
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Inventor after: Ren Dahong Inventor after: Lin Lizhao Inventor after: Liu Yanfen Inventor before: Ren Dahong |