CN111019840A - Method for producing cellulase, pectinase and xylanase by fermenting herbal tea residues by combining aspergillus niger and saccharomycetes - Google Patents

Method for producing cellulase, pectinase and xylanase by fermenting herbal tea residues by combining aspergillus niger and saccharomycetes Download PDF

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CN111019840A
CN111019840A CN201911389477.2A CN201911389477A CN111019840A CN 111019840 A CN111019840 A CN 111019840A CN 201911389477 A CN201911389477 A CN 201911389477A CN 111019840 A CN111019840 A CN 111019840A
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aspergillus niger
fermentation
xylanase
pectinase
herbal tea
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袁明贵
向蓉
彭新宇
徐志宏
余丹妮
田雅
周廷斤
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Guangdong Qianyan Animal Health Care Co ltd
Institute of Animal Health of Guangdong Academy of Agricultural Sciences
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Institute of Animal Health of Guangdong Academy of Agricultural Sciences
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Abstract

The invention discloses a process for producing cellulase, pectinase and xylanase by fermenting cold tea residues by combining aspergillus niger and saccharomycetes. The invention relates to a process for producing cellulase, xylanase and pectinase by solid-state fermentation of cold tea leaves by using aspergillus niger and saccharomycetes, and discloses a high-efficiency and low-cost method for producing the cellulase, xylanase and pectinase by solid-state fermentation of the cold tea leaves by using the aspergillus niger and the saccharomycetes for the first time.

Description

Method for producing cellulase, pectinase and xylanase by fermenting herbal tea residues by combining aspergillus niger and saccharomycetes
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a process for producing cellulase, pectinase and xylanase by fermenting herbal tea residues jointly with aspergillus niger and saccharomycetes.
Background
In order to retain the flavor components in the herbal tea as much as possible, the herbal tea is extracted at a lower temperature mostly, and the extraction times are few, so that the herbal tea residues and the original medicinal materials have similar nutrients and active components. Therefore, the herb tea residue has better nutritional and medicinal values and is a biological resource which is not fully utilized. In recent years, with the rapid development of the herbal tea industry, the discharge of the herbal tea residues is increased year by year, and the daily yield of the herbal tea residues reaches 680 tons. The traditional herb tea dregs are mostly treated by direct stacking, landfill and other modes, which causes serious resource waste and environmental pollution. Due to the limitation of the field, the herb tea residue cannot be processed in time, and even the herb tea residue has great influence on the production of enterprises. The recycling and high-value utilization of herb tea dregs become important problems which must be faced by enterprises and society.
Aspergillus niger (Aspergillus niger) belongs to the family of Moniliaceae, the subdivision Ascomycetes, Aphyllophorales, a common, safe and non-toxic fungus of the genus Aspergillus, and is widely distributed in grains, vegetable products and soil all over the world. Aspergillus niger is a main industrial strain for making sauce, wine, vinegar and saccharified feed, is a main industrial strain for producing enzyme preparation and single-cell protein, and is also an important feed additive. The yeast is widely applied to the food industry and can be used as an additive for meat, jam, soup, cheese, bread food, vegetables and seasonings.
Cellulase, xylanase and pectinase are main enzymes for degrading plant cell walls and are important enzymes for improving the utilization rate of feed and food, but the high price of enzyme preparations limits the wide application of the enzyme preparations in industry and agriculture. According to the existing problems, the invention aims to develop a process for producing cellulase, pectinase and xylanase by fermenting herb tea residues by combining aspergillus niger and saccharomycetes.
Disclosure of Invention
The invention aims to provide a process for producing cellulase, pectinase and xylanase by fermenting herbal tea residues jointly by aspergillus niger and saccharomycetes.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a method for producing cellulase, pectinase and xylanase, comprising the steps of: mixing Aspergillus niger and yeast to prepare a suspension, and inoculating the suspension on a herbal tea residue culture medium; the herb tea residue comprises flos Plumeriae Acutifoliae, flos Lonicerae, flos Chrysanthemi, herba mesonae chinensis, Prunellae Spica, Glycyrrhrizae radix and folium Microcorii Paniculati.
According to the embodiment of the invention, the method also comprises the steps of pre-activating and subculturing the Aspergillus niger and the yeast, wherein the Aspergillus niger and the yeast are respectively inoculated into the PDA solid culture medium for culture.
According to the embodiment of the invention, the Aspergillus niger activation condition is 50-90% of humidity and is culture for 120-160 h at 28-37 ℃.
According to an embodiment of the invention, the yeast is selected from candida utilis, saccharomyces cerevisiae, torulopsis delbrueckii, pichia pastoris, saccharomyces boulardii.
According to the embodiment of the invention, the yeast activation condition is that the yeast is cultured for 24-30 h at 28-37 ℃ and with the humidity of 50-90%.
According to the embodiment of the invention, the bacterial concentration of the bacterial suspension is 105～109cfu/mL, preferably at a bacterial suspension concentration of 2X 107cfu/mL。
According to the embodiment of the invention, the inoculation amount is 5-25%, the fermentation temperature is 28-40 ℃, and the fermentation time is 2-10 days.
According to the embodiment of the invention, the passage is 1-3 times.
According to the embodiment of the invention, the water content during fermentation is 50-90%, and the pH of the soak solution is 7-10.
According to an embodiment of the present invention, the herbal tea grounds medium comprises: 1-5 parts of cold tea residue powder, 0.04-0.20 part of ammonium sulfate, 0-0.1 part of glucose, 0.005-0.02 part of monopotassium phosphate, 0.002-0.020 part of dipotassium hydrogen phosphate and 5.0-9.0 parts of water.
According to the embodiment of the invention, 2 parts of cold tea leaves powder are added with 0.08 part of ammonium sulfate, 0.04 part of glucose, 0.01 part of monopotassium phosphate, 0.008 part of dipotassium phosphate and 8 parts of water.
According to the embodiment of the invention, the preparation method of the cool tea residue powder comprises the following steps: mixing flos Plumeriae Acutifoliae, flos Lonicerae, flos Chrysanthemi, herba mesonae chinensis, Prunellae Spica, Glycyrrhrizae radix and folium Microcorii Paniculati, decocting to obtain herbal tea residue, drying, and pulverizing.
The invention has the beneficial effects that:
the invention discloses a process for producing cellulase, pectinase and xylanase by fermenting cold tea residues by combining aspergillus niger and saccharomycetes. The invention relates to a process for producing cellulase, xylanase and pectinase by solid-state fermentation of cold tea leaves by using aspergillus niger and saccharomycetes. Meanwhile, the method is also beneficial to the degradation of the cool tea dregs and has important significance in the process of solving the problem of pollution of the cool tea dregs.
Detailed Description
The following embodiments are further illustrated by reference to the following specific examples:
preliminary preparation
1. Activating strains:
(1) activating Aspergillus niger on PDA solid culture medium, culturing at 30 deg.C with 80% humidity in electrothermal constant-temperature constant-humidity incubator for 120 hr, and continuously passaging for 2 times;
(2) activating yeast (Candida utilis) on PDA solid culture medium, culturing at 30 deg.C with 80% humidity in electrothermal constant temperature and humidity incubator for 25 hr, and continuously passaging for 2 times;
PDA solid medium: peeling 200g of potatoes, cutting into blocks, boiling for 30min, filtering by using gauze, adding 20g of cane sugar, and supplementing water to 1000mL after dissolving. Adding agar 20g, heating to dissolve, and wet-heat sterilizing at 121 deg.C for 20 min.
2. Preparing a herbal tea residue culture medium:
taking 6-8 parts of water, adding 0.08 part of ammonium sulfate, 0.04 part of glucose, 0.01 part of potassium dihydrogen phosphate and 0.008 part of dipotassium hydrogen phosphate, adjusting the pH to 5.0-9.0 by using potassium hydroxide, adding 2 parts of cold tea leaves, and carrying out damp-heat sterilization at 121 ℃ for 20 minutes;
cooling tea leaves: mixing 7 kinds of plant medicines including flos Plumeriae Acutifoliae, flos Lonicerae, flos Chrysanthemi, herba mesonae chinensis, Prunellae Spica, Glycyrrhrizae radix and folium Microcoris Paniculatae, and decocting to obtain herb tea residue.
The specific operations of examples 1 to 8 were as follows:
mixing Aspergillus niger and yeast (Candida utilis) to prepare mixed suspension with concentration of 2 × 107cfu/mL, inoculating the mixed suspension on a herb tea residue culture medium, wherein the specific fermentation conditions are shown in Table 1.
Calculating the activity of cellulase, xylanase and pectinase after fermentation:
1. after fermentation, adding 15mL of physiological saline into the fermentation product;
2. centrifuging the physiological saline suspension for 10 minutes at 15000g, taking the precipitate, drying and weighing;
the enzyme-labeling instrument is utilized, and a 3, 5-dinitrosalicylic acid color development method is adopted to rapidly test the activity of the cellulase, the xylanase and the pectinase.
And (3) determining the content of reducing sugar: taking 0.1mL of fermentation supernatant, adding 0.4mL of distilled water, then adding 0.5mL of DNS, mixing uniformly, carrying out boiling water bath for 10min, and measuring the absorbance at 530 nm. Standard curves were prepared with glucose at final concentrations of 0, 20, 40, 60, 80, 100. mu.g/mL to calculate the reducing sugar content.
And (3) cellulase activity determination: 0.3mL of a sodium carboxymethylcellulose solution (6.25mg/mL, pH 5.0) and 0.1mL of a 0.1mol/L disodium hydrogenphosphate-citric acid buffer solution (pH 5.0) were taken, and 0.1mL of the fermentation supernatant was mixed and then applied in a water bath at 40 ℃ for 10 min. Adding DNS 0.5mL, mixing, boiling water bath for 10min, and measuring absorbance at 530 nm. And (3) preparing a standard curve by using glucose with final concentrations of 0, 20, 40, 60, 80 and 100 mu g/mL, and calculating the enzyme activity by using a sample supernatant which is not fermented by adding the strain as a reference.
And (3) xylanase activity determination: taking 0.2mL of 1% xylan solution, 0.2mL of 0.1mol/L disodium hydrogen phosphate-citric acid buffer solution (pH 5.0), and 0.1mL of fermentation supernatant, mixing uniformly, and carrying out water bath at 40 ℃ for 10 min. Adding DNS 0.5mL, mixing, boiling water bath for 10min, and measuring absorbance at 550 nm. And (3) establishing a standard curve by using xylose with final concentrations of 0, 100, 200, 300, 400, 500 and 600 mu g/mL, and calculating the enzyme activity by using a sample supernatant which is not fermented by adding the strain as a reference.
And (3) measuring the activity of the pectinase: taking 0.2mL of 1% pectin solution, 0.2mL of 0.1mol/L disodium hydrogen phosphate-citric acid buffer solution (pH 5.0), and 0.1mL of fermentation supernatant, mixing uniformly, and carrying out water bath at 40 ℃ for 10 min. Adding DNS 0.5mL, mixing, boiling water bath for 10min, and measuring absorbance at 540 nm. And (3) establishing a standard curve by using galacturonic acid with final concentrations of 0, 20, 40, 60, 80, 100, 120, 140 and 160 mu g/mL, and calculating the enzyme activity by using a sample supernatant obtained by fermenting without adding the strain as a reference.
The enzyme activity calculation method comprises the following steps: the amount of enzyme that produced 1. mu.g of product per minute at 40 ℃ was 1 enzyme activity unit (U).
The formula is as follows: the enzyme kinetics is (a1-a0) × V2/(t × m × V1).
In the formula: a1 represents the quality of the reducing sugar detected (μ g) from the standard curve, A0 represents the quality of the reducing sugar already present in the fermentation supernatant; v1 represents the volume of the aspirated enzyme solution (mL); v2 represents the total volume of enzyme solution (mL); t represents enzymatic reaction time (min); m represents the mass (g) of the fermentation sample after drying.
The settings and enzyme activities of examples 1-8 are shown in Table 1:
TABLE 1 fermentation factor settings and three enzyme activities of examples 1-8
Figure BDA0002344514330000041
Note: the factor A is the water content which is the percentage content of water when the system only contains the cold tea leaves and the water;
the factor B is the pH value of the soaking solution, and the soaking solution is a solution which is prepared by adding potassium dihydrogen phosphate and dipotassium hydrogen phosphate into a certain volume of water according to the proportion requirement and then adjusting the solution to a fixed pH value by using 3 mol/L of potassium hydroxide;
factor C is fermentation temperature;
factor D is fermentation time in days.
Example 9
Mixing Aspergillus niger and yeast (Candida utilis) to prepare mixed suspension with concentration of 2 × 107cfu/mL, inoculating the mixed suspension on a herbal tea residue culture medium (the inoculation amount is 10 percent), controlling the water content in a fermentation system to be 60 percent, controlling the pH value of an immersion liquid to be 7, controlling the fermentation temperature to be 37 ℃ and controlling the fermentation time to be 5 days.
As a result: the analytical processing test results showed that the cellulase activity per unit mass of the fermentation product was (0.89. + -. 0.15). times.10 after the method of example 9 was used3U, xylanase activity is (5.32 +/-0.59) multiplied by 103U, the pectase activity is (5.74 +/-0.64) multiplied by 103U, the average of the three is (3.98 + -0.46) × 103U。
Example 10
Mixing Aspergillus niger and yeast (Candida utilis) to prepare mixed suspension with concentration of 2 × 107cfu/mL, inoculating the mixed suspension on a herbal tea residue culture medium (the inoculation amount is 10 percent), wherein the water content in a fermentation system is 60 percent, the pH value of an immersion liquid is 8, the fermentation temperature is 37 ℃, and the fermentation time is 5 days.
As a result: the analytical processing test results showed that the cellulase activity per unit mass of the fermentation product was (0.84. + -. 0.0.09). times.10 after the method of example 10 was used3U, xylanase activity is (4.82 +/-1.38) multiplied by 103U, the pectase activity is (2.81 +/-0.30) multiplied by 103U, the average of the three is (2.85 + -0.36) × 103U。
According to the above examples, it was found that the effects of examples 9 and 10 were the best, and the cellulose films, xylan and pectinase produced were the strongest in enzyme activity.

Claims (10)

1. A method for producing cellulase, pectinase and xylanase, comprising the steps of: mixing Aspergillus niger and yeast to prepare suspension, and inoculating the suspension on a herbal tea residue culture medium for fermentation; the herb tea residue comprises flos Plumeriae Acutifoliae, flos Lonicerae, flos Chrysanthemi, herba mesonae chinensis, Prunellae Spica, Glycyrrhrizae radix and folium Microcorii Paniculati.
2. The method according to claim 1, further comprising activating and passaging Aspergillus niger and yeast in advance, wherein the activating comprises inoculating Aspergillus niger and yeast into PDA solid culture medium respectively.
3. The method according to claim 2, wherein the Aspergillus niger activation condition is 50-90% humidity and culturing at 28-37 ℃ for 120-160 h.
4. The method according to claim 2, wherein the yeast activation condition is 50-90% humidity, and culturing is carried out at 28-37 ℃ for 24-30 h.
5. The method of claim 1, wherein said bacterial suspension is at a concentration of 105～109cfu/mL; preferably, the concentration of the bacterial suspension is 2X 107cfu/mL。
6. The method of claim 1, wherein the amount of inoculation is between 5% and 25%; the water content during fermentation is 50-90%, the pH value of the soak solution is 5-10, the fermentation temperature is 28-40 ℃, and the fermentation time is 2-10 days.
7. The method of claim 1, wherein the amount of inoculum is 10%; the water content during fermentation is 60%, the pH of the soaking solution is 7, the fermentation temperature is 37 ℃, and the fermentation time is 5 days.
8. The method of claim 1, wherein the herbal tea pomace medium comprises: 1-5 parts of cold tea residue powder, 0.04-0.20 part of ammonium sulfate, 0-0.1 part of glucose, 0.005-0.02 part of monopotassium phosphate, 0.002-0.020 part of dipotassium hydrogen phosphate and 5.5-8.5 parts of water.
9. The method of claim 1, wherein the herbal tea pomace medium comprises: 2 parts of cold tea residue powder, 0.08 part of ammonium sulfate, 0.04 part of glucose, 0.01 part of monopotassium phosphate, 0.008 part of dipotassium hydrogen phosphate and 8 parts of water.
10. The method according to claim 8 or 9, wherein the cold tea leaf powder is: mixing flos Plumeriae Acutifoliae, flos Lonicerae, flos Chrysanthemi, herba mesonae chinensis, Prunellae Spica, Glycyrrhrizae radix and folium Microcorii Paniculati, and decocting to obtain herbal tea residue.
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CN102187939A (en) * 2011-05-10 2011-09-21 成都信息工程学院 Method for producing health feed by using notoginseng residues fermenting
CN106085881A (en) * 2016-07-12 2016-11-09 西北农林科技大学 A kind of Aspergillus niger strain and application thereof
CN109825487A (en) * 2017-11-23 2019-05-31 朝阳华星生物工程有限公司 A kind of industrial fermentation process of co-producing xylanase and other coenzyme
CN110338289A (en) * 2019-07-30 2019-10-18 华南农业大学 It is a kind of to improve the herbal tea dreg fodder for fattening meat quality and its application

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Publication number Priority date Publication date Assignee Title
CN101628828A (en) * 2009-08-18 2010-01-20 广东加多宝饮料食品有限公司 Organic fertilizer prepared by using dregs of WangLoKAT herb residue and preparation method thereof
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