CN111778182B - Microbial compound microbial inoculum for ramie biological degumming pretreatment and preparation method thereof - Google Patents
Microbial compound microbial inoculum for ramie biological degumming pretreatment and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological degumming, in particular to a microbial composite microbial inoculum for ramie biological degumming pretreatment and a preparation method thereof. The preparation method comprises the following steps: respectively inoculating the Zygomyces bifidus, Rhizopus pachyticus, Erwinia and Pantoea dispersa into a culture medium for culturing to obtain Zygomyces bifidus bacterial liquid, Rhizopus pachytropsis bacterial liquid, Erwinia bacterial liquid and Pantoea dispersa bacterial liquid, and mixing the four bacterial liquids to obtain a composite bacterial agent seed liquid; and inoculating the composite microbial inoculum seed solution into a composite microbial inoculum culture medium for mixed fermentation to obtain the microbial composite microbial inoculum. The preparation method provided by the invention is simple, has low cost, and can effectively realize biological degumming of ramie.
Description
Technical Field
The invention relates to the technical field of biological degumming, in particular to a microbial composite microbial inoculum for ramie biological degumming pretreatment and a preparation method thereof.
Background
Ramie (with the scientific name of Boehmeria nivea (L.) Gaudich.) is a subfbushy or shrub plant of the genus Ramie of the family Urticaceae), whose fibers are characterized by high strength and high modulus, is widely used in industrial composites. China is the country with the largest ramie yield, and accounts for about 90% of the total yield all over the world. The ramie contains 20-30% of colloid in raw ramie, the colloid broadly refers to non-cellulose components in the raw ramie, and comprises hemicellulose, pectin, lignin, water-soluble substances, lipid wax, ash and the like, and the degumming process in the actual production mainly comprises the steps of destroying the hemicellulose and pectin components to separate the fibers into single fibers so as to be suitable for the spinning process. In the traditional textile industry, a chemical method is mainly adopted, high-temperature boiling-off of acid, alkali and oxides is carried out, and physical and mechanical means such as water washing, fiber beating and the like are used for realizing separation of colloid and fiber. The development of modern textile industry is severely restricted by the problem that the whole process needs repeated water washing steps and a large amount of acid, alkali and oxidant are used, so that the serious waste and pollution to water resources are caused.
The biological degumming realizes the decomposition of colloid components under mild conditions through the action of enzyme or microorganism, and has the advantages of low water consumption, light pollution and no damage to fiber, so the development and industrial application of the biological degumming technology become the inevitable trend of the development of the textile industry.
The existing biological degumming pretreatment process usually uses a single microorganism, the decomposition speed of the single microorganism is slow due to the complex colloid components, and meanwhile, the degumming effect is seriously influenced by the pollution of mixed bacteria in the environment due to higher requirement on the sterility degree. In the process of natural retting, the degumming effect is good under the action of natural compound flora, but the proliferation speed of the natural flora is slow, so that the traditional retting is not suitable for large-scale industrial production.
Disclosure of Invention
In view of the above, the invention provides a microbial composite inoculant for ramie biological degumming pretreatment, which can be industrially produced in a large scale and has a good decomposition effect on colloid components, and solves the problems of water resource waste and environmental pollution in the traditional chemical degumming process in the textile industry; the invention also provides a preparation method of the microbial composite inoculant, which is simple in preparation method, low in cost and capable of effectively realizing biological degumming of ramie.
The invention provides a preparation method of a microbial compound microbial inoculum for ramie biological degumming pretreatment, which comprises the following steps: respectively inoculating podophyllum japonicum (Dipodascus klebahnii), Rhizopus pachytrichoides (Rhizopus chlamydosporus), Erwinia amylovora (Erwinia amylovora) and Pantoea dispersa (Pantoea dispersa) into respective specific culture media for culture to obtain podophyllum japonicum liquid, Rhizopus pachytrichoides liquid, Erwinia amylovora liquid and Pantoea dispersa liquid, and mixing the four types of liquid to obtain a composite microbial inoculum seed liquid; and inoculating the composite microbial inoculum seed solution into a composite microbial inoculum culture medium for mixed fermentation to obtain the microbial composite microbial inoculum.
Furthermore, the cell numbers of the Zygomyces bifidus, Rhizopus pachyticus, Erwinia and Pantoea dispersa in the composite microbial inoculum seed liquid are respectively 30-40%, 10-15% and 10-20% of the total cell number in the composite microbial inoculum seed liquid in sequence.
Further, the preparation process of the complex microbial inoculum culture medium comprises the following steps: weighing 5-10 parts of peptone, 3-5 parts of yeast powder, 2-7 parts of sodium chloride and (NH)4)2SO40.5-2 parts of dipotassium hydrogen phosphate, 0.5-1.5 parts of rice flour, 5-10 parts of ramie raw ramie and 950-1050 parts of distilled water, and sterilizing at 121 ℃ for 20min to obtain the composite microbial inoculum culture medium.
Further, the pH value of the complex microbial inoculum culture medium is 7.0-7.2.
Further, the temperature of mixed fermentation of the composite microbial inoculum seed liquid is 28-32 ℃, and the time is 72-128 hours.
Further, the Zygomycetes bifidus is inoculated into a wort culture medium and cultured for 48-72 hours under the temperature condition of 22-30 ℃ and the aerobic condition to obtain Zygomyces bifidus bacterial liquid.
Further, the rhizopus pachytrichoides is inoculated into a wort culture medium and cultured for 48-72 hours under the temperature condition of 22-30 ℃ and the aerobic condition to obtain rhizopus pachyticus liquid.
Further, the preparation process of the wort culture medium is as follows: weighing 2-4 parts of yeast powder, 2-4 parts of malt extract, 3-6 parts of peptone, 7-15 parts of glucose and 950-1050 parts of distilled water, mixing, and sterilizing at 121 ℃ for 20min to obtain the malt extract culture medium.
Further, the pH value of the wort medium is 7.0-7.2.
Further, the Erwinia is inoculated into an LB culture medium and cultured for 24-48 hours under the temperature condition of 30-38 ℃ and the aerobic condition to obtain an Erwinia bacterial liquid.
Further, the pantoea dispersa is inoculated into an LB culture medium and cultured for 24-48 hours under the temperature condition of 30-38 ℃ and the aerobic condition to obtain the pantoea dispersa bacterial liquid.
Further, the preparation process of the LB culture medium comprises the following steps: weighing 5-15 parts of peptone, 3-10 parts of yeast extract, 5-10 parts of sodium chloride and 950-1050 parts of distilled water, mixing, and sterilizing at 121 ℃ for 20min to obtain the LB culture medium.
Further, the pH value of the LB culture medium is 7.0-7.2.
The invention also provides a microbial composite microbial inoculum prepared by the preparation method and used for the biological degumming pretreatment of ramie.
According to the invention, the pediococcus bifidus, the rhizopus pachyticus, the erwinia bacterium and the pantoea dispersa are respectively cultured, so that the pediococcus bifidus, the rhizopus pachyticus, the erwinia bacterium and the pantoea dispersa are propagated in a large quantity, and after the pediococcus bifidus, the rhizopus pachyticus, the erwinia bacterium liquid and the pantoea dispersa bacterium liquid are obtained after the relatively high activity and growth state are achieved; and then, mixing the bacterial liquids according to a specific proportion, and further mixing and fermenting in a complex microbial inoculum culture medium to obtain a microbial complex microbial inoculum, wherein various bacteria in the microbial complex microbial inoculum have synergistic effects, so that the efficient decomposition of colloid components in the ramie can be realized.
The technical scheme provided by the invention has the beneficial effects that: when the microbial composite inoculant provided by the invention is used for ramie biological degumming pretreatment, the energy consumption and the water consumption are low, and the problems of water resource waste and environmental pollution in the traditional chemical degumming process of the textile industry are solved; the microbial composite inoculant provided by the invention has the beneficial effects that the effective decomposition of colloid components in ramie can be realized under the synergistic action of various microorganisms, the biological degumming speed is improved, the degumming effect is good, the fibrilia is not damaged, and the problem of low proliferation speed of natural floras is solved through the application of the inoculant.
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FIG. 1 is a schematic flow chart of a preparation method of a microbial composite inoculant for ramie biological degumming pretreatment.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the following examples and accompanying drawings.
In the following examples, Zygomyces bipinnata, Rhizopus pachyticus, Erwinia and Pantoea dispersa were purchased from Minzhou organisms.
Preferably, the product of Zygomyces bifidus is ATCC 42397, the product of Rhizopus pachyticus is ACCC30302, the product of Erwinia is ATCC 51855, and the product of Pantoea dispersa is BMZ 068228.
Example 1:
the embodiment 1 of the invention provides a preparation method of a microbial compound microbial inoculum for ramie biological degumming pretreatment, which comprises the following steps:
(1) preparing a wort culture medium with a pH value of 7.0: weighing 30g of yeast powder, 30g of malt extract, 45g of peptone, 115g of glucose and 10L of distilled water, mixing, and sterilizing at 121 ℃ for 20 min;
(2) preparing an LB culture medium with the pH value of 7.0: weighing 100g of peptone, 80g of yeast extract, 75g of sodium chloride and 10L of distilled water, mixing, and sterilizing at 121 ℃ for 20 min;
(3) preparing a complex microbial inoculum culture medium with the pH value of 7.0: 80g of peptone, 40g of yeast powder, 55g of sodium chloride and (NH) are weighed4)2SO4Mixing 15g, dipotassium hydrogen phosphate 10g, rice flour 70g, ramie raw hemp 80g and distilled water 10L, and sterilizing at 121 ℃ for 20 min;
(4) preparing a microbial compound microbial inoculum: respectively inoculating the podophyllum and the rhizopus pachyrhizus to a wort culture medium, and culturing for 60 hours at the temperature of 25 ℃ under aerobic conditions to obtain a podophyllum bacterial liquid and a rhizopus pachyrhizus bacterial liquid; respectively inoculating the Erwinia and Pantoea dispersa into an LB culture medium, and culturing for 36 hours at the temperature of 35 ℃ under aerobic conditions to obtain Erwinia liquid and Pantoea dispersa liquid; mixing Zygomyces bifidus bacterial liquid, Rhizopus pachyticus bacterial liquid, Erwinia bacterium liquid and Pantoea dispersa bacterial liquid to obtain a composite bacterial agent seed liquid; inoculating the seed solution of the compound microbial inoculum into a culture medium of the compound microbial inoculum, and culturing for 96 hours at the temperature of 30 ℃ under aerobic conditions to obtain the microbial compound microbial inoculum.
The cell numbers of the Zygomyces bifidus, the Rhizopus pachyticus, the Erwinia and the Pantoea dispersa in the composite microbial inoculum seed liquid are respectively 35%, 12% and 18% of the total cell number in the composite microbial inoculum seed liquid in sequence.
In example 1, one portion per 10g is used.
The procedure for the preparation of example 1 is shown in FIG. 1.
Example 1, a control experiment is adopted to analyze the microbial degumming process, specifically, an experimental group I and control groups I to six are set, the experimental group I is to add ramie into the prepared composite microbial inoculum culture medium, and inoculate the microbial composite microbial inoculum prepared in example 1; the first control group is prepared by adding ramie into the prepared composite microbial inoculum culture medium, and inoculating Zygomyces bifidus and Rhizopus pachyces; adding ramie into the prepared composite microbial inoculum culture medium, and inoculating Erwinia and Pantoea dispersa in a control group II; a third control group is to add ramie into the prepared composite microbial inoculum culture medium and inoculate Zygomyces bifidus, Erwinia and Pantoea dispersa; adding ramie into the prepared composite microbial inoculum culture medium, and inoculating rhizopus pachytrichoides, erwinia bacterium and Pantoea dispersa; a fifth control group is that ramie is added into the prepared composite microbial inoculum culture medium, and the Zygomyces bifidus, Rhizopus pachyticus and Erwinia are inoculated; and a sixth control group is to add ramie into the prepared composite microbial inoculum culture medium and inoculate Zygomyces bifidus, Rhizopus pachyces and Pantoea dispersa.
In the experiment I and the control groups I to VI, the ramie adding proportion is 1-5g of ramie per 100ml of culture medium, and the inoculation amount of the microbial inoculum is 1-5%.
Culturing the experimental group I and the control groups I to six for 72h under the temperature condition of 30 ℃ and the aerobic condition, taking out the experimental group I and the control groups I to six, drying the experimental group I and the control groups I to six at 80 ℃ to constant weight, and determining the ramie components (lipid wax content, water soluble substance content, pectin substance content, hemicellulose content and cellulose content), wherein the ramie component analysis method refers to the national standard GB 5889-1986 Ramie chemical component quantitative analysis method. The gum degradation rate (original gum residue rate-72 h sample gum residue rate)/original gum residue rate × 100, and the measurement results are shown in table 1.
Table 1: ramie component determination
Centrifuging the culture solution for 5min at 10000rpm for 72h, collecting supernatant, and respectively measuring the activity of the mannose enzyme, the activity of the xylanase enzyme (referring to the measurement of the activity of the xylanase of the GB/T23874-.
Table 2: results of enzyme Activity measurement
From the table 1 and the table 2, under the synergistic effect of various bacteria, the microbial compound bacteria of the experimental group I have the highest enzyme activities of pectinase, mannase and xylanase, have the best decomposition effect on pectin, hydrosoluble substances and hemicellulose in the ramie, have the highest colloid degradation rate of 62.52 percent and realize the effective degumming on the ramie; meanwhile, while the colloid component is removed, the cellulose component in the ramie is well kept, and the cellulose content is increased to 87.82 percent after degumming pretreatment.
Example 2:
the embodiment 2 of the invention provides a preparation method of a microbial compound microbial inoculum for ramie biological degumming pretreatment, which comprises the following steps:
(1) preparing a wort culture medium with a pH value of 7.2: weighing 25g of yeast powder, 25g of malt extract, 55g of peptone, 85g of glucose and 10L of distilled water, mixing, and sterilizing at 121 ℃ for 20 min;
(2) preparing an LB culture medium with a pH value of 7.2: weighing 120g of peptone, 60g of yeast extract, 65g of sodium chloride and 10L of distilled water, mixing, and sterilizing at 121 ℃ for 20 min;
(3) preparing a complex microbial inoculum culture medium with the pH value of 7.2: weighing 65g of peptone, 35g of yeast powder, 45g of sodium chloride and (NH)4)2SO4Mixing 18g, 8g of dipotassium phosphate, 60g of rice flour, 70g of ramie raw hemp and 10L of distilled water, and sterilizing at 121 ℃ for 20 min;
(4) preparing a microbial compound microbial inoculum: respectively inoculating the podophyllum and the rhizopus pachyrhizus into a wort culture medium, and culturing for 60 hours under the temperature condition of 28 ℃ and the aerobic condition to obtain a podophyllum liquid and a rhizopus pachyrhizus liquid; respectively inoculating the Erwinia and Pantoea dispersa into an LB culture medium, and culturing for 36 hours at the temperature of 37 ℃ under aerobic conditions to obtain Erwinia liquid and Pantoea dispersa liquid; mixing Zygomyces bifidus bacterial liquid, Rhizopus pachyticus bacterial liquid, Erwinia bacterium liquid and Pantoea dispersa bacterial liquid to obtain a composite bacterial agent seed liquid; inoculating the seed solution of the compound microbial inoculum into a culture medium of the compound microbial inoculum, and culturing for 84 hours at the temperature of 30 ℃ under aerobic conditions to obtain the microbial compound microbial inoculum.
The cell numbers of the Zygomyces bifidus, the Rhizopus pachyticus, the Erwinia and the Pantoea dispersa in the composite microbial inoculum seed liquid are respectively 32%, 38%, 14% and 16% of the total cell number in the composite microbial inoculum seed liquid in sequence.
In example 2, one portion per 10g is used.
Adding ramie into the prepared composite microbial inoculum culture medium (the adding proportion of the ramie is 1-5g per 100ml of the culture medium), inoculating the microbial composite microbial inoculum prepared in the example 2 according to the inoculation amount of 1-5%, culturing for 72 hours at the temperature of 30 ℃ and under the aerobic condition, taking out, drying at 80 ℃ to constant weight, and measuring the components (lipid wax content, water soluble substance content, pectin substance content, hemicellulose content and cellulose content) of the ramie, wherein the measurement results are shown in table 3.
Centrifuging the culture solution for 5min at 10000rpm for 72h, collecting supernatant, and respectively measuring the activity of the mannose enzyme, the activity of the xylanase enzyme (referring to the measurement of the activity of the xylanase of the GB/T23874-.
Table 3: test results of the microbial composite inoculum prepared in example 2
Example 3:
the embodiment 3 of the invention provides a preparation method of a microbial compound microbial inoculum for ramie biological degumming pretreatment, which comprises the following steps:
(1) preparing a wort culture medium with a pH value of 7.0: weighing 35g of yeast powder, 35g of malt extract, 55g of peptone, 75g of glucose and 10L of distilled water, mixing, and sterilizing at 121 ℃ for 20 min;
(2) preparing an LB culture medium with the pH value of 7.0: weighing 135g of peptone, 90g of yeast extract, 95g of sodium chloride and 10L of distilled water, mixing, and sterilizing at 121 ℃ for 20 min;
(3) preparing a complex microbial inoculum culture medium with the pH value of 7.0: weighing 95g of peptone, 45g of yeast powder, 65g of sodium chloride and (NH)4)2SO4Mixing 18g, dipotassium hydrogen phosphate 12g, rice flour 85g, ramie raw hemp 85g and distilled water 10L, and sterilizing at 121 ℃ for 20 min;
(4) preparing a microbial compound microbial inoculum: respectively inoculating the podophyllum and the rhizopus pachyrhizus to a wort culture medium, and culturing for 66 hours at the temperature of 25 ℃ under aerobic conditions to obtain a podophyllum bacterial liquid and a rhizopus pachyrhizus bacterial liquid; respectively inoculating the Erwinia and Pantoea dispersa into an LB culture medium, and culturing for 48 hours at the temperature of 35 ℃ under aerobic conditions to obtain Erwinia liquid and Pantoea dispersa liquid; mixing Zygomyces bifidus bacterial liquid, Rhizopus pachyticus bacterial liquid, Erwinia bacterium liquid and Pantoea dispersa bacterial liquid to obtain a composite bacterial agent seed liquid; inoculating the seed solution of the compound microbial inoculum into a culture medium of the compound microbial inoculum, and culturing for 120 hours at the temperature of 30 ℃ under aerobic conditions to obtain the microbial compound microbial inoculum.
The cell numbers of the Zygomyces bifidus, Rhizopus pachyticus, Erwinia and Pantoea dispersa in the composite microbial inoculum seed liquid are respectively 32%, 36%, 14% and 18% of the total cell number in the composite microbial inoculum seed liquid in sequence.
In example 3, one portion per 10g is used.
Adding ramie into the prepared composite microbial inoculum culture medium (the adding proportion of the ramie is 1-5g per 100ml of the culture medium), inoculating the microbial composite microbial inoculum prepared in the example 3 according to the inoculation amount of 1-5%, culturing for 72 hours at the temperature of 30 ℃ and under the aerobic condition, taking out, drying at 80 ℃ to constant weight, and measuring the components (lipid wax content, water soluble substance content, pectin substance content, hemicellulose content and cellulose content) of the ramie, wherein the measurement results are shown in table 4.
Centrifuging the culture solution for 5min at 10000rpm for 72h, collecting supernatant, and respectively measuring the activity of the mannose enzyme, the activity of the xylanase enzyme (referring to the measurement of the activity of the xylanase of the GB/T23874-.
Table 4: test results of the microbial composite inoculum prepared in example 3
The features of the embodiments and embodiments described herein above may be combined with each other without conflict.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A preparation method of a microbial compound microbial inoculum for ramie biological degumming pretreatment is characterized by comprising the following steps: a strain of Zygomyces bipinnatus having a commercial product number of ATCC 42397 (Dipodascus klebahnii) Rhizopus pachytrichoides of commodity No. ACCC30302 (A)Rhizopus chlamydosporus) Erwinia, commercially available under the trade designation ATCC 51855 (Erwinia amylovora) Goods and merchandisePantoea dispersa of BMZ068228 (Pantoea dispersa) Respectively inoculating the strain into a culture medium for culture to obtain a Zygomyces bipinnata bacterial liquid, a Rhizopus pachyticus bacterial liquid, an Erwinia bacterium bacterial liquid and a Pantoea dispersa bacterial liquid, and mixing the four bacterial liquids to obtain a composite bacterial agent seed liquid; inoculating the composite microbial inoculum seed solution into a composite microbial inoculum culture medium for mixed fermentation to obtain a microbial composite microbial inoculum; the cell numbers of the pediococcus bifidus, the rhizopus pachyticus, the erwinia bacterium and the pantoea dispersa in the composite microbial inoculum seed liquid are respectively 30-40%, 10-15% and 10-20% of the total cell number in the composite microbial inoculum seed liquid in sequence.
2. The method for preparing the microbial composite inoculum for the ramie biological degumming pretreatment according to claim 1, wherein the preparation process of the composite inoculum culture medium is as follows: weighing 5-10 parts of peptone, 3-5 parts of yeast powder, 2-7 parts of sodium chloride and (NH)4)2SO40.5-2 parts of dipotassium hydrogen phosphate, 0.5-1.5 parts of rice flour, 5-10 parts of ramie raw ramie and 950-1050 parts of distilled water, and sterilizing at 121 ℃ for 20min to obtain the composite microbial inoculum culture medium.
3. The preparation method of the microbial composite inoculant for the biological degumming pretreatment of ramie according to claim 1, wherein the temperature of the mixed fermentation of the seed liquid of the composite inoculant is 28-32 ℃ and the time is 72-128 hours.
4. The method for preparing the microbial composite inoculant for the biological degumming pretreatment of ramie according to claim 1, wherein the pediococcus dunaliensis and the rhizopus pachyma are respectively inoculated into a wort culture medium, and cultured for 48-72 hours under the aerobic condition and the temperature condition of 22-30 ℃ to obtain pediococcus dunaliensis bacteria liquid and rhizopus pachyma bacteria liquid.
5. The method for preparing the microbial composite inoculant for the biological degumming pretreatment of ramie according to claim 4, wherein the preparation process of the wort culture medium is as follows: weighing 2-4 parts of yeast powder, 2-4 parts of malt extract, 3-6 parts of peptone, 7-15 parts of glucose and 950-1050 parts of distilled water, mixing, and sterilizing at 121 ℃ for 20min to obtain the malt extract culture medium.
6. The preparation method of the microbial composite inoculant for the biological degumming pretreatment of ramie according to claim 1, wherein erwinia and pantoea dispersa are respectively inoculated into an LB culture medium and cultured for 24-48 hours under aerobic conditions at a temperature of 30-38 ℃ to obtain the erwinia bacterial liquid and the pantoea dispersa bacterial liquid.
7. The method for preparing the microbial composite inoculant for the biological degumming pretreatment of ramie according to claim 6, wherein the preparation process of the LB culture medium is as follows: weighing 5-15 parts of peptone, 3-10 parts of yeast extract, 5-10 parts of sodium chloride and 950-1050 parts of distilled water, mixing, and sterilizing at 121 ℃ for 20min to obtain the LB culture medium.
8. The microbial compound microbial inoculum prepared by the preparation method of any one of claims 1 to 7 and used for the biological degumming pretreatment of ramie.
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