CN112266883A - Zymophyte liquid for whole-plant fermentation of hybrid paper mulberry, and preparation method and application thereof - Google Patents

Zymophyte liquid for whole-plant fermentation of hybrid paper mulberry, and preparation method and application thereof Download PDF

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CN112266883A
CN112266883A CN202011154142.5A CN202011154142A CN112266883A CN 112266883 A CN112266883 A CN 112266883A CN 202011154142 A CN202011154142 A CN 202011154142A CN 112266883 A CN112266883 A CN 112266883A
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许标
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Abstract

The invention belongs to the technical field of agriculture, and particularly relates to a zymophyte liquid for whole-plant fermentation of a hybrid paper mulberry, and a preparation method and application thereof. The fermentation bacteria liquid of the whole strain fermentation of the hybrid paper mulberry is prepared by mixing and fermenting probiotics such as pichia pastoris, torula yeast, lactobacillus acidophilus, bifidobacterium and the like and sugar. The zymophyte liquid can effectively degrade lignin and cellulose in the paper mulberry.

Description

Zymophyte liquid for whole-plant fermentation of hybrid paper mulberry, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of agriculture, and particularly relates to a zymophyte liquid for whole-plant fermentation of a hybrid paper mulberry, and a preparation method and application thereof.
Background
The crude protein content of the hybrid paper mulberry is up to 26-32%, the hybrid paper mulberry is rich in various amino acids, vitamins, trace elements and flavonoid physiologically active substances, and the leaf thickness of the hybrid paper mulberry is higher than that of the wild paper mulberry. However, the current fermentation technology of the hybrid broussonetia papyrifera is limited to the fermentation of fresh and tender branches and leaves, the fermentation degree is not thorough, the utilization degree is low, and the fermentation has little effect on high stems with relatively high lignification degree. Meanwhile, the work of picking fresh and tender branches and leaves must be finished manually, the efficiency is low, the cost is high, large-scale industrial application is difficult to carry out, a fermentation utilization technology which can be suitable for all stems and branches and leaves of the hybrid paper mulberry is urgently needed, the manual picking is changed into mechanical harvesting, all harvested products are fully fermented and utilized, the efficiency is improved, and the cost is reduced.
Disclosure of Invention
The invention aims to provide a zymophyte liquid for fermenting whole hybrid broussonetia papyrifera.
The present invention also provides a method for preparing the above-mentioned fermented bacterial liquid.
Still another object of the present invention is to provide a method for fermenting whole hybrid broussonetia papyrifera.
According to the embodiment of the invention, the zymophyte liquid for the whole hybrid broussonetia papyrifera fermentation is prepared by fermenting the following components: 14-17% (w/w) of carbohydrate, 90000-100000 cfu/ml of pichia pastoris, 75000-90000 cfu/ml of torula yeast, 80000-100000 cfu/ml of lactobacillus acidophilus, 60000-80000 cfu/ml of bifidobacterium, 50000-70000 cfu/ml of lactococcus lactis, 70000-90000 cfu/ml of lactobacillus casei and the balance of water.
In the zymophyte liquid, the pichia pastoris can effectively decompose cellulose and lignin in the paper mulberry, the mycoprotein content is high, and the fermented product can be further used for preparing feed; the lactobacillus acidophilus, the bifidobacterium, the lactococcus lactis and the lactobacillus casei form a probiotic lactobacillus flora, and generate a great amount of lactic acid and organic acid while generating nutrient substances, so that the growth of mixed bacteria is inhibited, and the decomposition and utilization of cellulose and lignin by pichia pastoris are promoted; the torula yeast is fermented to decompose the polysaccharide, the palatability of the feed can be improved by producing flavor substances, the protein content of the thalli is high, the torula yeast can be continuously used for preparing animal feed, meanwhile, the fermentation of the torula yeast can provide nutrition for the growth of pichia pastoris and probiotic lactic acid bacteria, and the torula yeast can promote the proliferation of lactic acid bacteria; therefore, the saccharomyces cerevisiae and the probiotic lactic acid bacteria group have synergistic effect, the pichia pastoris is effectively promoted to be converted and decomposed into the original substances in the paper mulberry, and the zymogen liquid is beneficial to subsequent fermentation processing and is convenient to improve the meat quality and the flavor of the raised animals.
The substances produced by the probiotic lactic acid bacteria comprise acidic metabolites (mainly lactic acid, acetic acid, propionic acid, butyric acid, isobutyric acid and the like), extracellular polysaccharide EPS (comprising capsular polysaccharide, mucopolysaccharide and the like), lactein, gamma aminobutyric acid, vitamins, conjugated linoleic acid, polypeptide substances and the like. The acidic metabolite can effectively inhibit pathogenic microorganisms and can also be used as a synthesis precursor of certain nutrient substances; and the lactein also has bacteriostatic action.
Therefore, the strains in the composite zymophyte liquid synergistically decompose and convert cellulose and lignin in the paper mulberry into available polysaccharides, decompose crude plant protein into small molecular peptide fragments and amino acids, synthesize mycoprotein, and enable flavonoid compounds in the paper mulberry to be easier to absorb through decomposition and conversion.
According to the fermentation bacteria liquid for the whole hybrid paper mulberry plant fermentation, the saccharide substances comprise, by weight, 5-8 parts of brown sugar, 3-6 parts of white granulated sugar, 1-3 parts of fructose and 1-3 parts of honey.
According to the embodiment of the invention, the preparation method of the zymophyte liquid for the whole hybrid broussonetia papyrifera fermentation comprises the following steps:
(1) taking sterilized purified water, and adding 14-17% of saccharides;
(2) adding pichia pastoris 90000-100000 cfu/ml, torula yeast 75000-90000 cfu/ml, lactobacillus acidophilus 80000-100000 cfu/ml, bifidobacterium 60000-80000 cfu/ml, lactococcus lactis 50000-70000 cfu/ml and lactobacillus casei 70000-90000 cfu/ml respectively, and stirring uniformly;
(3) anaerobic culture is carried out for 15 days at the temperature of 35-40 ℃.
In the preparation method, the growth speed and activity of each flora in the mixed strain can be well balanced under the anaerobic environment at 35-40 ℃, and if the fermentation temperature is changed, the growth speed and activity of each flora are influenced by different degrees, so that the proportion of the flora is changed, and the fermentation effect is finally influenced.
According to the preparation method of the zymophyte liquid for the whole hybrid paper mulberry plant fermentation, according to the specific embodiment of the invention, the sugar substances comprise 7 parts by weight of brown sugar, 5 parts by weight of white granulated sugar, 2 parts by weight of fructose and 2 parts by weight of honey.
According to the preparation method of the zymogen liquid for the whole hybrid broussonetia papyrifera fermentation, in the step (3), when the colony number of lactococcus lactis is more than 1000000cfu/ml, the fermentation is stopped to obtain the zymogen liquid.
After the lactococcus lactis meets the requirements, nutrient substances in the zymocyte liquid are limited within the range, and other flora can spontaneously form an equilibrium state.
According to a specific embodiment of the present invention, the whole plant fermentation method of the hybrid broussonetia papyrifera comprises the following steps: adding a zymophyte liquid accounting for 5-35% of the weight of the hybrid paper mulberry and 1-3% of sugar into the hybrid paper mulberry, placing the hybrid paper mulberry into a closed container, and fermenting for 10-20 days at 35-40 ℃, wherein the zymophyte liquid is prepared by fermenting 14-17% (w/w) of carbohydrate substances, 90000-100000 cfu/ml of pichia pastoris, 75000-90000 cfu/ml of torula yeast, 80000-100000 cfu/ml of lactobacillus acidophilus, 60000-80000 cfu/ml of bifidobacterium, 50000-70000 cfu/ml of lactococcus lactis and 70000-90000 cfu/ml of lactobacillus casei.
According to the whole-plant fermentation method of the hybrid paper mulberry, the sugar comprises brown sugar, white granulated sugar, fructose and/or honey, and the sugar can also be maltose, sucrose, lactose and the like.
According to the whole strain fermentation method of the hybrid broussonetia papyrifera, the hybrid broussonetia papyrifera comprises fresh hybrid broussonetia papyrifera, silage hybrid broussonetia papyrifera and/or dried hybrid broussonetia papyrifera.
According to the whole plant fermentation method of the hybrid broussonetia papyrifera, the hybrid broussonetia papyrifera is a whole plant hybrid broussonetia papyrifera and comprises broussonetia papyrifera leaves, branches and trunks.
According to the full-plant fermentation method of the broussonetia papyrifera, 5-35% of zymogen liquid and 1% of sugar are added into the broussonetia papyrifera, and then 1-10% of corn flour, 1-10% of bean flour and 1-10% of bean pulp powder are added for fermentation.
The invention has the beneficial effects that:
in the fermentation broth, on one hand, the pichia pastoris can effectively decompose cellulose and lignin in the paper mulberry, the probiotic lactobacillus group consisting of lactobacillus acidophilus, bifidobacterium, lactococcus lactis and lactobacillus casei can inhibit the growth of mixed bacteria and promote the pichia pastoris to decompose the cellulose and the lignin, the torula pastoris provides nutrition for the pichia pastoris and the probiotic lactobacillus group, and all the floras are synergistically effective in decomposing the whole paper mulberry; on the other hand, the pichia pastoris, the torula yeast and the probiotic lactobacillus flora are beneficial to subsequent feed processing, and can effectively improve the meat quality and flavor of the fed animals.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
EXAMPLE 1 preparation of a fermentation broth
1.1 taking sterilized purified water, adding 7 percent of brown sugar, 5 percent of white granulated sugar, 2 percent of fructose and 2 percent of honey by weight, respectively adding 95000cfu/ml of pichia pastoris, 80000cfu/ml of torula yeast, 90000cfu/ml of lactobacillus acidophilus, 70000cfu/ml of bifidobacterium, 60000cfu/ml of lactococcus lactis and 80000cfu/ml of lactobacillus casei, uniformly stirring, placing on a shaking bed, carrying out anaerobic culture at 38 ℃ for 15 days, and then measuring day by day.
Stopping fermentation when the colony number of the lactococcus lactis is more than 1000000cfu/ml to obtain a fermentation stock solution.
Before use, the fermentation stock solution is diluted by 8 times by using sterile water to obtain fermentation liquor.
1.2 taking sterilized purified water, adding 5% of brown sugar, 3% of white granulated sugar, 1% of fructose and 5% of honey by weight, respectively adding 90000cfu/ml of pichia pastoris, 75000cfu/ml of torula yeast, 80000cfu/ml of lactobacillus acidophilus, 60000cfu/ml of bifidobacterium, 50000cfu/ml of lactococcus lactis and 70000cfu/ml of lactobacillus casei, uniformly stirring, placing on a shaking bed, carrying out anaerobic culture at 35 ℃ for 15 days, and then measuring day by day.
Stopping fermentation when the colony number of the lactococcus lactis is more than 1000000cfu/ml to obtain a fermentation stock solution.
Before use, the fermentation stock solution is diluted by 8 times by using sterile water to obtain fermentation liquor.
1.3 taking sterilized purified water, adding 6 percent of brown sugar, 6 percent of white granulated sugar, 3 percent of fructose and 2 percent of honey by weight, respectively adding 100000cfu/ml of pichia pastoris, 90000cfu/ml of torula yeast, 100000cfu/ml of lactobacillus acidophilus, 80000cfu/ml of bifidobacterium, 70000cfu/ml of lactococcus lactis and 90000cfu/ml of lactobacillus casei, uniformly stirring, placing on a shaking bed, carrying out anaerobic culture for 15 days at 40 ℃, and then measuring day by day.
Stopping fermentation when the colony number of the lactococcus lactis is more than 1000000cfu/ml to obtain a fermentation stock solution.
Before use, the fermentation stock solution is diluted by 8 times by using sterile water to obtain fermentation liquor.
Example 2 treatment of Broussonetia papyrifera
2.1 fresh treatment
Fresh harvesting and mincing whole hybrid paper mulberry plants to directly ferment.
2.2 silage treatment
The whole hybrid paper mulberry plant is harvested, minced, ensiled for a period of time, and fermented.
2.3 drying treatment
Harvesting whole hybrid paper mulberry, drying to water content of less than 10%, mincing, and fermenting.
EXAMPLE 3 Whole plant fermentation of hybrid Broussonetia papyrifera
3.1 for fresh Broussonetia papyrifera
Adding fermentation liquor accounting for 5% of the weight of the fresh harvested hybrid paper mulberry and 1% of brown sugar into the freshly harvested hybrid paper mulberry, placing the hybrid paper mulberry in a closed container, and fermenting for 15 days at the temperature of 35-40 ℃;
derivative fermentation: adding fermentation liquor which is 10 percent of the weight of the newly harvested hybrid paper mulberry into the newly harvested hybrid paper mulberry and 1 percent of brown sugar, optionally additionally adding 1-10 percent of corn flour, 1-10 percent of bean flour and 1-10 percent of soybean meal powder, placing the mixture in a closed container, and fermenting for 15 days at 35-40 ℃.
3.2 for Broussonetia papyrifera
Adding fermentation liquor accounting for 20 percent of the weight of the paper mulberry and brown sugar accounting for 2 percent of the weight of the paper mulberry into the hybrid paper mulberry subjected to ensiling treatment, placing the hybrid paper mulberry in a closed container, and fermenting for 15 days at the temperature of 35-40 ℃;
derivative fermentation: adding fermentation liquor accounting for 15% of the weight of the paper mulberry and white granulated sugar accounting for 2% of the weight of the paper mulberry into the hybrid paper mulberry subjected to ensilage treatment, additionally adding 1-10% of corn flour, 1-10% of bean flour and 1-10% of bean pulp powder, placing the mixture in a closed container, and fermenting for 15 days at 35-40 ℃.
3.3 dried hybrid Broussonetia papyrifera
Adding 35% of fermentation liquor and 3% of brown sugar into the dried hybrid paper mulberry, placing the hybrid paper mulberry in a closed container, and fermenting for 15 days at the temperature of 35-40 ℃.
Derivative fermentation: and adding 30% of fermentation liquor and 3% of fructose into the dried hybrid paper mulberry, and additionally adding 1-10% of corn flour, placing the hybrid paper mulberry in a closed container, and fermenting for 15 days at 35-40 ℃.
Example 4
The results of comparing the unfermented broussonetia papyrifera with the whole fermentation product obtained by fermentation in example 1 of the present invention are shown in table 1.
TABLE 1 Change in nutrients before and after fermentation of Broussonetia papyrifera
Crude protein (%) Crude fiber (%) Lignin (%)
Not fermented 14.995 19.91 5.9
Fermentation of whole plant 14.485 10.5 3.1
Phase difference (%) 3 47.3 47.5
As shown in table 1, after 15 days of fermentation, the crude fiber content of the broussonetia papyrifera was reduced to 10.5%, and the degradation amount of the crude fiber was increased by 47.3% compared with the unfermented broussonetia papyrifera (% (fermented whole crude fiber% -unfermented whole crude fiber%)/unfermented crude fiber%); after the broussonetia papyrifera is fermented by the whole plant, the content of lignin is reduced to 3.1%, and compared with the unfermented broussonetia papyrifera, the degradation amount of crude fiber is improved by 47.5% ((fermented whole plant lignin% -unfermented whole plant lignin%)/unfermented lignin%). Therefore, the zymophyte liquid can effectively degrade crude fiber and lignin in the paper mulberry.
The commercial pig feed and the whole broussonetia papyrifera fermented feed obtained in example 3.1 of the present invention were added to the commercial pig feed in amounts of 10%, 20%, and 30%, respectively, and fed for 10 days to examine apparent digestibility of each group. The results are shown in table 2:
TABLE 2 apparent digestibility obtained by feeding different feeds
Control 10% addition 20% of addition 30% addition SEM P value
Total phosphorus (%) 72.2Bc 86.1Aa 79.1Bb 72.9Bc 1.42 <0.01
Inorganic phosphorus (%) 64.5Bc 84.2Aa 76.1Ab 75.8Ab 2.13 <0.01
Calcium (%) 69.7b 78.4a 76.2ab 73.5ab 2.46 0.149
Copper (%) 79.5B 98.5A 98.1A 97.4A 0.59 <0.01
Zinc (%) 77.7C 90.8A 87.9A 82.8B 1.02 <0.01
Iron (%) 70.5B 79.4A 74.7AB 70.8B 1.56 0.014
Manganese (%) 69.7Bb 79.7Aa 73.5ABb 67.5Bb 1.81 <0.01
The results are shown in table 2, and the apparent digestibility of the fattening pigs on total phosphorus, inorganic phosphorus, calcium, copper, iron, zinc and manganese is improved by adding 10%, 20% and 30% of the feed ration of the fermented paper mulberry. (Note: P less than 0.01 is very different, and the letter indices in the table above are understood that 70.5B and 79.4A differ significantly so the indices differ in letter but 74.7ABAnd 70.5BThe contrast is not significant and has the same B, 74.7ABAnd 79.4AThe difference is not significant and so there is the same corner mark a. The rest of the data are processed in the same way. )
Setting a plurality of experimental groups, wherein the strains used by each experimental group are different, respectively carrying out whole-plant fermentation on the paper mulberry, wherein,
experimental group 1: pichia pastoris;
experimental group 2: pichia pastoris and torula pastoris;
experimental group 3: pichia pastoris, torula yeast, lactobacillus acidophilus and bifidobacterium;
experimental group 4: pichia pastoris, torula yeast, lactobacillus acidophilus, bifidobacterium, lactobacillus lactis and lactobacillus casei;
experimental group 5: torula, lactobacillus acidophilus, bifidobacterium, lactobacillus lactis and lactobacillus casei;
the resulting fermentation broth from each group was mixed with paper mulberry and fermented under the fermentation conditions of example 3 of the present invention. The results of the fermentation of each group were analyzed by comparison, and the specific results are shown in table 3:
TABLE 3 influence of different fermentation strains on fermentation of whole broussonetia papyrifera
Figure RE-GDA0002807251860000071
Figure RE-GDA0002807251860000081
As shown in Table 3, only Pichia pastoris and no synergistic enhancement of other strains are contained in the experimental group 1, the decomposition effect of crude fiber and lignin in the paper mulberry is poor, and the overall fermentation efficiency is low; the experiment group 5 has no pichia pastoris, so that the improvement of the fermentation efficiency cannot be fully reflected even if other strains build an environment; compared with the experimental group 1, the crude fiber in the experimental group 2 is reduced by 0.6%, and the content of lignin is even slightly increased; the experiment group 3 adds two kinds of lactic acid bacteria on the basis of the experiment group 2, the content of crude fiber is reduced by 1.6 percent, and the content of lignin is reduced by only 0.4 percent; in the experimental group 4, under the combined action of the pichia pastoris, the torula yeast and the 4 lactic acid bacteria, the content of crude fibers is reduced by 5.0%, the content of lignin is greatly reduced by 1.5%, and the content of lignin and the content of crude fibers are both greatly reduced, so that the synergistic effect of the pichia pastoris, the torula yeast and the 4 probiotic lactic acid bacteria is demonstrated, and the fermentation degradation efficiency of the whole broussonetia papyrifera is greatly improved.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (10)

1. The zymophyte liquid for whole strain fermentation of the hybrid paper mulberry is characterized by being prepared by fermenting 14-17% (w/w) of carbohydrate, 90000-100000 cfu/ml of pichia pastoris, 75000-90000 cfu/ml of torula pastoris, 80000-100000 cfu/ml of lactobacillus acidophilus, 60000-80000 cfu/ml of bifidobacterium, 50000-70000 cfu/ml of lactococcus lactis and 70000-90000 cfu/ml of lactobacillus casei.
2. The zymophyte liquid for fermentation of the whole hybrid broussonetia papyrifera as claimed in claim 1, wherein the sugar substances comprise 5-8 parts by weight of brown sugar, 3-6 parts by weight of white granulated sugar, 1-3 parts by weight of fructose and 1-3 parts by weight of honey.
3. The method of preparing a fermentation broth for the whole fermentation of hybrid broussonetia papyrifera as claimed in claim 1, comprising the steps of:
(1) taking sterilized purified water, and adding 14-17% (w/w) of saccharides;
(2) adding 90000-100000 cfu/ml of pichia pastoris, 75000-90000 cfu/ml of torula yeast, 80000-100000 cfu/ml of lactobacillus acidophilus, 60000-80000 cfu/ml of bifidobacterium, 50000-70000 cfu/ml of lactococcus lactis and 70000-90000 cfu/ml of lactobacillus casei into the purified water, and uniformly stirring;
(3) anaerobic culture is carried out for 15 days at the temperature of 35-40 ℃.
4. The method for preparing zymogen liquid for fermentation of whole hybrid broussonetia papyrifera as claimed in claim 3, wherein the sugar substances comprise 7 parts by weight of brown sugar, 5 parts by weight of white granulated sugar, 2 parts by weight of fructose and 2 parts by weight of honey.
5. The method according to claim 3, wherein the fermentation broth is obtained by stopping the fermentation when the number of lactococcus lactis colonies is larger than 1000000cfu/ml in the step (3).
6. A whole plant fermentation method of a hybrid paper mulberry, which is characterized by comprising the following steps: adding 5-35% of zymophyte liquid and 1-3% of sugar into the hybrid paper mulberry, and fermenting for 10-20 days at 35-40 ℃ in a closed container, wherein the zymophyte liquid is prepared by fermenting 14-17% (w/w) of sugar substances, 90000-100000 cfu/ml of pichia pastoris, 75000-90000 cfu/ml of torula yeast, 80000-100000 cfu/ml of lactobacillus acidophilus, 60000-80000 cfu/ml of bifidobacterium, 50000-70000 cfu/ml of lactococcus lactis and 70000-90000 cfu/ml of lactobacillus casei.
7. The method for fermenting whole hybrid broussonetia papyrifera as claimed in claim 6, wherein the sugar comprises brown sugar, white sugar, fructose and/or honey.
8. The method for fermenting whole hybrid broussonetia papyrifera according to claim 6, wherein the hybrid broussonetia papyrifera comprises fresh hybrid broussonetia papyrifera, silage hybrid broussonetia papyrifera and/or dried hybrid broussonetia papyrifera.
9. The method for fermenting whole hybrid broussonetia papyrifera according to claim 6, wherein the hybrid broussonetia papyrifera is a whole hybrid broussonetia papyrifera which comprises broussonetia papyrifera leaves, branches and trunks.
10. The method for fermenting the whole hybrid paper mulberry according to claim 6, wherein the hybrid paper mulberry is added with 5-35% of zymophyte liquid and 1% of sugar, and then added with 1-10% of corn flour, 1-10% of bean flour and 1-10% of bean pulp powder for fermentation.
CN202011154142.5A 2020-10-26 2020-10-26 Zymophyte liquid for whole-plant fermentation of hybrid paper mulberry, and preparation method and application thereof Pending CN112266883A (en)

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