CN112970932A - Method for preparing concentrated protein powder and xylo-oligosaccharide by using broussonetia papyrifera leaves and application - Google Patents
Method for preparing concentrated protein powder and xylo-oligosaccharide by using broussonetia papyrifera leaves and application Download PDFInfo
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Abstract
The invention relates to a method for preparing concentrated protein powder and xylo-oligosaccharide by using broussonetia papyrifera leaves and application thereof, wherein the method comprises the following steps: crushing and extruding broussonetia papyrifera leaves to obtain extrusion liquid rich in protein and residues rich in cellulose, lignin and hemicellulose. Spray drying the extrusion liquid to obtain concentrated protein powder of broussonetia papyrifera leaves; and carrying out microbial fermentation, homogenate and enzymolysis on the residues to obtain the broussonetia papyrifera leaf xylooligosaccharide rich in xylooligosaccharide, and applying the broussonetia papyrifera leaf xylooligosaccharide to animal culture, so that the broussonetia papyrifera leaf xylooligosaccharide has good effects of promoting growth and enhancing immunity, and has excellent economic value.
Description
Technical Field
The invention belongs to the technical field of feed additives, and particularly relates to a method for preparing concentrated protein powder and xylo-oligosaccharide by using broussonetia papyrifera leaves and application of the concentrated protein powder and the xylo-oligosaccharide.
Background
The broussonetia papyrifera leaves contain rich protein, the content of crude protein in dry substances of the broussonetia papyrifera leaves can reach 26-32%, the content of amino acid is 4.5 times that of rice, 2.5 times that of corn and 1.8 times that of soybeans, and the content of vitamins and trace elements of the broussonetia papyrifera leaves is several times that of most fruits and vegetables, so that the broussonetia papyrifera leaves are also used as pig raising feed by common people. However, the problems exist in the culture process, such as palatability problem of the broussonetia papyrifera leaf powder, and the feed intake of animals is reduced due to the fact that the broussonetia papyrifera leaf powder is high in fiber content and poor in palatability.
Xylo-oligosaccharide is also called xylo-oligosaccharide, is an important functional food and feed additive, has good physicochemical properties of low heat, stability, safety, no toxicity and the like, has unique physiological functions of promoting the reproduction of beneficial bacteria in intestinal tracts, particularly bifidobacterium and inhibiting the growth of harmful bacteria, has attracted wide attention all over the world, and is widely applied to human health care products and animal feeds. At present, the xylo-oligosaccharide is mostly prepared by taking cellulose substances such as corncobs, bagasse, straws and the like as raw materials.
However, reports that concentrated protein powder and xylo-oligosaccharide are prepared by using broussonetia papyrifera leaves and are applied to the field of animal breeding are not found at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for preparing concentrated protein powder and xylo-oligosaccharide by using broussonetia papyrifera leaves and application thereof.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the method for preparing the concentrated protein powder and the xylo-oligosaccharide by using the broussonetia papyrifera leaves comprises the following steps:
(1) crushing the fresh broussonetia papyrifera leaves after the impurities are removed to 5-200 meshes;
(2) mixing the crushed fresh broussonetia papyrifera leaves with water according to the mass ratio of 1 (0.1-3), and then squeezing and dehydrating, preferably using a screw press to obtain a squeezing liquid and squeezing residues; spray drying the squeezed solution to obtain concentrated protein powder of broussonetia papyrifera leaves;
(3) mixing and fermenting the squeezed residues with bacillus subtilis and candida utilis at 25-37 ℃ for 1-3 weeks to obtain fermented paper mulberry leaf residues;
(4) mixing fermented paper mulberry leaf residues with water according to the mass ratio of 1 (1-10), homogenizing, and performing enzymolysis reaction on the slurry obtained after homogenizing by using bacillus subtilis xylanase to obtain an enzymolysis reaction material; the mass percentage concentration of the bacillus subtilis xylanase in the enzymolysis reaction is 0.3-0.7%, the enzymolysis reaction time is 24-48 h, the enzymolysis temperature is 50-60 ℃, and the pH value of an enzymolysis system is 5.0-6.3;
(5) centrifuging the enzymolysis reaction material at 3000-12000 r/min, taking supernatant, concentrating, and spray drying to obtain the xylo-oligosaccharide.
Preferably, the mixed fermentation of the pressing residue by using the bacillus subtilis and the candida utilis in the step (3) is to prepare the pressing residue, brown sugar, urea and monopotassium phosphate into a solid fermentation base material according to the mass ratio of 975:10:10: 5; preparing bacillus subtilis liquid and candida utilis liquid, wherein the bacterium concentration of the bacillus subtilis liquid and the candida utilis liquid is 1010CFU/mL, mixing the bacillus subtilis liquid and the candida utilis liquid according to the volume ratio of 1:1, inoculating the mixture to a solid fermentation base material according to the mass ratio of 3%, and performing mixed fermentation for 1-3 weeks at the temperature of 25-37 ℃ to obtain fermented paper mulberry leaf residues.
Preferably, the control parameters for concentration and spray drying in the step (5) are as follows: the vacuum pressure is-0.1-0.15 MPa, the air inlet temperature of spray drying is 180-200 ℃, the air outlet temperature is 70-80 ℃, and the spray feeding flow rate is 580-620 mL/h.
The concentrated protein powder and xylooligosaccharide prepared by the method can be used for preparing animal feed. Preferably, concentrated protein powder and/or xylo-oligosaccharide are/is added into the animal complete formula feed, the mass percentage content of the concentrated protein powder in the animal complete formula feed is 1-10%, and the mass percentage content of the xylo-oligosaccharide in the animal complete formula feed is 0.1-5%. The animal can be pig, chicken, duck, cattle, sheep, goose, rabbit, and quail.
The invention is further illustrated below:
the method comprises the steps of taking fresh broussonetia papyrifera leaves as raw materials, removing protein in the broussonetia papyrifera leaves by crushing and extruding to obtain concentrated protein of the broussonetia papyrifera leaves, then breaking fiber and hemicellulose structures of the broussonetia papyrifera leaves by using a microbial fermentation and homogenate method, and performing enzymolysis by using xylanase to obtain the xylooligosaccharide of the broussonetia papyrifera leaves.
Specifically, the invention firstly removes impurities from fresh broussonetia papyrifera leaves and then crushes the fresh broussonetia papyrifera leaves. Mixing the pulverized fresh folium Broussonetiae with water (tap water, the same below), squeezing and dehydrating with screw squeezer (Zhongtian/ZTY), and spray drying the obtained squeezed solution to obtain folium Broussonetiae concentrated protein powder. Removing the protein residue from folium Broussonetiae, and preparing xylooligosaccharide from folium Broussonetiae.
And (3) mixing and fermenting the squeezed residues by using bacillus subtilis PL83 (purchased from China center for type culture Collection (Wuhan), the preservation number of the strain is CCTCC NO: M2013412) and candida utilis (purchased from China center for industrial microorganism preservation, the preservation number of the strain is CICC NO:31430), decomposing cellulose, lignin and hemicellulose of the paper mulberry leaves by using cellulase and hemicellulase generated by the bacillus subtilis and the candida utilis, and breaking the fibrous structure of the paper mulberry leaves (5-200 meshes), so that the subsequent enzymolysis of the xylanase is facilitated. Adding 1-5% (mass ratio) of brown sugar as a carbon source and 1-5% (mass ratio) of urea as a nitrogen source in the fermentation process for shortening the fermentation time; controlling the fermentation temperature to be 25-37 ℃ and the fermentation time to be 1-3 weeks; the structure of the broussonetia papyrifera leaves is crushed through the steps, so that more cellulose, hemicellulose, polysaccharide, oligosaccharide and the like are exposed, and the degradation of xylanase is facilitated.
And mixing the fermented paper mulberry leaf residues with water by using a high-pressure homogenizer for homogenization, and carrying out enzymolysis reaction on the homogenized slurry by using bacillus subtilis xylanase: the enzyme concentration is: 0.3-0.7%, carrying out enzymolysis reaction for 24-48 h, controlling the enzymolysis temperature at 50-60 ℃, and controlling the pH value of an acetic acid-acetate buffer solution of an enzymolysis system at 5.0-6.3 to obtain an enzymolysis reaction material. And centrifuging the enzymolysis reaction material, concentrating the supernatant, and spray drying to obtain xylooligosaccharide (centrifuging to remove impurities and particulate matter, spray drying the supernatant to dehydrate the supernatant, and changing the liquid into solid for storage and use).
The broussonetia papyrifera leaves are fermented, homogenized and subjected to enzymolysis, so that the xylooligosaccharide content and digestibility are increased, the problem of palatability of the broussonetia papyrifera leaves can be solved, the xylooligosaccharide content can be increased, the effects of enhancing the immune function of animals, promoting growth, reducing the diarrhea rate of the animals and reducing the occurrence of diarrhea can be achieved.
Compared with the prior art, the invention has the following advantages and effects:
(1) in the prior art, dry materials such as wheat bran, corncobs, rice husks and the like are used as raw materials, and fresh broussonetia papyrifera leaves are used, so that the drying cost can be saved;
(2) the invention adopts an extrusion mode suitable for fresh leaves to remove protein, and the prior art adopts an ultrasonic mode or the like or does not adopt the step of removing protein;
(3) the invention adopts microbial fermentation and homogenate method to break the structure of cellulose, hemicellulose and lignin, so as to facilitate the subsequent enzymolysis of xylanase, and the prior art has no design;
(4) fresh broussonetia papyrifera leaves are extruded and spray-dried to prepare concentrated broussonetia papyrifera leaf protein powder for animal breeding, and the product does not exist at present;
(5) the concentrated protein powder and xylo-oligosaccharide product of the broussonetia papyrifera leaves treated by the method effectively solve the problem of poor palatability of the broussonetia papyrifera leaves as feed, and can be added into the feed in large dose.
(6) The concentrated broussonetia papyrifera leaf protein powder and the xylo-oligosaccharide product treated by the method are added into feed by 1-10%, the diarrhea rate of piglets is reduced by 30-50%, the feed conversion ratio of pigs, chickens and ducks is reduced by 5-10%, the growth speed of animals is increased by 5-10%, and the litter size of sows is increased by 10-30%.
In a word, the invention provides a preparation method for producing the broussonetia papyrifera leaf concentrated protein powder and the broussonetia papyrifera leaf xylo-oligosaccharide by using the broussonetia papyrifera leaves, and the method is easy and convenient to operate. The method solves the palatability problem of folium Broussonetiae as feed, and can increase xylooligosaccharide content to enhance animal immunity and promote growth, improve animal intestinal health, and reduce epidemic disease. The invention also provides application of the paper mulberry leaf concentrated protein powder and xylo-oligosaccharide in animal breeding. The broussonetia papyrifera leaves are fermented, homogenized and subjected to enzymolysis, so that the xylooligosaccharide content and digestibility are increased, the palatability problem of the broussonetia papyrifera leaves can be solved, the xylooligosaccharide content can be increased, the effects of enhancing the immune function of animals and promoting growth are achieved, and the broussonetia papyrifera leaves have great application value in promoting the industrial development of the broussonetia papyrifera leaves.
The specific implementation mode is as follows:
example 1:
preparing a bacillus subtilis PL83 fermentation liquid:
A. taking 2ml of strain A (Bacillus subtilis PL83) with viable bacteria concentration of 1010CFU/ml；
B. Inoculating into 100ml LB medium (composed of tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000ml, pH 7.2), shake-flask fermentation culturing for 24 hours.
C. The fermentation temperature is 28 or 30 or 32 or 35 or 37 ℃, the pH value is 7.2, the rotation speed is 200-300 r/min, and the fermentation time is 24 or 28 or 32 or 36 h. The shake flask fermentation medium is an LB medium and comprises the following components: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000ml of distilled water, wherein the pH value is 7.2.
D. After the shake flask fermentation is finished, carrying out fermentation tank amplification culture, inoculating 100ml of shake flask fermentation seed liquid into a 10L fermentation tank, wherein the liquid loading amount is 6L, the fermentation temperature is 30 or 33 or 35 or 37 ℃, the pH value is 7.2, the stirring speed is 200-300 r/min, and the fermentation is carried out for 24 or 27 or 30 or 33 or 36 h. The components of the pilot culture medium of the fermentation tank are consistent with those of the shake flask fermentation culture medium, and the culture medium is placed at 4 ℃ for standby after the fermentation is finished. A Bacillus subtilis PL83 fermentation broth was obtained and used for the microbial fermentation in example 3.
Preparing candida utilis fermentation liquor:
A. taking 2ml of strain B (Candida utilis), wherein the viable bacteria concentration is 1010 CFU/ml;
B. inoculating into 100ml culture medium, and performing shake flask fermentation at 26 or 28 or 30 deg.C, pH 6.6, and rotation speed of 200 r/min;
C. the fermentation time is 24 or 27 or 30 or 33 or 36 or 39 or 42 or 45 or 48 h. The shaking flask fermentation medium is an YPD medium and comprises the following components: tryptone 20g, yeast extract 10g, glucose 20g, distilled water 1000ml, pH 6.8-7.2.
D. After the shake flask fermentation is finished, carrying out the enlarged culture of a fermentation tank, inoculating 100ml of shake flask fermentation seed liquid into a 20L fermentation tank, filling 6L of liquid, fermenting at the temperature of 28 or 30 or 32 or 34 or 36 or 37 ℃ and the pH value of 6.6 or 6.7 or 6.8 at the stirring speed of 200-300 r/min for 36 or 40 or 44 or 48 or 52 or 58 or 61 or 67 or 72 h. The components of the pilot culture medium of the fermentation tank are consistent with those of the shake flask fermentation culture medium, and the culture medium is placed at 4 ℃ for standby after the fermentation is finished. Candida utilis fermentation broth was obtained and used for the microbial fermentation in example 3.
The method for preparing the concentrated protein powder and the xylo-oligosaccharide by using the broussonetia papyrifera leaves comprises the following steps:
A. crushing the broussonetia papyrifera leaves 1 by using a grass powder crusher, wherein the crushing granularity is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 130, 150, 180 or 200 meshes;
B. and C, extruding the crushed broussonetia papyrifera leaves in the step A and water in a ratio of 1:1 by using a screw press to obtain a pressed liquid 3, centrifuging at 3000 rpm, and performing spray drying on the supernatant, wherein the vacuum degree is as follows: -0.1MPa, spray drying inlet air temperature: 180 ℃, air outlet temperature 70 ℃, spray feed flow rate: 600 ml/h; obtaining the concentrated protein powder 9 of the broussonetia papyrifera leaves.
C. Carrying out microbial fermentation on the squeezed residues, and preparing a solid fermentation base material according to the following formula: 975kg of squeezed residue; 10kg of brown sugar, 10kg of urea and 5kg of monopotassium phosphate. The bacterial liquid concentrations of Bacillus subtilis PL83 (the fermentation liquid prepared above) and Candida utilis (the fermentation liquid prepared above) were adjusted to 1010CFU/ml, mixing according to the volume ratio of 1:1, inoculating into the fermentation base material according to the mass ratio of 3%, fully mixing, bagging, and fermenting for 7 days. Through fermentation, the structure of the broussonetia papyrifera leaves is broken, and cellulose, hemicellulose, glycan and the like are degraded.
D. Homogenizing the fermented material by a high-pressure homogenizer at a ratio of material to water of 1:3 for 6 at 30MPA for 10 minutes until the particle diameter reaches 10 microns to obtain homogenate. And then, carrying out enzymolysis reaction on the obtained homogenate by using xylanase, wherein the optimized conditions of the enzymolysis reaction are that the mass concentration of a substrate is 6%, the mass concentration of enzyme in the system is 0.4%, the enzymolysis reaction time is 24h, the enzymolysis temperature is 60 ℃, and the pH value of an acetic acid-acetate buffer solution of the enzymolysis system is 5. And then centrifuging the enzymolysis reaction product, concentrating the supernatant (at the temperature of 60 ℃) and carrying out spray drying 8 to obtain the paper mulberry leaf xylo-oligosaccharide 10. The centrifugation time is 10min, the rotation speed of the centrifuge is 5000r/min, the vacuum degree is-0.1 MPa, the air inlet temperature of spray drying is 80 ℃, the air outlet temperature is 70 ℃, and the spray feeding flow rate is 600 ml/h. Homogenizing and enzymolysis to decompose polysaccharide in folium Broussonetiae into xylooligosaccharide, spray drying to dehydrate liquid xylooligosaccharide into solid xylooligosaccharide, and facilitate transportation and application.
Example 2:
the concentrated protein powder and xylo-oligosaccharide prepared in example 1 can be used in the preparation of animal feed: adding 1-10% of the concentrated protein powder of broussonetia papyrifera leaves into the complete formula feed, mixing uniformly, and feeding animals. Adding 0.1-5% of paper mulberry leaf xylo-oligosaccharide into the complete compound feed, mixing well, and feeding animals.
Example 3: pig raising experiment of paper mulberry leaf concentrated protein powder and xylo-oligosaccharide
The similar 21-day-old weaned piglets are randomly selected to form a control group and a test group, wherein each group has 30 heads, each group is divided into 3 columns, and each column has 10 heads. The control group was fed a basal diet, with the formula shown in table 1:
TABLE 1
Premix compound*Each kilogram of the copper alloy contains 10.00mg of copper; 100.00mg iron; 0.30mg sodium; 100.00mg zinc; 10.00mg magnesium; 386.0IU vitamin D3; 3086.0 IU vitamin A; 15.4IU vitamin E; 2.30mg vitamin K; 3.90mg riboflavin; 15.40mg D-pantothenic acid; 23.00mg niacin; 77.00mg choline; and 15.4. mu.g vitamin B12.
The test group is 2 groups, one group is obtained by adding 3 percent (mass ratio) of broussonetia papyrifera leaf concentrated protein on the basis of unchanging the formula, and the other group is obtained by adding 3 percent (mass ratio) of broussonetia papyrifera leaf xylo-oligosaccharide on the basis of unchanging the formula.
After 21-day feeding test, the statistical results of the number of heads, the weight increment, the feed intake and the feed-meat ratio of the test group and the control group are shown in the table 2:
TABLE 2
| Control group | 3% Broussonetia papyrifera leaf concentrated proteome | 3% of broussonetia papyrifera leaf xylo-oligosaccharide group | |
| Total head number (head) | 26 | 26 | 26 |
| Are all heavy (kilogram) | 5.73±0.07 | 5.71±0.07 | 5.73±0.11 |
| All over the whole length (get) | 12.88±0.47a | 13.53±0.37b | 13.77±0.53b |
| Average daily gain of head (g) | 340±21.13a | 372±13.37b | 383±12.73b |
| First average daily food intake (g) | 581±43.03 | 577±38.13 | 579±36.19 |
| Meat ratio of materials | 1.71±0.11a | 1.55±0.07b | 1.52±0.27b |
| Diarrhea Rate (%) | 17.83 | 6.13 | 6.33 |
The result shows that 3% of the broussonetia papyrifera leaf concentrated protein or the broussonetia papyrifera leaf xylo-oligosaccharide is added, so that the piglet production performance can be improved, the feed conversion ratio can be reduced, and the piglet diarrhea rate can be obviously reduced.
Claims (5)
1. A method for preparing concentrated protein powder and xylo-oligosaccharide by using broussonetia papyrifera leaves is characterized by comprising the following steps:
(1) crushing the fresh broussonetia papyrifera leaves after the impurities are removed to 5-200 meshes;
(2) mixing the crushed fresh broussonetia papyrifera leaves with water according to the mass ratio of 1 (0.1-3), and then squeezing and dehydrating to obtain a squeezing liquid and squeezing residues; spray drying the squeezed solution to obtain concentrated protein powder of broussonetia papyrifera leaves;
(3) mixing and fermenting the squeezed residues with bacillus subtilis and candida utilis at 25-37 ℃ for 1-3 weeks to obtain fermented paper mulberry leaf residues;
(4) mixing fermented paper mulberry leaf residues with water according to the mass ratio of 1 (1-10), homogenizing, and performing enzymolysis reaction on the slurry obtained after homogenizing by using bacillus subtilis xylanase to obtain an enzymolysis reaction material; the mass percentage concentration of the bacillus subtilis xylanase in the enzymolysis reaction is 0.3-0.7%, the enzymolysis reaction time is 24-48 h, the enzymolysis temperature is 50-60 ℃, and the pH value of an enzymolysis system is 5.0-6.3;
(5) centrifuging the enzymolysis reaction material at 3000-12000 r/min, taking supernatant, concentrating, and spray drying to obtain the xylo-oligosaccharide.
2. The method for preparing concentrated protein powder and xylo-oligosaccharide from broussonetia papyrifera leaves as claimed in claim 1, wherein the mixed fermentation of the pressing residue with bacillus subtilis and candida utilis in the step (3) is to prepare the pressing residue, brown sugar, urea and potassium dihydrogen phosphate into a solid fermentation base material according to a mass ratio of 975:10:10: 5; preparing bacillus subtilis solution and candida utilis solutionThe bacterial concentration in the bacillus subtilis bacterial liquid and the candida utilis bacterial liquid is 1010CFU/mL, mixing the bacillus subtilis liquid and the candida utilis liquid according to the volume ratio of 1:1, inoculating the mixture to a solid fermentation base material according to the mass ratio of 3%, and performing mixed fermentation for 1-3 weeks at the temperature of 25-37 ℃ to obtain fermented paper mulberry leaf residues.
3. The method for preparing the concentrated protein powder and the xylo-oligosaccharide by using the broussonetia papyrifera leaves as claimed in claim 1, wherein the control parameters during the concentration and the spray drying in the step (5) are as follows: the vacuum pressure is-0.1-0.15 MPa, the air inlet temperature of spray drying is 180-200 ℃, the air outlet temperature is 70-80 ℃, and the spray feeding flow rate is 580-620 mL/h.
4. Use of a protein concentrate powder and xylo-oligosaccharides produced by the method according to any one of claims 1 to 3 for the preparation of animal feed.
5. The application of claim 3, wherein the application is to add concentrated protein powder and/or xylo-oligosaccharide into the animal complete formula feed, the mass percentage content of the concentrated protein powder in the animal complete formula feed is 1-10%, and the mass percentage content of the xylo-oligosaccharide in the animal complete formula feed is 0.1-5%.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110053224A1 (en) * | 2008-01-25 | 2011-03-03 | Yangming Martin Lo | Novel composition of matter and method for stimulating the growth of beneficial microorganisms |
| CN103652307A (en) * | 2013-12-03 | 2014-03-26 | 山西医科大学 | Method for extracting leaf proteins from broussonetia papyrifera leaf dry powder and comprehensive utilization of waste |
| CN105533160A (en) * | 2015-12-08 | 2016-05-04 | 安徽中科安岳林业科技发展有限公司 | Hybrid paper mulberry tree leaf and shoot fermentation feed and preparation method thereof |
| CN112175846A (en) * | 2020-10-22 | 2021-01-05 | 河南省科学院生物研究所有限责任公司 | Candida utilis strain UCY-11 and application thereof in preparation of fermented hybrid broussonetia papyrifera feed |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110053224A1 (en) * | 2008-01-25 | 2011-03-03 | Yangming Martin Lo | Novel composition of matter and method for stimulating the growth of beneficial microorganisms |
| CN103652307A (en) * | 2013-12-03 | 2014-03-26 | 山西医科大学 | Method for extracting leaf proteins from broussonetia papyrifera leaf dry powder and comprehensive utilization of waste |
| CN105533160A (en) * | 2015-12-08 | 2016-05-04 | 安徽中科安岳林业科技发展有限公司 | Hybrid paper mulberry tree leaf and shoot fermentation feed and preparation method thereof |
| CN112175846A (en) * | 2020-10-22 | 2021-01-05 | 河南省科学院生物研究所有限责任公司 | Candida utilis strain UCY-11 and application thereof in preparation of fermented hybrid broussonetia papyrifera feed |
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