CN103478413A - Method for producing protein feed by mixed-strain solid-state fermentation of ginkgo leaf residues - Google Patents
Method for producing protein feed by mixed-strain solid-state fermentation of ginkgo leaf residues Download PDFInfo
- Publication number
- CN103478413A CN103478413A CN201310427156.3A CN201310427156A CN103478413A CN 103478413 A CN103478413 A CN 103478413A CN 201310427156 A CN201310427156 A CN 201310427156A CN 103478413 A CN103478413 A CN 103478413A
- Authority
- CN
- China
- Prior art keywords
- ginkgo leaf
- state fermentation
- liquid spawn
- inoculated
- housed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention relates to a method for producing protein feed by using ginkgo leaf residues as raw materials through mixed bacteria solid state fermentation. Respectively carrying out activation and amplification culture on candida tropicalis, aspergillus oryzae, bacillus subtilis and lactobacillus plantarum to obtain four liquid strains; drying and crushing ginkgo leaf residues, adding a certain amount of water and a nitrogen source, mixing, and sterilizing to obtain a solid fermentation culture medium; and inoculating the four liquid strains into a solid fermentation culture medium according to a certain proportion, fully and uniformly mixing, fermenting at the temperature of 28-30 ℃ under the natural pH value for 72-120 h, and drying to obtain the ginkgo leaf residue protein feed. The protein feed has high crude protein content up to 46%, comprehensive nutrient components, good palatability, simple preparation method, low cost, and can effectively improve the production performance and feed conversion rate of livestock and poultry, and realize large-scale production. The invention also provides a way for utilizing the waste ginkgo leaf residues in forestry processing.
Description
Technical field
The invention belongs to agriculture and forestry organic waste material process and utilization technology field, particularly relate to and a kind ofly take the ginkgo leaf slag and carry out the method that mixed culture solid state fermentation is produced protein feed as raw material.
Background technology
Fast development along with China's animal husbandry and feed industry, China increases the demand of feedstuff is anxious, especially protein resource is more in short supply to make the feed resource of China, according to statistics by 2012, whole nation protein feed can be 0.22 hundred million ton for amount, demand is 0.66 hundred million ton, and the quantity delivered of visible China protein feed is wretched insufficiency.On the one hand China's protein feed resources lacks, and on the other hand due to the long-term use of feeding antibiotic, existing feed nutritive value is mostly lower and be not easy to be digested and assimilated by animal, causes security, quality and the utilization rate of feed all lower.Therefore actively utilize the agriculture and forestry organic waste material exploitation to there is the plant protein fodder of bioenzyme activity, for the deficiency that makes up protein feed on market and the security and the utilization rate that improve feed, there is very important meaning.
Ginkgo is the distinctive rare tree of China, and China's gingko resource is abundant, accounts for more than 80% of world's total amount.Because ginkgo leaf has various bioactivators, be widely used in medicine, biological pesticide, health food and cosmetic industry.Ginkgo leaf produces a large amount of ginkgo leaf slags after extracting active matter, and annual probably have the ginkgo leaf slag of 30,000 tons of left and right to be thrown away or burn as discarded object, not only causes the serious waste of resource, and pollute the environment.Contain rich in protein and amino acid in the ginkgo leaf slag, content reaches more than 30%, contains 9 kinds of essential amino acids, also contains in addition abundant polysaccharide, VC, VE, carrotene, mineral matter and trace element etc. in the ginkgo leaf slag.Therefore the ginkgo leaf slag of take prepares animal feeding-stuff containing somatic protein as raw material, not only can turn waste into wealth, and makes the ginkgo leaf resource go on the green circulatory development, and be the exploitation injection new vitality of ginkgo leaf resource, has wide future develop.
The bio-transformation of animal feeding-stuff containing somatic protein mostly adopts the single bacterial strain of microorganism or combination strain fermentation, and from current research with should be used for, the effect of mixed culture fermentation is more effective than single strain fermentation.Mixed culture fermentation has utilized the characteristic of cooperation between microbial species, collaborative alternate, between a plurality of bacterial classifications, can compensate mutually its defect, common growth.The method of microorganism fermenting and producing feed mainly comprises two kinds of liquid state fermentation and solid state fermentations, solid state fermentation has advantages of that liquid state fermentation does not have, as few as waste water and dregs, easily process, energy consumption is low, and culture medium raw material mostly is natural substrates or waste residue, and environmental pollution is little, therefore, now mostly adopting the mode of solid state fermentation aspect fermented feed production.
Only have at present both at home and abroad and take ginkgo leaf and prepare the patent report of biological feedstuff as raw material, and the patent report for preparing biological feedstuff about the ginkgo leaf slag seldom.Reported and take ginkgo leaf as raw material as patent 201210549464.9, adopted the single culture bacillus to carry out solid state fermentation and prepare biology feed additive; Patent 200910031310.9 has been reported and take ginkgo leaf as raw material, adopts the single culture candida utili to carry out solid state fermentation and prepares biology feed additive; Patent 200910031310.9 has reported that take ginkgo leaf, waste edible fungus bacterium slag, dregs of beans is raw material, adopts the single culture aspergillus niger to carry out the fermenting and preparing biological feed addictive.The problems such as these patents all adopt single culture to be fermented, and the biological feedstuff prepared exists that protein content is low, nutritional labeling is single, it is poor to digest and assimilate and palatability is poor.The ginkgo leaf slag prepare aspect biological feedstuff only have patent 201010295193.x to report the ginkgo dregs of a decoction, stalk are added to water-stop after direct fermentation, the method is not added any bacterial classification direct fermentation, the fermented feed protein content prepared is low, be of low nutritive value, and the method adopts liquid state fermentation, produce a large amount of waste water, processing is difficult, energy consumption is high, and contaminated environment.
For the deficiency of above invention, the present invention proposes take the ginkgo leaf slag and produces protein feed as raw material adopts the method for mixed culture solid state fermentation.The method adopts fermented by mixed bacterium, and in the ginkgo leaf residue protein feed prepared, not only protein content is high, and, containing multivitamin, various active enzyme, several amino acids and various trace elements, nutritional labeling is comprehensive, and feed is digested and assimilated, good palatability; Adopt the mode of production of solid state fermentation, waste water and dregs is few, easily processes, and energy consumption is low, and environmental pollution is little.This invention utilizes this huge resource of ginkgo leaf slag, and fermentation prepares the protein feed of comprehensive nutrition, not only can turn waste into wealth, and make ginkgo leaf go on the road of green development capable of circulation, and the method will provide technical support for the Application and Development of ginkgo leaf slag.
Summary of the invention
The object of the present invention is to provide and a kind ofly take the ginkgo leaf slag and carry out the method that mixed culture solid state fermentation is produced protein feed as raw material, the method energy consumption is low, environmental pollution is little, and green safety is simple to operate, production cost is low, and the ginkgo leaf residue protein forage protein content prepared is high, and nutritional labeling is comprehensive, and feed is digested and assimilated, good palatability, be applicable to suitability for industrialized production.
The present invention is achieved by the following technical solutions.
A kind of mixed culture solid state fermentation ginkgo leaf slag is produced the method for protein feed, and concrete steps are as follows:
(1) bacterial classification is cultivated
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that the 5mL malt extract medium is housed after the candida tropicalis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mL malt extract medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the candida tropicalis liquid spawn.
B. the amplification cultivation of aspergillus oryzae: will be inoculated in the Boiling tube that 5mLCzapek ' s fluid nutrient medium is housed after the aspergillus oryzae activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that the 5mLBPY fluid nutrient medium is housed after the bacillus subtilis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLBPY fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that the 5mLMRS fluid nutrient medium is housed after the Lactobacillus plantarum activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLMRS fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the Lactobacillus plantarum liquid spawn.
(2) prepare solid-state fermentation culture medium
After the ginkgo leaf slag is carried out to drying, pulverizing, the water that adds 2～4 times of ginkgo leaf slag weight, then add the nitrogenous source of ginkgo leaf slag weight 5～12%, after mixing, at 120～125 ℃ of lower steam sterilizing 20～30min, obtain ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation prepares protein feed
Above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, bacillus subtilis liquid spawn and Lactobacillus plantarum liquid spawn are accessed respectively in ginkgo leaf slag solid-state fermentation culture medium in certain bacterial classification ratio, the inoculation total amount of four kinds of liquid spawns is 8～12% of solid-state fermentation culture medium weight, after inoculation, fully mix, then under 28～30 ℃ of conditions of temperature, static fermentation 72～120h under the nature pH value, finally tunning is dried to constant weight under 50 ℃ of conditions, obtain ginkgo leaf residue protein feed.
Described ginkgo leaf slag be by 3～6 years living ginkgo spires the leaf slag after extracting GINKGO BILOBA EXTRACT carry out air-dry after gained.
Described nitrogenous source kind is urea or ammonium sulfate.
The access bacterial classification ratio of described Candida liquid spawn, aspergillus oryzae liquid spawn, bacillus subtilis liquid spawn and Lactobacillus plantarum liquid spawn is 1～2:2:1～2:1.
In the ginkgo leaf residue protein feed that described solid state fermentation prepares, crude protein content is 45～48%, and lactic acid content is 2.0～2.5%, and crude fiber content is 6～8%.
The fermented bacterium that the present invention adopts is the composite bacteria that candida tropicalis, aspergillus oryzae, bacillus subtilis and four kinds of bacterial classifications of Lactobacillus plantarum form.Respectively four kinds of bacterial classifications have been carried out to amplification cultivation and prepared liquid spawn, concrete preparation method is as follows:
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that the 5mL malt extract medium is housed after the candida tropicalis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mL malt extract medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the candida tropicalis liquid spawn.
B. the amplification cultivation of aspergillus oryzae: will be inoculated in the Boiling tube that 5mLCzapek ' s fluid nutrient medium is housed after the aspergillus oryzae activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that the 5mLBPY fluid nutrient medium is housed after the bacillus subtilis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLBPY fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that the 5mLMRS fluid nutrient medium is housed after the Lactobacillus plantarum activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLMRS fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the Lactobacillus plantarum liquid spawn.
The present invention investigates fermentation bacteria used thereby kind kind, has carried out respectively single culture fermentation test and composite bacteria fermentation test.The impact on the tunning crude protein content of candida tropicalis, candida utili, geotrichum candidum, aspergillus oryzae, aspergillus niger, Trichoderma viride, bacillus subtilis and Lactobacillus plantarum different strain has been investigated respectively in the single culture fermentation test; Higher saccharomycete, mould and Lactobacillus plantarum, the bacillus subtilis of crude protein content filtered out in single bacterium fermentation test carried out to the composite fermentation test of 2 bacterium, 3 bacterium and 4 bacterium, measure crude protein content in the composite fermentation product.Result shows, 4 kinds of bacterial classifications that in the single culture tunning, crude protein content is the highest are candida tropicalis, aspergillus oryzae, bacillus subtilis and Lactobacillus plantarum; The crude protein content of composite bacteria tunning will be higher than the tunning of single culture, the four bacterium combined fermentation product crude protein contents that strain combination is candida tropicalis+aspergillus oryzae+bacillus subtilis+Lactobacillus plantarum are the highest, therefore, the fermented bacterium that the present invention adopts is the composite bacteria that candida tropicalis, aspergillus oryzae, bacillus subtilis and four kinds of bacterial classifications of Lactobacillus plantarum form.
The present invention be take the ginkgo leaf slag as raw material, by adding a certain amount of water and nitrogenous source, prepares solid-state fermentation culture medium.After the ginkgo leaf slag is carried out to drying, pulverizing, the water that adds 2～4 times of ginkgo leaf slag weight, then the nitrogenous source that adds ginkgo leaf slag weight 5～12%, after mixing, at 120～125 ℃ of lower steam sterilizing 20～30min, obtain ginkgo leaf slag solid-state fermentation culture medium after cooling, described nitrogenous source is urea or ammonium sulfate.
Moisture is microorganism one of the necessary base substance that ferments, every kind of microorganism all has the moisture of its growth of optimum, this is because water is the important component part of microbial cell, is the medium that microorganism carries out metabolic activity, also participates in a part of biochemical reaction simultaneously.
The present invention investigates the material-water ratio of solid-state fermentation culture medium, material-water ratio is got respectively 1:1,1:2,1:3,1:4,1:5 carries out mixed culture solid state fermentation, measure the crude protein content of tunning, result shows that working as material-water ratio is in 1:2～1:4 scope, crude protein content in tunning all can reach more than 45%, when material-water ratio is 1; 5 o'clock, in tunning, crude protein content was on a declining curve.When wherein material-water ratio is 1:3, the crude protein content in tunning reaches and is up to 48.13%.Therefore preferably material-water ratio is 1:3.
Its nitrogen content of ginkgo leaf slag is lower, and component mostly is insoluble carbohydrate, restriction due to the fermentation substrate self reason, if do not add certain nitrogenous source in test and actual production, be difficult to meet the needs of growth of microorganism, also can't reach the requirement of producing protein feed, so need to add a certain amount of nitrogenous source in solid-state fermentation culture medium simultaneously.
The present invention investigates nitrogenous source kind and addition, choose respectively urea and ammonium sulfate as culture media nitrogen source, the nitrogenous source addition is respectively 3%, 5%, 8%, 10%, 12%, measure the crude protein content of product after mixed culture solid state fermentation, result shows, urea to the amplification of tunning crude protein content higher than ammonium sulfate.When the urea addition is 8%, in tunning, crude protein content reaches the highest.Therefore the preferred nitrogen source category is urea, and the nitrogenous source addition is 8%.
The present invention adopts mixed culture solid state fermentation to prepare ginkgo leaf residue protein feed.Candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, bacillus subtilis liquid spawn and Lactobacillus plantarum liquid spawn are accessed respectively in ginkgo leaf slag solid-state fermentation culture medium in certain bacterial classification ratio, the inoculation total amount of four kinds of liquid spawns is solid-state fermentation culture medium 8～12%, after inoculation, fully mix, then under 28～30 ℃ of conditions of temperature, static fermentation 72～120h under the nature pH value, finally tunning is dried to constant weight under 50 ℃ of conditions, obtain ginkgo leaf residue protein feed.The access bacterial classification ratio of described candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, bacillus subtilis liquid spawn and Lactobacillus plantarum liquid spawn is 1～2:2:1～2:1.
The present invention investigates the composite bacteria ratio, select inoculative proportion to be respectively 1: 1: 2:1,2: 2: 1:1,1: 2: 2:1,2: 1: 1:1,2: 1: 2:1,1: 2: 1:1, measure the crude protein content of tunning, result shows, when the aspergillus oryzae ratio is 2, the crude protein content of tunning all can reach more than 45%, when wherein the inoculative proportion of candida tropicalis, aspergillus oryzae, bacillus subtilis and Lactobacillus plantarum is 2:2:1:1, in tunning, crude protein content is the highest, reaches 47.85%.Therefore, the best inoculative proportion of preferred candida tropicalis, aspergillus oryzae, bacillus subtilis and Lactobacillus plantarum is 2: 2: 1:1.
The present invention investigates the inoculum concentration of composite bacteria, presses respectively 6%, 8%, 10%, 12%, 15% inoculum concentration access solid fermentation culture medium.Result shows that working as inoculum concentration is in 8～12% scopes, and the crude protein content in tunning all can reach more than 45%, and when inoculum concentration is 15%, in tunning, crude protein content is on a declining curve.When wherein inoculum concentration is 10%, in tunning, crude protein content is the highest, is 48.38%, and therefore preferably inoculum concentration is 10%.
The present invention investigated the solid state fermentation time, select fermentation time to be respectively 24h, 48h, 72h, 96h, 120h, result shows that fermentation time is in 72～120h scope, crude protein content in tunning all can reach more than 45%, when fermentation time is 72h, in tunning, crude protein content reaches and is up to 47.39%, continues to increase fermentation time, protein content held stationary trend in tunning.。Therefore preferably fermentation time is 72h.
The present invention is analyzed the ginkgo leaf residue protein feed index components of preparation, measures respectively its crude protein, lactic acid and crude fiber content, and compares with raw material.Result shows that in ginkgo leaf residue protein feed, crude protein content is 45～48%, and lactic acid content is 2.0～2.5%, and crude fiber content is 6～8%.With raw material, compare, the crude protein content of protein feed has had and has significantly improved, and crude fiber content has had obvious reduction, has increased in addition lactic component in tunning and can strengthen humoral immunity and the cellular immunity of animal, can also improve in addition the palatability of feed.
The present invention has carried out growth pig feeding experiment to ginkgo leaf residue protein feed, and the ginkgo leaf residue protein feed of preparation is substituted to the rapeseed dregs in basic diet fully, for feeding of the pig that grows, checks its impact on growth pig production performance and feed digestibility.
Result shows, with control group (basic diet interpolation rapeseed dregs), compare, the average daily gain of the growth pig of test group (basic diet adds ginkgo leaf residue protein feed) has improved 23.61%, the material anharmonic ratio has reduced by 18.44%, the CP digestibility of feed has improved 15.38%, and the dry digestibility has improved 10.45%.Illustrate that the ginkgo leaf residue protein Feed Energy of the gained of the present invention of feeding effectively improves production performance and the feed conversion rate of growth pig, it can substitute the rapeseed dregs in basic diet fully, for feeding of the pig that grows.
Beneficial effect of the present invention:
(1) to take forest resourceies processing waste ginkgo leaf slag be raw material in the present invention, adopts the method for bio-transformation to prepare protein feed, not only can turn waste into wealth, and can be the high-quality protein feed that China's animal husbandry provides safely, is of high nutritive value.
(2) the present invention adopts mixed bacteria to be fermented, with traditional single culture fermentation, compare, in the ginkgo leaf residue protein feed prepared, not only protein content is high, and containing multivitamin, various active enzyme, several amino acids and various trace elements, nutritional labeling is comprehensive, good palatability.
(3) the present invention adopts the method for solid state fermentation, and waste water and dregs is few, easily processes, and energy consumption is low, and environmental pollution is little, and technological operation is simple, and production cost is low, safe, is applicable to suitability for industrialized production.
(4) the ginkgo leaf residue protein Feed Energy that prepared by the present invention effectively improves production performance and the feed conversion rate of livestock and poultry, has good market prospects.
The accompanying drawing explanation
Fig. 1 ginkgo leaf slag mixed culture solid state fermentation prepares the process chart of protein feed.
The specific embodiment
The present invention is described in further detail for following examples, and the present invention is not limited.
Embodiment 1
Bacterial classification is cultivated
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that the 5mL malt extract medium is housed after the candida tropicalis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mL malt extract medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the candida tropicalis liquid spawn.
B. the amplification cultivation of aspergillus oryzae: will be inoculated in the Boiling tube that 5mLCzapek ' s fluid nutrient medium is housed after the aspergillus oryzae activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that the 5mLBPY fluid nutrient medium is housed after the bacillus subtilis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLBPY fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that the 5mLMRS fluid nutrient medium is housed after the Lactobacillus plantarum activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLMRS fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the Lactobacillus plantarum liquid spawn.
The present invention investigates fermentation bacteria used thereby kind kind, has carried out respectively single culture fermentation test and composite bacteria fermentation test.By the ginkgo leaf slag solid-state fermentation culture medium sterilizing that mixes, cooling after, access respectively 10% candida tropicalis, candida utili, geotrichum candidum, aspergillus oryzae, aspergillus niger, Trichoderma viride, bacillus subtilis and Lactobacillus plantarum, at 30 ℃, after the lower static fermentation 72h of nature pH value, measure crude protein content in tunning, the results are shown in Table 1; Higher saccharomycete, mould and Lactobacillus plantarum, the bacillus subtilis of crude protein content filtered out in single bacterium fermentation test carried out to 2 bacterium (inoculative proportion 1: 1), 3 bacterium (inoculative proportion 1: 1: 1) and 4 bacterium (inoculative proportion 1: 1: 1: composite fermentation test l), measure crude protein content in the composite fermentation product, the results are shown in Table 2.
The impact of the different single bacterium of table 1 on the tunning crude protein content
| Bacterial classification | Crude protein content (%) |
| Blank | 9.23 |
| Aspergillus niger | 21.67 |
| Aspergillus oryzae | 32.55 |
| Trichoderma viride | 10.41 |
| Geotrichum candidum | 13.29 |
| Lactobacillus plantarum | 27.97 |
| Candida tropicalis | 28.22 |
| Candida utili | 16.05 |
| Bacillus subtilis | 29.01 |
The impact of table 2 different composite strain combination on the tunning crude protein content
| Strain combination | Crude protein content (%) |
| Aspergillus oryzae+candida tropicalis | 34.86 |
| Aspergillus oryzae+bacillus subtilis | 32.29 |
| Aspergillus oryzae+Lactobacillus plantarum | 32.67 |
| Aspergillus oryzae+candida tropicalis+bacillus subtilis | 40.57 |
| Aspergillus oryzae+candida tropicalis+Lactobacillus plantarum | 38.25 |
| Aspergillus oryzae+bacillus subtilis+Lactobacillus plantarum | 35.91 |
| Aspergillus oryzae+candida tropicalis+bacillus subtilis+Lactobacillus plantarum | 45.26 |
As can be seen from Table 1,4 kinds of bacterial classifications that in the single culture tunning, crude protein content is the highest are candida tropicalis (28.22%), aspergillus oryzae (32.55), bacillus subtilis (29.01%) and Lactobacillus plantarum (27.97%); As can be seen from Table 2, the four bacterium combined fermentation product crude protein contents that strain combination is candida tropicalis+aspergillus oryzae+bacillus subtilis+Lactobacillus plantarum are the highest, therefore, the fermented bacterium that the present invention adopts is the composite bacteria that candida tropicalis, aspergillus oryzae, bacillus subtilis and four kinds of bacterial classifications of Lactobacillus plantarum form.
Embodiment 2
The preparation of solid-state fermentation culture medium
After the ginkgo leaf slag is carried out to drying, pulverizing, the water that adds 2～4 times of ginkgo leaf slag weight, then add the nitrogenous source of ginkgo leaf slag weight 5～12%, after mixing, at 120～125 ℃ of lower steam sterilizing 20～30min, obtain ginkgo leaf slag solid-state fermentation culture medium after cooling.
Described ginkgo leaf slag be by 3～6 years living ginkgo spires the leaf slag after extracting GINKGO BILOBA EXTRACT carry out air-dry after gained.
Described nitrogenous source is urea or ammonium sulfate.
Moisture is microorganism one of the necessary base substance that ferments, every kind of microorganism all has the moisture of its growth of optimum, this is because water is the important component part of microbial cell, is the medium that microorganism carries out metabolic activity, also participates in a part of biochemical reaction simultaneously.
The present invention investigates the material-water ratio of solid-state fermentation culture medium, and material-water ratio is got respectively 1:1,1:2,1:3,1:4,1:5 carries out mixed culture solid state fermentation, measures the crude protein content of tunning, the results are shown in Table 3.As can be seen from Table 3, in material-water ratio is 1:2～1:4 scope, the crude protein content in tunning all can reach more than 45%, when material-water ratio is 1; 5 o'clock, in tunning, crude protein content was on a declining curve.When wherein material-water ratio is 1:3, the crude protein content in tunning reaches and is up to 48.13%.Therefore preferably material-water ratio is 1:3.
The impact of the different material-water ratios of table 3 on the tunning crude protein content
| Material-water ratio | Crude protein content (%) |
| 1:1 | 39.14 |
| 1:2 | 45.48 |
| 1:3 | 48.13 |
| 1:4 | 45.14 |
| 1:5 | 35.46 |
Its nitrogen content of ginkgo leaf slag is lower, and component mostly is insoluble carbohydrate, restriction due to the fermentation substrate self reason, if do not add certain nitrogenous source in test and actual production, be difficult to meet the needs of growth of microorganism, also can't reach the requirement of producing protein feed, so need to add a certain amount of nitrogenous source in solid-state fermentation culture medium simultaneously.
The present invention investigates nitrogenous source kind and addition, chooses respectively urea and ammonium sulfate as culture media nitrogen source, and the nitrogenous source addition is respectively 3%, 5%, 8%, 10%, 12%, measures the crude protein content of product after mixed culture solid state fermentation, the results are shown in Table 4.As can be seen from Table 4, urea to the amplification of tunning crude protein content higher than ammonium sulfate.When the urea addition is 8%, in tunning, crude protein content reaches the highest.Therefore the preferred nitrogen source category is urea, and the nitrogenous source addition is 8%.
The impact on the tunning crude protein of table 4 different nitrogen sources and addition
Embodiment 3
Solid state fermentation prepares protein feed
Candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, bacillus subtilis liquid spawn and Lactobacillus plantarum liquid spawn are accessed respectively in ginkgo leaf slag solid-state fermentation culture medium in certain bacterial classification ratio, the inoculation total amount of four kinds of liquid spawns is solid-state fermentation culture medium 8～12%, after inoculation, fully mix, then under 28～30 ℃ of conditions of temperature, static fermentation 72～120h under the nature pH value, finally tunning is dried to constant weight under 50 ℃ of conditions, obtain ginkgo leaf residue protein feed.
The access bacterial classification ratio of described candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, bacillus subtilis liquid spawn and Lactobacillus plantarum liquid spawn is 1～2:2:1～2:1.
The present invention investigates the composite bacteria ratio, access respectively the bacterium liquid of saccharomycete, mould, bacillus subtilis and the Lactobacillus plantarum of different proportion by 10% inoculum concentration in the solid fermentation culture medium, carry out fermentation test, inoculative proportion is respectively 1: 1: 2:1,2: 2: 1:1,1: 2: 2:1,2: 1: 1:1,2: 1: 2:1,1: 2: 1:1, measure the crude protein content of tunning, the results are shown in Table 5.As shown in Table 5, when the aspergillus oryzae ratio is 2, the crude protein content of tunning all can reach more than 45%, when wherein the inoculative proportion of candida tropicalis, aspergillus oryzae, bacillus subtilis and Lactobacillus plantarum is 2:2:1:1, in tunning, crude protein content is the highest, reaches 47.85%.Therefore, the best inoculative proportion of preferred candida tropicalis, aspergillus oryzae, bacillus subtilis and Lactobacillus plantarum is 2: 2: 1:1.
The impact of table 5 different strain ratio on the tunning crude protein content
| The bacterial classification ratio | Crude protein content (%) |
| 1∶1∶2：1 | 40.59 |
| 2∶2∶1：1 | 47.85 |
| 1∶2∶2：1 | 46.03 |
| 2∶1∶1：1 | 44.02 |
| 2∶1∶2：1 | 43.23 |
| 1∶2∶1：1 | 45.01 |
The present invention investigates the inoculum concentration of composite bacteria, by the composite bacteria that filters out respectively by 6%, 8%, 10%, 12%, 15% inoculum concentration access solid fermentation culture medium, sterilizing, after cooling, at 30 ℃, after the lower static fermentation 72h of nature pH value, the tunning crude protein content is respectively 34.59%, 45.08%, 48.38%, 47.29%, 37.21%.Result shows that working as inoculum concentration is in 8～12% scopes, and the crude protein content in tunning all can reach more than 45%, and when inoculum concentration is 15%, in tunning, crude protein content is on a declining curve.When wherein inoculum concentration is 10%, in tunning, crude protein content is the highest, is 48.38%, and therefore preferably inoculum concentration is 10%.
The present invention investigated the solid state fermentation time, by the composite bacteria that filters out respectively by 10% inoculum concentration access solid fermentation culture medium, sterilizing, after cooling, at 30 ℃, under nature pH value, the static fermentation 24h of difference, 48h, 72h, 96h, 120h, the tunning crude protein content is respectively 25.34%, 30.28%, 47.39%, 47.03%, 46.28%.Result shows that fermentation time is in 72～120h scope, crude protein content in tunning all can reach more than 45%, and when fermentation time is 72h, in tunning, crude protein content reaches and is up to 47.39%, continue to increase fermentation time, protein content held stationary trend in tunning.Therefore preferably fermentation time is 72h.
Embodiment 4
Ginkgo leaf residue protein feed index components is analyzed
The present invention measures respectively crude protein, lactic acid and crude fiber content in the ginkgo leaf residue protein feed prepared, and compares with raw material, the results are shown in Table 6.Result shows that in ginkgo leaf residue protein feed, crude protein content is 45～48%, and lactic acid content is 2.0～2.5%, and crude fiber content is 6～8%.With raw material, compare, the crude protein content of protein feed has had and has significantly improved, and crude fiber content has had obvious reduction, has increased in addition lactic component in tunning and can strengthen humoral immunity and the cellular immunity of animal, can also improve in addition the palatability of feed.
The constituent analysis of table 6 tunning
| Fermentate | Crude protein (%) | Lactic acid content (%) | Crude fibre (%) |
| Fermentation raw material | 8.41 | 0 | 17.24 |
| Tunning | 45～48 | 2.0～2.5 | 6～8 |
Embodiment 5
Ginkgo leaf residue protein feed is to the test of growth pig feeding effect
The present invention substitutes the rapeseed dregs in basic diet fully by the ginkgo leaf residue protein feed of preparation, for feeding of the pig that grows, checks its impact on growth pig production performance and feed digestibility.
Choose and grow normally, age in days is close, 24 of the growth pigs of body weight 25kg left and right, and by the feeding experiment requirement, and by the weight of initial pig, identical or phase approximately principle is divided into 2 groups at random, establishes 2 repetitions for every group, and each repeats 6 (male and female half and half).One group is control group (basic diet adds 8% rapeseed dregs), and another group is test group (basic diet adds 8% ginkgo leaf residue protein feed).The formal test phase is 28d altogether, raises all management and immunity routinely of rules, and weigh the morning that prerun starts day on an empty stomach, just trying in the phase, when every two weeks and off-test, stop hello 24h after morning weigh on an empty stomach.Feed intake is observed, recorded to duration of test every day, calculates average feed intake, daily gain and the material anharmonic ratio of test pig, the results are shown in Table 7.In addition experimental period within last 4 days, carry out digestion trial, utilize indicator method, collect swine excrement, proportion sample is preserved, and measures crude protein and indicator content, calculates the apparent digestibility of feed, the results are shown in Table 8.
Result shows, with control group, compares, and the average daily gain of the growth pig of test group has improved 23.61%, and the material anharmonic ratio has reduced by 18.44%, and the CP digestibility of feed has improved 15.38%, and the dry digestibility has improved 10.45%.Illustrate that the ginkgo leaf residue protein Feed Energy of the gained of the present invention of feeding effectively improves production performance and the feed conversion rate of growth pig, it can substitute the rapeseed dregs in basic diet fully, for feeding of the pig that grows.
The impact of table 7 ginkgo leaf residue protein feed on growth pig production performance
The impact of table 8 ginkgo leaf residue protein feed on the feed nutrient apparent digestibility
| Project | The CP digestibility | The dry digestibility |
| Control group (basic diet adds 8% rapeseed dregs) | 74.82 | 80.84 |
| Test group (basic diet adds 8% ginkgo leaf residue protein feed) | 86.33 | 89.29 |
Embodiment 6:
The preparation of ginkgo leaf residue protein feed
(1) bacterial classification is cultivated
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that the 5mL malt extract medium is housed after the candida tropicalis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mL malt extract medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the candida tropicalis liquid spawn.
B. the amplification cultivation of aspergillus oryzae: will be inoculated in the Boiling tube that 5mLCzapek ' s fluid nutrient medium is housed after the aspergillus oryzae activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that the 5mLBPY fluid nutrient medium is housed after the bacillus subtilis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLBPY fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that the 5mLMRS fluid nutrient medium is housed after the Lactobacillus plantarum activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLMRS fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the Lactobacillus plantarum liquid spawn.
(2) prepare solid-state fermentation culture medium
Get ginkgo leaf slag 1kg, dry, pulverize after, add the water of 2000mL, then add the urea of 80g, after mixing, at 120 ℃ of lower steam sterilizing 30min, obtain ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation prepares protein feed
Ratio by above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn in 2:2:1:1 accesses respectively in ginkgo leaf slag solid-state fermentation culture medium, the inoculation total amount of four kinds of liquid spawns is 12% of solid-state fermentation culture medium weight, after inoculation, fully mix, then under 28 ℃ of conditions of temperature, static fermentation 72h under the nature pH value, finally tunning is dried to constant weight under 50 ℃ of conditions, obtain ginkgo leaf residue protein feed.Its crude protein content is 46.78% after measured, and crude fiber content is 6.06%, and lactic acid content is 2.45%.
Embodiment 7:
The preparation of ginkgo leaf residue protein feed
(1) bacterial classification is cultivated
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that the 5mL malt extract medium is housed after the candida tropicalis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mL malt extract medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the candida tropicalis liquid spawn.
B. the amplification cultivation of aspergillus oryzae: will be inoculated in the Boiling tube that 5mLCzapek ' s fluid nutrient medium is housed after the aspergillus oryzae activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that the 5mLBPY fluid nutrient medium is housed after the bacillus subtilis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLBPY fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that the 5mLMRS fluid nutrient medium is housed after the Lactobacillus plantarum activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLMRS fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the Lactobacillus plantarum liquid spawn.
(2) prepare solid-state fermentation culture medium
Get ginkgo leaf slag 1kg, dry, pulverize after, add the water of 3500mL, then add the urea of 50g, after mixing, at 125 ℃ of lower steam sterilizing 30min, obtain ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation prepares protein feed
Ratio by above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn in 1:2:2:1 accesses respectively in ginkgo leaf slag solid-state fermentation culture medium, the inoculation total amount of four kinds of liquid spawns is 10% of solid-state fermentation culture medium weight, after inoculation, fully mix, then under 28 ℃ of conditions of temperature, static fermentation 86h under the nature pH value, finally tunning is dried to constant weight under 50 ℃ of conditions, obtain ginkgo leaf residue protein feed.Its crude protein content is 48.24% after measured, and crude fiber content is 7.05%, and lactic acid content is 2.51%.
Embodiment 8:
The preparation of ginkgo leaf residue protein feed
(1) bacterial classification is cultivated
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that the 5mL malt extract medium is housed after the candida tropicalis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mL malt extract medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the candida tropicalis liquid spawn.
B. the amplification cultivation of aspergillus oryzae: will be inoculated in the Boiling tube that 5mLCzapek ' s fluid nutrient medium is housed after the aspergillus oryzae activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that the 5mLBPY fluid nutrient medium is housed after the bacillus subtilis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLBPY fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that the 5mLMRS fluid nutrient medium is housed after the Lactobacillus plantarum activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLMRS fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the Lactobacillus plantarum liquid spawn.
(2) prepare solid-state fermentation culture medium
Get ginkgo leaf slag 1kg, dry, pulverize after, add the water of 4000mL, then add the ammonium sulfate of 100g, after mixing, at 125 ℃ of lower steam sterilizing 30min, obtain ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation prepares protein feed
Ratio by above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn in 1:2:1:1 accesses respectively in ginkgo leaf slag solid-state fermentation culture medium, the inoculation total amount of four kinds of liquid spawns is 9% of solid-state fermentation culture medium weight, after inoculation, fully mix, then under 28 ℃ of conditions of temperature, static fermentation 110h under the nature pH value, finally tunning is dried to constant weight under 50 ℃ of conditions, obtain ginkgo leaf residue protein feed.Its crude protein content is 45.92% after measured, and crude fiber content is 8.02%, and lactic acid content is 2.02%.
Embodiment 9:
The preparation of ginkgo leaf residue protein feed
(1) bacterial classification is cultivated
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that the 5mL malt extract medium is housed after the candida tropicalis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mL malt extract medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the candida tropicalis liquid spawn.
B. the amplification cultivation of aspergillus oryzae: will be inoculated in the Boiling tube that 5mLCzapek ' s fluid nutrient medium is housed after the aspergillus oryzae activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that the 5mLBPY fluid nutrient medium is housed after the bacillus subtilis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLBPY fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that the 5mLMRS fluid nutrient medium is housed after the Lactobacillus plantarum activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLMRS fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the Lactobacillus plantarum liquid spawn.
(2) prepare solid-state fermentation culture medium
Get ginkgo leaf slag 1kg, dry, pulverize after, add the water of 3200mL, then add the urea of 90g, after mixing, at 120 ℃ of lower steam sterilizing 30min, obtain ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation prepares protein feed
Ratio by above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn in 2:2:1:1 accesses respectively in ginkgo leaf slag solid-state fermentation culture medium, the inoculation total amount of four kinds of liquid spawns is 10% of solid-state fermentation culture medium weight, after inoculation, fully mix, then under 28 ℃ of conditions of temperature, static fermentation 96h under the nature pH value, finally tunning is dried to constant weight under 50 ℃ of conditions, obtain ginkgo leaf residue protein feed.Its crude protein content is 47.08% after measured, and crude fiber content is 7.76%, and lactic acid content is 2.12%.
Embodiment 10:
The preparation of ginkgo leaf residue protein feed
(1) bacterial classification is cultivated
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that the 5mL malt extract medium is housed after the candida tropicalis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mL malt extract medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the candida tropicalis liquid spawn.
B. the amplification cultivation of aspergillus oryzae: will be inoculated in the Boiling tube that 5mLCzapek ' s fluid nutrient medium is housed after the aspergillus oryzae activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that the 5mLBPY fluid nutrient medium is housed after the bacillus subtilis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLBPY fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that the 5mLMRS fluid nutrient medium is housed after the Lactobacillus plantarum activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLMRS fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the Lactobacillus plantarum liquid spawn.
(2) prepare solid-state fermentation culture medium
Get ginkgo leaf slag 1kg, dry, pulverize after, add the water of 3600mL, then add the ammonium sulfate of 120g, after mixing, at 125 ℃ of lower steam sterilizing 30min, obtain ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation prepares protein feed
Ratio by above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn in 1:2:2:1 accesses respectively in ginkgo leaf slag solid-state fermentation culture medium, the inoculation total amount of four kinds of liquid spawns is 8% of solid-state fermentation culture medium weight, after inoculation, fully mix, then under 28 ℃ of conditions of temperature, static fermentation 120h under the nature pH value, finally tunning is dried to constant weight under 50 ℃ of conditions, obtain ginkgo leaf residue protein feed.Its crude protein content is 46.18% after measured, and crude fiber content is 7.46%, and lactic acid content is 2.35%.
Claims (5)
1. a mixed culture solid state fermentation ginkgo leaf slag is produced the method for protein feed, it is characterized in that, take the ginkgo leaf slag as raw material, adopt candida tropicalis, aspergillus oryzae, bacillus subtilis and Lactobacillus plantarum to carry out mixed culture solid state fermentation to the ginkgo leaf slag, comprise the following steps:
Bacterial classification is cultivated
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that the 5mL malt extract medium is housed after the candida tropicalis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mL malt extract medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the candida tropicalis liquid spawn;
B. the amplification cultivation of aspergillus oryzae: will be inoculated in the Boiling tube that 5mLCzapek ' s fluid nutrient medium is housed after the aspergillus oryzae activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the aspergillus oryzae liquid spawn;
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that the 5mLBPY fluid nutrient medium is housed after the bacillus subtilis activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLBPY fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the bacillus subtilis liquid spawn;
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that the 5mLMRS fluid nutrient medium is housed after the Lactobacillus plantarum activation, cultivate 24h for 28 ℃, then with 5% inoculum concentration, be inoculated in the triangular flask that the 500mLMRS fluid nutrient medium is housed, 28 ℃ of constant-temperature tables (180r/min) are cultivated 36h, obtain the Lactobacillus plantarum liquid spawn;
(2) prepare solid-state fermentation culture medium
After the ginkgo leaf slag is carried out to drying, pulverizing, the water that adds 2～4 times of ginkgo leaf slag weight, then add the nitrogenous source of ginkgo leaf slag weight 5～12%, after mixing, at 120～125 ℃ of lower steam sterilizing 20～30min, obtain ginkgo leaf slag solid-state fermentation culture medium after cooling;
(3) mixed culture solid state fermentation prepares protein feed
Above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, bacillus subtilis liquid spawn and Lactobacillus plantarum liquid spawn are accessed respectively in ginkgo leaf slag solid-state fermentation culture medium in certain bacterial classification ratio, the inoculation total amount of four kinds of liquid spawns is 8～12% of solid-state fermentation culture medium weight, after inoculation, fully mix, then under 28～30 ℃ of conditions of temperature, static fermentation 72～120h under the nature pH value, finally tunning is dried to constant weight under 50 ℃ of conditions, obtain ginkgo leaf residue protein feed.
2. a kind of mixed culture solid state fermentation ginkgo leaf slag as claimed in claim 1 is produced the method for protein feed, it is characterized in that: described ginkgo leaf slag be by the leaf slag of 3～6 years livings ginkgo spires after the extraction GINKGO BILOBA EXTRACT carry out air-dry after gained.
3. the method that a kind of mixed culture solid state fermentation ginkgo leaf slag as claimed in claim 1 is produced protein feed, it is characterized in that: the nitrogenous source described in step (2) is urea or ammonium sulfate.
4. the method that a kind of mixed culture solid state fermentation ginkgo leaf slag as claimed in claim 1 is produced protein feed, it is characterized in that: the access bacterial classification ratio of the candida tropicalis liquid spawn described in step (3), aspergillus oryzae liquid spawn, bacillus subtilis liquid spawn and Lactobacillus plantarum liquid spawn is 1～2:2:1～2:1.
5. the method that a kind of mixed culture solid state fermentation ginkgo leaf slag as claimed in claim 3 is produced protein feed, it is characterized in that: in the ginkgo leaf residue protein feed that described solid state fermentation prepares, crude protein content is 45～48%, lactic acid content is 2.0～2.5%, and crude fiber content is 6～8%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310427156.3A CN103478413B (en) | 2013-09-18 | 2013-09-18 | Method for producing protein feed by mixed-strain solid-state fermentation of ginkgo leaf residues |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310427156.3A CN103478413B (en) | 2013-09-18 | 2013-09-18 | Method for producing protein feed by mixed-strain solid-state fermentation of ginkgo leaf residues |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103478413A true CN103478413A (en) | 2014-01-01 |
| CN103478413B CN103478413B (en) | 2014-10-29 |
Family
ID=49819276
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310427156.3A Active CN103478413B (en) | 2013-09-18 | 2013-09-18 | Method for producing protein feed by mixed-strain solid-state fermentation of ginkgo leaf residues |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103478413B (en) |
Cited By (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104286593A (en) * | 2014-11-08 | 2015-01-21 | 宁波市江东农茹生物科技有限公司 | Special feed additive for silver carp culture |
| CN104472856A (en) * | 2014-12-29 | 2015-04-01 | 江苏千药堂国医研究院有限公司 | Method for preparing protein feed by utilizing waste liquid fermentation in production of ginkgo leaf extracts |
| CN104472862A (en) * | 2014-12-29 | 2015-04-01 | 江苏千药堂国医研究院有限公司 | Biological feed additive and preparation method thereof |
| CN104472868A (en) * | 2014-12-29 | 2015-04-01 | 江苏千药堂国医研究院有限公司 | Processing method of biologic feed additive |
| CN104489243A (en) * | 2014-12-29 | 2015-04-08 | 邳州市东方养殖有限公司 | Method for preparing biofeed by using rabbit dung |
| CN104489242A (en) * | 2014-12-29 | 2015-04-08 | 邳州市东方养殖有限公司 | Processing method of biologically fermented rabbit dung feed |
| CN104489281A (en) * | 2014-12-29 | 2015-04-08 | 江苏千药堂国医研究院有限公司 | Method for preparing single-cell protein feed additive by utilizing ginkgo leaf processing wastes |
| CN104543391A (en) * | 2014-12-29 | 2015-04-29 | 江苏千药堂国医研究院有限公司 | Preparation method of liquid feed additive |
| CN104560805A (en) * | 2014-12-29 | 2015-04-29 | 江苏千药堂国医研究院有限公司 | Method for preparing cell protein through fermentation of liquid waste in ginkgo leaf extract production |
| CN105211555A (en) * | 2015-10-22 | 2016-01-06 | 山东省农业科学院农业资源与环境研究所 | One utilizes edible fungi residue fermenting and producing cow protein feed new method |
| CN105410357A (en) * | 2015-12-31 | 2016-03-23 | 四川金雨农业股份有限公司 | Folium mori powder biological complete feed and production method thereof |
| CN106860654A (en) * | 2017-01-18 | 2017-06-20 | 南京中医药大学 | A kind of utilization fruit of Chinese magnoliavine dregs of a decoction coproduction lignanoid and the method for protein feed |
| CN107484549A (en) * | 2017-08-24 | 2017-12-19 | 南京林业大学 | A kind of method and its application of coronoid process dissipate capsule bacterium solid state fermentation ginkgo leaf |
| CN108260737A (en) * | 2018-02-24 | 2018-07-10 | 龙岩学院 | A kind of production technology for the ginkgo leaf compound fermentation preparation for enhancing broiler feeding benefit |
| CN109452448A (en) * | 2018-07-18 | 2019-03-12 | 焦作恒辉牧业有限公司 | A kind of feed additive preparation method containing sweet wormwood powder and chrysanthemum powder |
| CN110150461A (en) * | 2019-07-04 | 2019-08-23 | 徐州工程学院 | A kind of Composite fermentation type microbiological feed and preparation method thereof |
| CN110973354A (en) * | 2020-01-02 | 2020-04-10 | 四川农业大学 | Solid-state fermentation wheat bran feed and preparation method and application thereof |
| CN110973351A (en) * | 2019-12-19 | 2020-04-10 | 广东省农业科学院动物科学研究所 | Calliandra haematocephala fermentation product and preparation method and application thereof |
| CN111685233A (en) * | 2020-07-20 | 2020-09-22 | 巢湖学院 | Method for processing high-protein feed by using pear residues, pear leaves and pear branches |
| CN112021451A (en) * | 2020-08-07 | 2020-12-04 | 东北农业大学 | Fermentation method for increasing content of fatty and acid soluble proteins in wheat bran |
| CN112806477A (en) * | 2021-01-18 | 2021-05-18 | 天津农学院 | Method for producing feed by multi-strain solid-state fermentation of pumpkin seed meal |
| CN115176864A (en) * | 2022-07-04 | 2022-10-14 | 徐州工程学院 | Probiotic fermented compound feed and preparation method thereof |
| CN115553380A (en) * | 2022-10-13 | 2023-01-03 | 徐州工程学院 | Fermented feed and fermentation process thereof |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1653932A (en) * | 2004-12-10 | 2005-08-17 | 宝鸡市星星协力生物有限公司 | Highly efficient oligosaccharide high yielded enzyme composite animal microecological formulation without public hazard and its preparing method |
| CN1919044A (en) * | 2006-09-20 | 2007-02-28 | 南京林业大学 | Animal feedstuff additive and preparation method thereof |
| CN101463335A (en) * | 2009-01-16 | 2009-06-24 | 黑龙江泽农寒地生物工程有限公司 | Piggery fermentation bed composite microbial preparation and preparation thereof |
| CN101543259A (en) * | 2009-05-08 | 2009-09-30 | 南京林业大学 | Preparation method of biological feed additive |
| CN101965924A (en) * | 2010-09-28 | 2011-02-09 | 上海市奶牛研究所 | Method for preparing milk cow replacement cattle fermented TMR feed from gingko decoction dreg |
| CN102212494A (en) * | 2011-04-12 | 2011-10-12 | 王颖 | Organic matter decomposing inoculant, and preparation method and application thereof |
| CN102318743A (en) * | 2011-09-30 | 2012-01-18 | 南京林业大学 | Method for improving ginkgo leaf feeding palatability and increasing product bioactivity |
| CN103005159A (en) * | 2012-12-18 | 2013-04-03 | 南京林业大学 | Preparation method of ginkgo leaf biological feed additive |
| CN103141692A (en) * | 2013-03-20 | 2013-06-12 | 邹平泰康生物饲料有限公司 | Feed additive for ruminant animals and preparation method thereof |
| CN103168921A (en) * | 2012-06-18 | 2013-06-26 | 南开大学 | Method for producing straw feed |
| CN103283961A (en) * | 2013-05-10 | 2013-09-11 | 浙江工业大学 | Method for preparing health-benefiting fermented soybean meal |
-
2013
- 2013-09-18 CN CN201310427156.3A patent/CN103478413B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1653932A (en) * | 2004-12-10 | 2005-08-17 | 宝鸡市星星协力生物有限公司 | Highly efficient oligosaccharide high yielded enzyme composite animal microecological formulation without public hazard and its preparing method |
| CN1919044A (en) * | 2006-09-20 | 2007-02-28 | 南京林业大学 | Animal feedstuff additive and preparation method thereof |
| CN101463335A (en) * | 2009-01-16 | 2009-06-24 | 黑龙江泽农寒地生物工程有限公司 | Piggery fermentation bed composite microbial preparation and preparation thereof |
| CN101543259A (en) * | 2009-05-08 | 2009-09-30 | 南京林业大学 | Preparation method of biological feed additive |
| CN101965924A (en) * | 2010-09-28 | 2011-02-09 | 上海市奶牛研究所 | Method for preparing milk cow replacement cattle fermented TMR feed from gingko decoction dreg |
| CN102212494A (en) * | 2011-04-12 | 2011-10-12 | 王颖 | Organic matter decomposing inoculant, and preparation method and application thereof |
| CN102318743A (en) * | 2011-09-30 | 2012-01-18 | 南京林业大学 | Method for improving ginkgo leaf feeding palatability and increasing product bioactivity |
| CN103168921A (en) * | 2012-06-18 | 2013-06-26 | 南开大学 | Method for producing straw feed |
| CN103005159A (en) * | 2012-12-18 | 2013-04-03 | 南京林业大学 | Preparation method of ginkgo leaf biological feed additive |
| CN103141692A (en) * | 2013-03-20 | 2013-06-12 | 邹平泰康生物饲料有限公司 | Feed additive for ruminant animals and preparation method thereof |
| CN103283961A (en) * | 2013-05-10 | 2013-09-11 | 浙江工业大学 | Method for preparing health-benefiting fermented soybean meal |
Non-Patent Citations (1)
| Title |
|---|
| 王晓凡等: "多菌种复合固态发酵对菜籽粕硫甙去除效果的研究", 《食品科技》 * |
Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104286593A (en) * | 2014-11-08 | 2015-01-21 | 宁波市江东农茹生物科技有限公司 | Special feed additive for silver carp culture |
| CN104472856A (en) * | 2014-12-29 | 2015-04-01 | 江苏千药堂国医研究院有限公司 | Method for preparing protein feed by utilizing waste liquid fermentation in production of ginkgo leaf extracts |
| CN104472862A (en) * | 2014-12-29 | 2015-04-01 | 江苏千药堂国医研究院有限公司 | Biological feed additive and preparation method thereof |
| CN104472868A (en) * | 2014-12-29 | 2015-04-01 | 江苏千药堂国医研究院有限公司 | Processing method of biologic feed additive |
| CN104489243A (en) * | 2014-12-29 | 2015-04-08 | 邳州市东方养殖有限公司 | Method for preparing biofeed by using rabbit dung |
| CN104489242A (en) * | 2014-12-29 | 2015-04-08 | 邳州市东方养殖有限公司 | Processing method of biologically fermented rabbit dung feed |
| CN104489281A (en) * | 2014-12-29 | 2015-04-08 | 江苏千药堂国医研究院有限公司 | Method for preparing single-cell protein feed additive by utilizing ginkgo leaf processing wastes |
| CN104543391A (en) * | 2014-12-29 | 2015-04-29 | 江苏千药堂国医研究院有限公司 | Preparation method of liquid feed additive |
| CN104560805A (en) * | 2014-12-29 | 2015-04-29 | 江苏千药堂国医研究院有限公司 | Method for preparing cell protein through fermentation of liquid waste in ginkgo leaf extract production |
| CN105211555A (en) * | 2015-10-22 | 2016-01-06 | 山东省农业科学院农业资源与环境研究所 | One utilizes edible fungi residue fermenting and producing cow protein feed new method |
| CN105410357A (en) * | 2015-12-31 | 2016-03-23 | 四川金雨农业股份有限公司 | Folium mori powder biological complete feed and production method thereof |
| CN106860654A (en) * | 2017-01-18 | 2017-06-20 | 南京中医药大学 | A kind of utilization fruit of Chinese magnoliavine dregs of a decoction coproduction lignanoid and the method for protein feed |
| CN107484549A (en) * | 2017-08-24 | 2017-12-19 | 南京林业大学 | A kind of method and its application of coronoid process dissipate capsule bacterium solid state fermentation ginkgo leaf |
| CN108260737A (en) * | 2018-02-24 | 2018-07-10 | 龙岩学院 | A kind of production technology for the ginkgo leaf compound fermentation preparation for enhancing broiler feeding benefit |
| CN109452448A (en) * | 2018-07-18 | 2019-03-12 | 焦作恒辉牧业有限公司 | A kind of feed additive preparation method containing sweet wormwood powder and chrysanthemum powder |
| CN110150461A (en) * | 2019-07-04 | 2019-08-23 | 徐州工程学院 | A kind of Composite fermentation type microbiological feed and preparation method thereof |
| CN110973351A (en) * | 2019-12-19 | 2020-04-10 | 广东省农业科学院动物科学研究所 | Calliandra haematocephala fermentation product and preparation method and application thereof |
| CN110973354A (en) * | 2020-01-02 | 2020-04-10 | 四川农业大学 | Solid-state fermentation wheat bran feed and preparation method and application thereof |
| CN110973354B (en) * | 2020-01-02 | 2023-08-18 | 四川农业大学 | Solid-state fermentation wheat bran feed and preparation method and application thereof |
| CN111685233A (en) * | 2020-07-20 | 2020-09-22 | 巢湖学院 | Method for processing high-protein feed by using pear residues, pear leaves and pear branches |
| CN112021451A (en) * | 2020-08-07 | 2020-12-04 | 东北农业大学 | Fermentation method for increasing content of fatty and acid soluble proteins in wheat bran |
| CN112806477A (en) * | 2021-01-18 | 2021-05-18 | 天津农学院 | Method for producing feed by multi-strain solid-state fermentation of pumpkin seed meal |
| CN115176864A (en) * | 2022-07-04 | 2022-10-14 | 徐州工程学院 | Probiotic fermented compound feed and preparation method thereof |
| CN115553380A (en) * | 2022-10-13 | 2023-01-03 | 徐州工程学院 | Fermented feed and fermentation process thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103478413B (en) | 2014-10-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103478413B (en) | Method for producing protein feed by mixed-strain solid-state fermentation of ginkgo leaf residues | |
| CN101273749B (en) | Method of mixed fermentation treatment using vinasse as main feed raw material | |
| CN102934736B (en) | Method for preparing sweet potato skin/ sweet potato powder dreg fermented feed | |
| CN107801837B (en) | Biological fermentation process for forage-based utilization of chicken manure | |
| CN105053593A (en) | Traditional Chinese medicine residues prepared poultry feed additive and preparation method thereof | |
| CN103211084A (en) | Method for preparing biological enzyme-enriched camellia seed meal fermented feed | |
| CN103504123A (en) | Fermented soybean meal with function of complex enzymes and preparation method for fermented soybean meal | |
| CN102524544B (en) | Feed enzymic preparation prepared by using soy sauce residues and preparation method of feed enzymic preparation | |
| CN102972623A (en) | Method for producing protein feed through hypsizygus marmoreus fungus chaff solid state fermentation and protein feed being produced via method for producing protein feed through hypsizygus marmoreus fungus chaff solid state fermentation | |
| CN103704501B (en) | Method for producing high-efficiency broiler feed by utilization of corn straw fermentation | |
| CN103820339A (en) | Dehydrated solid composite microbial agent capable of increasing albumen level of cassava residue and preparation method of dehydrated solid composite microbial agent | |
| CN102488087A (en) | Biological detoxification method for camellia seed cakes | |
| CN103184174A (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN105543131A (en) | Compound bacteria/cottonseed meal fermented feed and preparation method thereof | |
| CN102919624A (en) | Microbial fermentation and detoxification method of rapeseed cake | |
| CN104855674A (en) | Production method for microbial fermentation complete feed by combining strain joint transformations | |
| CN106035990B (en) | Method for preparing biological feed by solid-state enzymolysis fermentation of citrus pulp, product and application thereof | |
| CN102860413A (en) | Nutritive feed and preparation method thereof | |
| CN107853452A (en) | A kind of production method of additive for microbe feedstuff | |
| CN101194668A (en) | Process for preparing blood meal biological modified peptide protein and application of the same | |
| CN101407762A (en) | Microbial solid inocula, and preparation and use thereof | |
| CN103719537A (en) | Nonreactive biological fermented feed and preparation method thereof | |
| CN103352016A (en) | Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha | |
| CN103229891A (en) | Method for producing protein feed by using cynomorium songaricum residues | |
| CN103642695B (en) | One Aspergillus oryzae and the application prepared at fermentable in fodder additives thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220428 Address after: 276800 an Zhuang Zhen Chen Guan Zhuang Cun, Ju county, Rizhao City, Shandong Province Patentee after: Rizhao Hengbang animal husbandry technology Co.,Ltd. Address before: 518000 1009, No.1, langrong Road, Xinshi community, Dalang street, Longhua District, Shenzhen City, Guangdong Province Patentee before: Shenzhen Hongtu Technology Service Co.,Ltd. Effective date of registration: 20220428 Address after: 518000 1009, No.1, langrong Road, Xinshi community, Dalang street, Longhua District, Shenzhen City, Guangdong Province Patentee after: Shenzhen Hongtu Technology Service Co.,Ltd. Address before: No. 16, No. five village, Xuanwu District, Nanjing, Jiangsu Province Patentee before: INSTITUTE OF CHEMICAL INDUSTRY OF FOREST PRODUCTS, CAF |
|
| TR01 | Transfer of patent right |