CN103478413B - 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 PDF

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CN103478413B
CN103478413B CN201310427156.3A CN201310427156A CN103478413B CN 103478413 B CN103478413 B CN 103478413B CN 201310427156 A CN201310427156 A CN 201310427156A CN 103478413 B CN103478413 B CN 103478413B
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ginkgo leaf
state fermentation
liquid spawn
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CN103478413A (en
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王成章
周昊
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Rizhao Hengbang Animal Husbandry Technology Co ltd
Shenzhen Hongtu Technology Service Co ltd
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Institute of Chemical Industry of Forest Products of CAF
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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 for 72-120 h at a natural pH value at the temperature of 28-30 ℃, 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

A kind of mixed culture solid state fermentation ginkgo leaf slag is produced the method for protein feed
Technical field
The invention belongs to agriculture and forestry organic waste material process and utilization technology field, particularly relate to a kind of method of carrying out mixed culture solid state fermentation production protein feed taking ginkgo leaf slag as raw material.
Background technology
Along with the fast development of 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, the wretched insufficiency of quantity delivered of visible China protein feed.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 agriculture and forestry organic waste material exploitation to there is the plant protein fodder of bioenzyme activity, there is very important meaning for the deficiency that makes up protein feed on market with the security and the utilization rate that improve feed.
Ginkgo is the distinctive rare tree of China, and China's gingko resource is abundant, accounts for the 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, probably has the ginkgo leaf slag of 30,000 tons of left and right to be thrown away or burn as discarded object every year, not only causes the serious waste of resource, and pollutes the environment.In ginkgo leaf slag, contain rich in protein and amino acid, 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 ginkgo leaf slag.Therefore prepare animal feeding-stuff containing somatic protein taking ginkgo leaf slag as raw material, not only can turn waste into wealth, make ginkgo leaf resource go on green circulatory development, and be the exploitation injection new vitality of ginkgo leaf resource, there is 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 multiple bacterial classifications, can compensate mutually its defect, jointly 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 in 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 in the mode that mostly adopts solid state fermentation aspect fermented feed production.
Only have at present both at home and abroad the patent report of preparing biological feedstuff taking ginkgo leaf as raw material, and it is little to prepare the patent report of biological feedstuff about ginkgo leaf slag.As patent 201210549464.9 has been reported taking ginkgo leaf as raw material, adopt single culture bacillus to carry out solid state fermentation and prepare biology feed additive; Patent 200910031310.9 has been reported taking ginkgo leaf as raw material, adopts single culture candida utili to carry out solid state fermentation and prepares biology feed additive; Patent 200910031310.9 has been reported taking ginkgo leaf, waste edible fungus bacterium slag, dregs of beans as raw material, adopts single culture aspergillus niger to carry out fermenting and preparing biological feed addictive.The problems such as these patents all adopt single culture to ferment, and the biological feedstuff preparing exists that protein content is low, nutritional labeling is single, it is poor to digest and assimilate and palatability is poor.Ginkgo leaf slag prepare only have patent 201010295193.x to report aspect biological feedstuff 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 for preparing 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 to adopt taking ginkgo leaf slag as raw material the method for mixed culture solid state fermentation to produce protein feed.The method adopts fermented by mixed bacterium, and in the ginkgo leaf residue protein feed preparing, 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; The mode of production that adopts 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 the protein feed of comprehensive nutrition is prepared in fermentation, 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 a kind of method of carrying out mixed culture solid state fermentation production protein feed taking ginkgo leaf slag 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 preparing is high, and nutritional labeling is comprehensive, and feed is digested and assimilated, good palatability, is applicable to suitability for industrialized production.
The present invention is achieved by the following technical solutions.
Mixed culture solid state fermentation ginkgo leaf slag is produced a 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 5mL malt extract medium is housed after candida tropicalis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mL malt extract medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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 aspergillus oryzae activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that 5mLBPY fluid nutrient medium is housed after bacillus subtilis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLBPY fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that 5mLMRS fluid nutrient medium is housed after Lactobacillus plantarum activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLMRS fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain Lactobacillus plantarum liquid spawn.
(2) prepare solid-state fermentation culture medium
After ginkgo leaf slag is dried, is pulverized, add the water of 2~4 times of ginkgo leaf slag weight, then add the nitrogenous source of ginkgo leaf slag weight 5~12%, after mixing, steam sterilizing 20~30min at 120~125 DEG C, obtains ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation is prepared 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 DEG C of conditions of temperature, static fermentation 72~120h under nature pH value, finally tunning is dried to constant weight under 50 DEG C of conditions, obtain ginkgo leaf residue protein feed.
Described ginkgo leaf slag be by 3~6 years raw 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 of candida tropicalis, aspergillus oryzae, bacillus subtilis and four kinds of bacterial classification compositions of Lactobacillus plantarum.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 5mL malt extract medium is housed after candida tropicalis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mL malt extract medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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 aspergillus oryzae activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that 5mLBPY fluid nutrient medium is housed after bacillus subtilis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLBPY fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that 5mLMRS fluid nutrient medium is housed after Lactobacillus plantarum activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLMRS fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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.Candida tropicalis, candida utili, geotrichum candidum, aspergillus oryzae, aspergillus niger, Trichoderma viride, bacillus subtilis and the impact of Lactobacillus plantarum different strain on tunning crude protein content have been investigated respectively in single culture fermentation test; Higher the crude protein content filtering out in single bacterium fermentation test saccharomycete, mould and Lactobacillus plantarum, bacillus subtilis are carried out to the composite fermentation test of 2 bacterium, 3 bacterium and 4 bacterium, measure crude protein content in composite fermentation product.Result shows, 4 kinds of bacterial classifications that in 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, strain combination is that four bacterium combined fermentation product crude protein contents of candida tropicalis+aspergillus oryzae+bacillus subtilis+Lactobacillus plantarum are the highest, therefore the fermented bacterium that, the present invention adopts is the composite bacteria of candida tropicalis, aspergillus oryzae, bacillus subtilis and four kinds of bacterial classification compositions of Lactobacillus plantarum.
The present invention is taking ginkgo leaf slag as raw material, by adding a certain amount of water and nitrogenous source to prepare solid-state fermentation culture medium.After ginkgo leaf slag is dried, is pulverized, add the water of 2~4 times of ginkgo leaf slag weight, then add the nitrogenous source of ginkgo leaf slag weight 5~12%, after mixing, steam sterilizing 20~30min at 120~125 DEG C, after cooling, obtain ginkgo leaf slag solid-state fermentation culture medium, 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 within the scope of 1:2~1:4, 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, due to the restriction of 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 cannot reach the requirement of producing protein feed, therefore in solid-state fermentation culture medium, need to add a certain amount of nitrogenous source simultaneously.
The present invention investigates nitrogenous source kind and addition, choose respectively urea and ammonium sulfate as culture media nitrogen source, nitrogenous source addition is respectively 3%, 5%, 8%, 10%, 12%, after mixed culture solid state fermentation, measure the crude protein content of product, result shows, urea to the amplification of tunning crude protein content higher than ammonium sulfate.In the time that urea addition is 8%, in tunning, crude protein content reaches the highest.Therefore preferred nitrogen source category is urea, and 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 DEG C of conditions of temperature, static fermentation 72~120h under nature pH value, finally tunning is dried to constant weight under 50 DEG C 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 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, in the time that 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, accesses solid fermentation culture medium respectively by 6%, 8%, 10%, 12%, 15% inoculum concentration.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 in the time that 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 within the scope of 72~120h, crude protein content in tunning all can reach more than 45%, in the time that 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 analyzes 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%.Compared with raw material, 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 and can strengthen humoral immunity and the cellular immunity of animal in tunning, 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 completely, for feeding of the pig that grows, checks its impact on growth pig production performance and feed digestibility.
Result shows, compared with control group (basic diet interpolation rapeseed dregs), the average daily gain of the growth pig of test group (basic diet adds ginkgo leaf residue protein feed) has improved 23.61%, material anharmonic ratio has reduced by 18.44%, the CP digestibility of feed has improved 15.38%, and 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 completely, for feeding of the pig that grows.
Beneficial effect of the present invention:
(1) the present invention, taking forest resourceies processing waste ginkgo leaf slag as raw material, 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 ferment, compared with fermenting with traditional single culture, in the ginkgo leaf residue protein feed preparing, 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.
Brief description of the drawings
Fig. 1 ginkgo leaf slag mixed culture solid state fermentation is prepared the process chart of protein feed.
Detailed description of the invention
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 5mL malt extract medium is housed after candida tropicalis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mL malt extract medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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 aspergillus oryzae activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that 5mLBPY fluid nutrient medium is housed after bacillus subtilis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLBPY fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that 5mLMRS fluid nutrient medium is housed after Lactobacillus plantarum activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLMRS fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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 mixing, cooling after, access respectively 10% candida tropicalis, candida utili, geotrichum candidum, aspergillus oryzae, aspergillus niger, Trichoderma viride, bacillus subtilis and Lactobacillus plantarum, at 30 DEG C, after the lower static fermentation 72h of nature pH value, measure crude protein content in tunning, the results are shown in Table 1; Higher the crude protein content filtering out in single bacterium fermentation test saccharomycete, mould and Lactobacillus plantarum, bacillus subtilis are 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 composite fermentation product, the results are shown in Table 2.
The impact of the different single bacterium of table 1 on 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 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 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, strain combination is that four bacterium combined fermentation product crude protein contents of candida tropicalis+aspergillus oryzae+bacillus subtilis+Lactobacillus plantarum are the highest, therefore the fermented bacterium that, the present invention adopts is the composite bacteria of candida tropicalis, aspergillus oryzae, bacillus subtilis and four kinds of bacterial classification compositions of Lactobacillus plantarum.
Embodiment 2
The preparation of solid-state fermentation culture medium
After ginkgo leaf slag is dried, is pulverized, add the water of 2~4 times of ginkgo leaf slag weight, then add the nitrogenous source of ginkgo leaf slag weight 5~12%, after mixing, steam sterilizing 20~30min at 120~125 DEG C, obtains ginkgo leaf slag solid-state fermentation culture medium after cooling.
Described ginkgo leaf slag be by 3~6 years raw 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, when material-water ratio is within the scope of 1:2~1:4, 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 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, due to the restriction of 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 cannot reach the requirement of producing protein feed, therefore in solid-state fermentation culture medium, need to add a certain amount of nitrogenous source simultaneously.
The present invention investigates nitrogenous source kind and addition, chooses respectively urea and ammonium sulfate as culture media nitrogen source, and 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.In the time that urea addition is 8%, in tunning, crude protein content reaches the highest.Therefore preferred nitrogen source category is urea, and nitrogenous source addition is 8%.
The impact on tunning crude protein of table 4 different nitrogen sources and addition
Embodiment 3
Solid state fermentation is prepared 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 DEG C of conditions of temperature, static fermentation 72~120h under nature pH value, finally tunning is dried to constant weight under 50 DEG C 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 composite bacteria ratio, to in solid fermentation culture medium respectively by the bacterium liquid of saccharomycete, mould, bacillus subtilis and the Lactobacillus plantarum of 10% inoculum concentration access different proportion, 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, the crude protein content of measuring tunning, the results are shown in Table 5.As shown in Table 5, in the time that 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 tunning crude protein content
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, the composite bacteria filtering out is accessed to solid fermentation culture medium by 6%, 8%, 10%, 12%, 15% inoculum concentration respectively, sterilizing, after cooling, at 30 DEG C, after the lower static fermentation 72h of nature pH value, 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 in the time that 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, the composite bacteria filtering out is accessed to solid fermentation culture medium by 10% inoculum concentration respectively, sterilizing, after cooling, at 30 DEG C, under nature pH value, static fermentation 24h, 48h, 72h, 96h, 120h respectively, tunning crude protein content is respectively 25.34%, 30.28%, 47.39%, 47.03%, 46.28%.Result shows that fermentation time is within the scope of 72~120h, crude protein content in tunning all can reach more than 45%, and in the time that 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 preparing, 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%.Compared with raw material, 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 and can strengthen humoral immunity and the cellular immunity of animal in tunning, 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 ginkgo leaf residue protein feed of preparation is substituted the rapeseed dregs in basic diet completely by the present invention, 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 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, 6 of each repetitions (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).Altogether 28d of formal test phase, raises all management and immunity routinely of code, and weighs the morning that prerun starts day on an empty stomach, is just trying in the phase, and when every two weeks and off-test, empty stomach is weighed to stop feeding after 24h morning.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, compared with control group, the average daily gain of the growth pig of test group has improved 23.61%, and material anharmonic ratio has reduced by 18.44%, and the CP digestibility of feed has improved 15.38%, and 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 completely, 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 feed nutrient apparent digestibility
Project CP digestibility 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 5mL malt extract medium is housed after candida tropicalis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mL malt extract medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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 aspergillus oryzae activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that 5mLBPY fluid nutrient medium is housed after bacillus subtilis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLBPY fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that 5mLMRS fluid nutrient medium is housed after Lactobacillus plantarum activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLMRS fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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, steam sterilizing 30min at 120 DEG C, obtains ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation is prepared protein feed
Above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn are accessed respectively in ginkgo leaf slag solid-state fermentation culture medium in the ratio of 2:2:1:1, 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 DEG C of conditions of temperature, static fermentation 72h under nature pH value, finally tunning is dried to constant weight under 50 DEG C 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 5mL malt extract medium is housed after candida tropicalis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mL malt extract medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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 aspergillus oryzae activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that 5mLBPY fluid nutrient medium is housed after bacillus subtilis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLBPY fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that 5mLMRS fluid nutrient medium is housed after Lactobacillus plantarum activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLMRS fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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, steam sterilizing 30min at 125 DEG C, obtains ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation is prepared protein feed
Above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn are accessed respectively in ginkgo leaf slag solid-state fermentation culture medium in the ratio of 1:2:2:1, 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 DEG C of conditions of temperature, static fermentation 86h under nature pH value, finally tunning is dried to constant weight under 50 DEG C 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 5mL malt extract medium is housed after candida tropicalis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mL malt extract medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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 aspergillus oryzae activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that 5mLBPY fluid nutrient medium is housed after bacillus subtilis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLBPY fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that 5mLMRS fluid nutrient medium is housed after Lactobacillus plantarum activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLMRS fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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, steam sterilizing 30min at 125 DEG C, obtains ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation is prepared protein feed
Above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn are accessed respectively in ginkgo leaf slag solid-state fermentation culture medium in the ratio of 1:2:1:1, 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 DEG C of conditions of temperature, static fermentation 110h under nature pH value, finally tunning is dried to constant weight under 50 DEG C 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 5mL malt extract medium is housed after candida tropicalis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mL malt extract medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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 aspergillus oryzae activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that 5mLBPY fluid nutrient medium is housed after bacillus subtilis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLBPY fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that 5mLMRS fluid nutrient medium is housed after Lactobacillus plantarum activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLMRS fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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, steam sterilizing 30min at 120 DEG C, obtains ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation is prepared protein feed
Above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn are accessed respectively in ginkgo leaf slag solid-state fermentation culture medium in the ratio of 2:2:1:1, 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 DEG C of conditions of temperature, static fermentation 96h under nature pH value, finally tunning is dried to constant weight under 50 DEG C 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 5mL malt extract medium is housed after candida tropicalis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mL malt extract medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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 aspergillus oryzae activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain aspergillus oryzae liquid spawn.
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that 5mLBPY fluid nutrient medium is housed after bacillus subtilis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLBPY fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain bacillus subtilis liquid spawn.
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that 5mLMRS fluid nutrient medium is housed after Lactobacillus plantarum activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLMRS fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of constant-temperature tables (180r/min) are cultivated 36h, obtain 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, steam sterilizing 30min at 125 DEG C, obtains ginkgo leaf slag solid-state fermentation culture medium after cooling.
(3) mixed culture solid state fermentation is prepared protein feed
Above-mentioned candida tropicalis liquid spawn, aspergillus oryzae liquid spawn, Lactobacillus plantarum liquid spawn and bacillus subtilis liquid spawn are accessed respectively in ginkgo leaf slag solid-state fermentation culture medium in the ratio of 1:2:2:1, 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 DEG C of conditions of temperature, static fermentation 120h under nature pH value, finally tunning is dried to constant weight under 50 DEG C 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. the method that mixed culture solid state fermentation ginkgo leaf slag is produced protein feed, it is characterized in that, taking 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 ginkgo leaf slag, comprise the following steps:
(1) bacterial classification is cultivated
A. the amplification cultivation of Candida tropicalis: will be inoculated in the Boiling tube that 5mL malt extract medium is housed after candida tropicalis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mL malt extract medium is housed with 5% inoculum concentration, 28 DEG C of cultivation 36h of the constant-temperature table that is 180r/min with rotating speed, obtain 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 aspergillus oryzae activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLCzapek ' s fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of cultivation 36h of the constant-temperature table that is 180r/min with rotating speed, obtain aspergillus oryzae liquid spawn;
C. the amplification cultivation of bacillus subtilis: will be inoculated in the Boiling tube that 5mLBPY fluid nutrient medium is housed after bacillus subtilis activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLBPY fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of cultivation 36h of the constant-temperature table that is 180r/min with rotating speed, obtain bacillus subtilis liquid spawn;
D. the amplification cultivation of Lactobacillus plantarum: will be inoculated in the Boiling tube that 5mLMRS fluid nutrient medium is housed after Lactobacillus plantarum activation, cultivate 24h for 28 DEG C, then be inoculated in the triangular flask that 500mLMRS fluid nutrient medium is housed with 5% inoculum concentration, 28 DEG C of cultivation 36h of the constant-temperature table that is 180r/min with rotating speed, obtain Lactobacillus plantarum liquid spawn;
(2) prepare solid-state fermentation culture medium
After ginkgo leaf slag is dried, is pulverized, add the water of 2~4 times of ginkgo leaf slag weight, then add the nitrogenous source of ginkgo leaf slag weight 5~12%, after mixing, steam sterilizing 20~30min at 120~125 DEG C, obtains ginkgo leaf slag solid-state fermentation culture medium after cooling;
(3) mixed culture solid state fermentation is prepared 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 DEG C of conditions of temperature, static fermentation 72~120h under nature pH value, finally tunning is dried to constant weight under 50 DEG C 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 3~6 years raw ginkgo spires the leaf slag after 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, 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, 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%.
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