CN100353858C - Method for manufacturing feedstuff protein by solid state fermentation of apple dregs liquid - Google Patents

Method for manufacturing feedstuff protein by solid state fermentation of apple dregs liquid Download PDF

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CN100353858C
CN100353858C CNB2005100418070A CN200510041807A CN100353858C CN 100353858 C CN100353858 C CN 100353858C CN B2005100418070 A CNB2005100418070 A CN B2005100418070A CN 200510041807 A CN200510041807 A CN 200510041807A CN 100353858 C CN100353858 C CN 100353858C
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fermentation
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CN1663421A (en
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宋纪蓉
徐抗震
黄洁
马海霞
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Northwest University
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Abstract

The present invention discloses a method for producing feed protein by the liquid and solid state fermentation of apple dregs. The method has the following processes that pulverized apple dregs are firstly prepared into a liquid state fermentation culture medium; then, mixed bacteria ripened by seed cultivation are added for liquid state submerged fermentation; finally, the submerged fermentation liquid is added in a solid state fermentation culture medium of the apple dregs for solid state fermentation. Compared with the independent solid state fermentation, the liquid and solid state fermentation technique has the advantages of less quantity of seed culture media, less pure cultivation of the bacteria and low production cost. The produced feed protein products have the advantages of high protein content, abundant nutrition and low cost. The present invention is suitable for the industrial scale production and the popularized application of the feed protein production of the fermentation of the apple dregs.

Description

The method of manufacturing feedstuff protein by solid state fermentation of apple dregs liquid
Technology neck city
The invention belongs to fermentation of apple pulp and produce the method for forage protein.
Background technology
Usually the forage protein of saying (Feeding protein) is single cell protein (Single cellprotein, SCP), claim microprotein or mycoprotein again, be meant saccharomycete, fungi, mould, and unicellular organism body such as non-characteristic of disease bacterium in contained protein, content is up to 45%-74%, and reasonable, the A wide selection of colours and designs of each seed amino acid collocation, and vitamin and trace element are abundant, do not contain cholesterol etc., these advantages all be general crops feed can not compare.
China is the maximum in the world apple place of production, more than 2,200 ten thousand tons of annual productions, Gross World Product 1/3rd more than.Shaanxi Province is again the apple production base of China's maximum, and the whole province's apple cultivation gross area reaches 6,020,000 mu at present, more than 550 ten thousand tons of gross annual output amounts, and the position is the first in the nation.Since the nineties in last century, the a large amount of AJC machining production lines of introducing in the whole nation, only Shaanxi Province just has more than 20, produce AJC in 2003 and reach 300,000 tons, almost account for 1/3rd of quantum of world trade, export more than 30 countries and regions, become the maximum AJC in the whole nation and concentrate the producing region.When producing a large amount of inspissated juices, more than 60 ten thousand tons pomace will be discharged in annual only Shaanxi.After drying on a small quantity, to add outside the utilization as simple feed, the overwhelming majority is because of the moisture height, and protein content is low, is of low nutritive value, and is very easily rotten smelly, can't all utilize.Particularly the fruit processing busy season, around the fruit juice processing factory, pile up like a mountain for slag crust, decomposes through microorganism, and acid is smelly unpleasant, serious environment pollution not only, and cause the waste of the plain and hemicellulose resource of GF.
Utilizing pomace to produce protein feed is the good approach of a twice laid, and it is many that current utilization (pomace) cellulose resource is converted into the research of protein feed.Joshi, and VK (Bioresourcetechnology.1996,56,251-255), Rahmat, H (World journal ofmicrobiology ﹠amp; Biotechnology.1995,11 (2): 168-170), Roussos, S. (Advances in solid-state fermentation.1997:257-271), Sandhu, D.K (Journal of Scientific ﹠amp; Industrial Research.1997, (56) 2:86-90) etc., and Zhang Yuzhen (food and fermentation industries, 1996,3:29-32), nationality is kept tie, and (bioengineering makes progress, 1999,19 (5): 30-33), Zhang Changxia (Shandong food fermentation, 2003,4:20-22), Gao Zaixing (Northwest University's journal, 2003,33 (6): 701-704), Li Juxiu (Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's journal, 2003,31 (increasing): 79-81), Chang Xianbo (Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's journal (natural science edition) 32 (1): 40-46), Zhang Naifeng (new agricultural technology, 2004,2:46-47), Wang Yanhua (Chinese feed, 2004,8:35-35), Luo Wen (science and technology and engineering, 2004,4 (5): 371-374) grade has reported that respectively they utilize the single bacterial classification or the mixed bacteria of screening, and adopting independent solid state fermentation is the research of primary raw material fermenting and producing forage protein with the pomace, because the initial incubation based component, adopt the difference of bacterial classification and independent solid-state fermentation process parameter, therefore the protein content of product is not quite similar, and from 15% to 35% all has report.The process that fermentation of apple pulp is produced forage protein is the concentration process of " nitrogen ", and for example the crude protein content of fermentation raw material from 20% ferments to 40% content, just needs the carbohydrate of half quality to become H during the fermentation 2O and CO 2, owing to the objective requirement of microbial growth environment, protein content can not be too high simultaneously, and protein content does not have comparativity at this.Simultaneously, be not quite similar, generally between 48-108h for choosing also of fermentation time.Disclose the method that a kind of pomace double-bacterium ferment prepares forage protein as Chinese patent application 03127919.8, its fermentation time also reaches 48-72h.Overall consideration, separately solid state fermentation exists that seed culture medium consumption is many, inoculum concentration big, equipment is used more relatively and fermentation time deficiency such as length, say from economic angle, investment is higher with production cost, for producing the lower product of this added value of forage protein, suitability for industrialized production is difficult to implement, and still is in experimental study and preliminary industrialization stage at present.
Summary of the invention
The object of the present invention is to provide that a kind of equipment investment is low, mixed bacteria symbiosis and cooperative ability is strong, fermentation time weak point, protein content etc. are of high nutritive value, low production cost, economically feasible, be fit to the method that the fermentation of apple pulp of suitability for industrialized production is produced forage protein.The seed culture medium consumption that this method can overcome existing independent manufacturing feedstuff protein by solid state fermentation is many, bacterial classification pure culture complexity, the deficiency that equipment investment and production cost height, fermentation time are relatively long.、
The method of manufacturing feedstuff protein by solid state fermentation of apple dregs liquid:
(1) at first the pomace of pulverizing is prepared burden into the liquid state fermentation culture medium;
(2) mixed bacteria that inserts the seed culture maturation then carries out liquid submerged fermentation;
(3) submerged fermentation liquid is linked into again carries out solid state fermentation in the pomace solid-state fermentation culture medium at last.
The liquid submerged fermentation parameter is:
Seed inoculum concentration: 5%-20% (v/v);
The liquid submerged fermentation time: 24-48h;
Liquid submerged fermentation temperature: 30-34 ℃.
The solid state fermentation parameter is:
Switching amount: 15-40 (the solid-state substrate of mL/100g);
Thickness of feed layer: 3-5.5cm;
Solid state fermentation temperature: 30-34 ℃;
The solid state fermentation time: 72-96h.
Liquid state fermentation culture medium and solid-state fermentation culture medium mainly are made up of pomace, urea, inorganic salts and the water pulverized.Solid state component and content are pomace 78%-91% in the liquid state fermentation culture medium, sucrose 5%-10%, urea 1%-4%, (NH 4) 2SO 41%-4%, KH 2PO 41%-4%, the water that adds 10-15 times of solid material becomes the liquid state fermentation culture medium.
Pomace solid medium composition and content are pomace 75%-85%, wheat bran 10%-20%, urea 2%-4%, (NH 4) 2SO 41%-3%, KH 2PO 42%-4%, adding water adjusting initial water content is 50%-75%.
Mixed bacteria contains candida utili, fruit wine yeast, Trichoderma viride.The volumetric mixture ratio of bacterial classification is a candida utili: Trichoderma viride: fruit wine yeast=(3-4.5): (2-4): (1-2); The volumetric mixture ratio of bacterial classification also can be a candida utili: Trichoderma viride: fruit wine yeast: healthy and free from worry wood is mould=(2-4): (1-2): (6-8): (1-3).
Pomace particle diameter liquid towards ferment effect has appreciable impact.Particle diameter is more little, and the dispersion of pomace in solution is good more, and its nutriment helps being utilized by microorganism more, and at increased logarithmic phase, it is more vigorous that growth is bred.The raw material suitable for producing that was shown 60 mesh sieves by experimental result is used.
Transfer when bacterial classification is in increased logarithmic phase, viable bacteria kind quantity is more, and merisis is vigorous, suitable switching.Through the mixed culture fermentation of deep layer liquid state, symbiosis between the bacterial classification and cooperative ability grow, in solid ferment process, grow vigorous, speed is fast, can shorten deadtime and fermentation time.Experimental result shows that crude protein content increases sharply before 36h, then tends towards stability.Promptly be preferable transit time this moment.
Reduce the switching amount and mean the increase of producing convenient and solid state fermentation output, should reduce inoculum concentration in allowed limits as far as possible.But very little, fermentation time is prolonged, protein content is low, does not bring into play the bacterial classification advantage simultaneously, infects other assorted bacterium easily, produces peculiar smell, influences the quality of tunning.Experiment shows that 15-40mL/100g solid state fermentation substrate can satisfy the production needs.
Fermentation time is the important parameter of production control.Time is short, does not reach desirable protein content; But oversize, the albumen increase is not remarkable, and the production cycle is elongated, and it is big that production cost becomes.Experimental result shows that crude protein content no longer includes obvious ascendant trend, i.e. the preferable selection of time of 72h after the fermentation 72h.And solid state fermentation needs more than the 96h separately, with the independent solid-state time shortening 24h that compares, obviously shortened the production cycle, and crude protein content increases to some extent.
Compare the major advantage of this liquid-solid state fermentation technology with independent solid state fermentation:
1. the seed culture medium consumption is few, and the bacterial classification pure culture is few, can reduce production costs.
2. the independent amplification that three or four bacterial classifications are carried out is cultivated and is changed the Mixed culture that becomes a big fermentation tank, can simplify production technology, reduces equipment investment.
3. in advance bacterial classification is mixed to amplify and cultivate, hybrid technique was become simply, make phase coadaptation in advance between the bacterial classification again, symbiosis and synergy ability grow.
4. when carrying out solid state fermentation, the unit inoculation becomes big and stronger symbiosis and the synergy ability of mixed bacteria, can obviously shorten fermentation time and production cycle, the utilization rate of equipment and installations height, and output is big.
5. the forage protein product of producing simultaneously, the protein content height, nutritious, cost is lower, is more suitable for fermentation of apple pulp and produces the industrial scale production of forage protein and apply, for the protein feed of shortage provides a new way.
Description of drawings
Accompanying drawing 1 is produced the liquid-solid state fermentation process chart of forage protein for fermentation of apple pulp.
The specific embodiment
Embodiment 1:
Seed culture medium: potato 200g, sucrose 20g, water is some; Its preparation method is: potato is cut into small pieces, adds an amount of running water and boil and boil 20-30min, filter, get filtrate and add running water to 1000mL.
Seed culture of strains: get the 300mL seed culture medium and insert the 500mL conical flask and keep the 20min sterilization down at 121 ℃, insert each slant strains after the cooling room temperature, 30-32 ℃ of air bath shaking table, 120-140rpm cultivates 36-54h, obtains cultivating ripe seed bacterial classification.
The liquid submerged fermentation culture medium consists of: dried pomace 90%, sucrose 5%, urea 2%, (NH 4) 2SO 42%, KH 2PO 41%.And add the water of 10 times of solid materials.
The mixed bacteria and the ratio that insert: candida utili: Trichoderma viride: fruit wine yeast=4: 3: 1.5,
Solid medium consists of: dried pomace 75%, wheat bran 20%, urea 2%, (NH 4) 2SO 42%, KH 2PO 41%, adding water adjusting initial water content is 60%.
Behind bright wet pomace process 75 ℃ of dryings of heated-air drying line and the crushing screening, getting 60 purpose raw materials is mixed with the liquid state fermentation culture medium and inserts large fermentation tank, insert the mixed bacteria of seed tank culture maturation simultaneously by the amount of 10% (v/v), under 32 ℃, liquid submerged fermentation 36h, pour into submerged fermentation liquid in the pomace solid medium for preparing by the amount of the solid-state substrate of 30mL/100g then, under initial water content 65% unsterilised condition, take out fermented sample behind 32 ℃ of solid-state cultivation 72h, drying, granulation, packing promptly obtain the apple dreg fodder protein product.
The independent solid state fermentation experiment of contrast: bright wet pomace is prepared burden into solid-state fermentation culture medium through 75 ℃ of dryings of heated-air drying line and after pulverizing, and it consists of: dried pomace 75%, wheat bran 20%, urea 2%, (NH 4) 2SO 42%, KH 2PO 41%.Insert mixed bacteria (the mixed proportion candida utili: Trichoderma viride: fruit wine yeast=4: 3: 1.5) of seed tank culture maturation by the amount of 10% (v/v), initial water content 65%, under the unsterilised condition, take out fermented sample behind 32 ℃ of solid-state cultivation 96h, drying, granulation, packing promptly obtain the apple dreg fodder protein product.
The product main component, trace element and the amino acid composition measuring result that carry out above-mentioned two kinds of different fermentations forms are shown in table 1, table 2 and table 3:
Table 1 main component content (%)
Crude protein True albumen Crude fat Crude fibre Ash content
The independent solid state fermentation product of fermentation raw material liquid-solid state fermentation product 16.28 28.43 29.08 10.02 26.59 26.63 8.94 13.47 13.56 16.68 10.84 10.32 4.25 6.52 7.85
Table 2 micronutrient levels (mg/kg)
Ca(％) Mg(％) Fe Zn Mn Cu Se Pb Cd As
The fermentation of apple pulp product 0.113 0.493 0.061 0.204 169.2 276.8 14.6 31.3 13.1 20.8 9.8 16.7 0.18 0.29 0.6 1.6 0.02 0.68 0.17 0.59
Table 3 amino acid content (%)
Project Pomace Fermentation raw material Independent solid state fermentation product The liquid-solid state fermentation product
Aspartic acid ASP threonine THR serine SER glutamic acid GLU proline PRO glycine GLY alanine ALA cysteine CYR valine VAL methionine MET isoleucine ILE leucine LEU tyrosine TYR phenylalanine PHE lysine LYS histidine HIS arginine ARG 0.4887 0.1987 0.1779 0.8055 0.2782 0.2347 0.2014 0.0607 0.3136 0.0594 0.2261 0.4382 0.1958 0.2700 0.1846 0.0711 0.2477 0.848 0.3391 0.3727 1.68 3 0.5266 0.5057 0.4838 0.0843 0.9772 0.1482 0.2364 0.5368 0.4149 0.6014 0.2753 0.194 0.5048 1.6950 0.7533 0.8395 2.4060 0.8500 0.8489 0.9595 0.1050 0.9767 0.1846 0.8545 1.2120 0.5996 0.8269 1.0010 0.3935 0.8729 1.8640 0.8757 0.8895 2.6730 0.7421 0.8987 0.9700 0.2539 1.3340 0.2244 0.8364 1.3070 0.7523 0.8229 1.0330 0.3890 0.9563
Total amount 4.4523 8.7322 15.3789 16.8222
Assay method:
The mensuration of crude protein: adopt GB GB/T6432-86, promptly micro-Kjeldahl.
The mensuration of true albumen: adopt improved micro-Kjeldahl---take by weighing solid state fermentation product 5g,, fully stir with 40mL 75% ethanol lixiviate 24h, suction filtration, filter residue is used 40mL 75% ethanol cyclic washing 2 times again, with flush away inorganic nitrogen and urea, and the while precipitating proteins.To be deposited under 65 ℃ and be dried to constant weight, and accurately take by weighing and carry out nitratedly about 1g, measure promptly above-mentioned GB GB/T6432-86 again with micro-Kjeldahl.
The mensuration of crude fat content: adopt GB GB/T6433-86, promptly Soxhlet is put forward the method for taking out.
The mensuration of crude fiber content: adopt GB GB/T6434-86, i.e. the acid-alkali washing method.
The mensuration of content of ashes: adopt GB GB/T6438-86, i.e. ashing method.
Amino acid whose mensuration: measure by the 121-MB of Institute of Analysis of Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology type amino-acid analyzer.
By the said determination result as can be known, with the pomace is primary raw material, behind the mixed bacteria liquid-solid state fermentation, significant change all takes place in the product main component, crude protein, true albumen, crude fat, content of ashes has improved 78.62% respectively, 165.76%, 51.67% and 84.70%, crude fibre has then reduced by 38.13%, determined amino acid analysis through 17 kinds of necessity, its content is increased sharply and is abundant, complete, especially for the long crucial serine of fowl dirty swine, lysine, tyrosine and arginine etc. respectively than fermentation raw material gaining rate up to 162.3%, 275.2%, 81.3% and 89.4%, nutritive value significantly increases.
By the fermented product main component of two kinds of different process forms and amino acid content more as can be known, liquid-solid state fermentation increases to some extent than independent solid state fermentation nutritional labeling, illustrates once more that also liquid-solid state fermentation is better than independent solid state fermentation.
By determination of trace elements as can be known, beneficial element content is abundant, and contents of heavy metal elements meets the requirement of national similar feed base-material sanitary standard.Detect fermented product through feeding quality supervision inspection center of the authoritative department Ministry of Agriculture (Xi'an), salmonella does not all detect, AFB 10.0034mg/kg be far smaller than the requirement of similar feed base-material such as wheat bran, corn national forage health standards (GB13078-91) such as (0.05mg/kg).Carry out the toxicity check through the disease prevention and control center, Shaanxi Province, the nontoxic level in the true border of product can be used as protein feed and is widely used in the breed field.
Embodiment 2:
Bright wet pomace through 65 ℃ of dryings of heated-air drying line and crushing screening after, got 40 purpose raw materials and inserted large fermentation tank, the consisting of of liquid state fermentation culture medium: dried pomace 90%, sucrose 4%, urea 2%, (NH by the liquid state fermentation culture medium 4) 2SO 42%, KH 2PO 42%, and add the moisture of 12 times of solid materials.Insert the mixed bacteria of seed tank culture maturation simultaneously by the amount of 8% (v/v), the bacterial classification ratio of its access: candida utili: Trichoderma viride: fruit wine yeast: healthy and free from worry wood is mould=and 2: 1.5: 6: 3, under 33 ℃, liquid submerged fermentation 36h, pour zymotic fluid into prepare pomace solid medium by the amount of the solid-state substrate of 20mL/100g then, the consisting of of culture medium: dried pomace 84%, wheat bran 10%, urea 3%, (NH 4) 2SO 42%, KH 2PO 41%.Under water content 70% unsterilised condition, 33 ℃ are taken out fermented sample behind the solid-state cultivation 78h down, and drying, granulation, packing promptly obtain the apple dreg fodder protein product.Its crude protein content can reach 27.69%, true protein content 25.21%.
Assay method is the same.

Claims (1)

1, the method for manufacturing feedstuff protein by solid state fermentation of apple dregs liquid is characterized in that:
(1) at first the pomace of pulverizing is prepared burden into the liquid state fermentation culture medium;
Solid state component and content are in the liquid state fermentation culture medium: pomace 78%-91%, sucrose 5%-10%, urea 1%-4%, (NH 4) 2SO 41%-4%, KH 2PO 41%-4%, the water that adds 10-15 times of solid material becomes the liquid state fermentation culture medium;
(2) mixed bacteria that inserts the seed culture maturation then carries out liquid submerged fermentation;
Described mixed bacteria contains candida utili, fruit wine yeast, Trichoderma viride;
The volumetric mixture ratio of bacterial classification is a candida utili: Trichoderma viride: fruit wine yeast=(3-4.5): (2-4): (1-2), perhaps the volumetric mixture ratio of bacterial classification is a candida utili: Trichoderma viride: fruit wine yeast: healthy and free from worry wood is mould=(2-4): (1-2): (6-8): (1-3);
The liquid submerged fermentation parameter is: seed inoculum concentration 5%-20% (v/v); Fermentation time 24-48h; Fermentation temperature 30-34 ℃;
(3) submerged fermentation liquid is linked into again carries out solid state fermentation in the pomace solid-state fermentation culture medium at last;
Pomace solid medium composition and content are: pomace 75%-85%, wheat bran 10%-20%, urea 2%-4%, (NH 4) 2SO 41%-3%, KH 2PO 42%-4%, adding water adjusting initial water content is 50%-75%;
The solid state fermentation parameter is: the solid-state substrate of switching amount 15-40mL/100g; Thickness of feed layer 3-5.5cm; Fermentation temperature 30-34 ℃; Fermentation time 72-96h.
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CN101214006B (en) * 2008-01-03 2010-08-04 陕西恒兴食品科技有限公司 Method for using EM original liquid fermentation apple pomace to produce protein feed
CN101411393B (en) * 2008-11-28 2011-12-14 李绍章 Method for preparing active wet mixing feed fermented from liquid state to solid-state containing probiotics
CN101558819B (en) * 2009-05-25 2012-05-02 姜志德 Biological feed produced by fermenting waste residues of fruits and vegetables and production method thereof
CN102523914A (en) * 2010-11-30 2012-07-04 国立大学法人信州大学 Mushroom culture medium capable of being reused in livestock feed and method for making the same
CN102823725B (en) * 2012-09-07 2013-08-14 甘肃昆仑生化有限责任公司 Method for producing biologic protein feed by using solid-state fermentation fiber dreg
CN103048417A (en) * 2013-01-07 2013-04-17 黑龙江省麒麟工贸公司 Measurement method for true protein in feed
CN103262963B (en) * 2013-06-19 2015-06-10 陕西杨凌富仕特饲料有限公司 Live pig fattening feed taking fermented agricultural and sideline products as raw materials
CN106666077A (en) * 2016-12-29 2017-05-17 四川省旺达饲料有限公司 Preparation method of fruit-flavored fermented rice bran
CN116179371A (en) * 2023-05-04 2023-05-30 鲁东大学 Application of apple pomace as resource

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