CN103820339A - Dehydrated solid composite microbial agent capable of increasing albumen level of cassava residue and preparation method of dehydrated solid composite microbial agent - Google Patents
Dehydrated solid composite microbial agent capable of increasing albumen level of cassava residue and preparation method of dehydrated solid composite microbial agent Download PDFInfo
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Abstract
The invention belongs to the technical field of bio-fermentation, and in particular relates to a dehydrated solid composite microbial agent capable of increasing albumen level of cassava residue and a preparation method of the dehydrated solid composite microbial agent. The technology key point of the leavening agent is that the preparation method comprises the following steps: screening aspergillus oryzae, fragile mucoraceae, yeast, and bacterium composition thereof, and preparing bran yeast thereof; determining optimal adding ratio for fermenting the cassava residue; uniformly mixing according to the ratio to form composite solid leavening agent containing sufficient activated yeast cells, aspergillus oryzae spores, and mucoraceae spores; when the solid substrate of the cassava residue fermented by the composite microbial agent contains 15-20% of bran, the bio-fermentation product is an excellent feed raw material. The microbial agent is cheap in manufacturing cost, and convenient to use, and qualified fermentation product can be obtained by adding a proper amount of composite leavening agents into a cassava residue solid substrate according to the actual situation and conducting proper cultivation; the fermentation product can be used as the feed raw material, so that the food is saved, cultivation cost is reduced, and the economic benefit of cultivation is improved.
Description
Technical field
The invention belongs to biological fermentation feed field, particularly the recycling of plant refuse and biological fodder field.
Background technology
Cassava has another name called cassava, tree turns over potato, be one of world's staple food grain, there is the laudatory title of " king of starch ", " underground grain ", mainly originate in tropical and subtropical region, be collectively referred to as the large tuber crops in the world three with sweet potato, potato, the output of cassava ranked seventh position in food crop.Current, approximately there is more than 90 countries and regions cultivation cassava in the whole world, and cultivated area exceedes 1,400 ten thousand hectares, and ultimate production is about 1.3 hundred million tons.Cassava is widely used in starch producing, alcohol and feed, and is further applied to the aspects such as food, medicine, papermaking, building materials.The main growing area of China cassava in Guangxi, the province such as Guangdong, Hainan, Yunnan, Fujian.In Chinese Cassava Starch Production district, the output in Guangxi occupies the first, accounts for more than 1/2nd, and annual Guangxi cassava slag output is about 400,000 tons.
Manioc waste is the mixture that cassava has been extracted the later tankage of starch, physical and chemical composition analysis shows: in manioc waste, the content of non-nitrogen compound is the highest, can reach 78.7%, its main component is Zulkovsky starch compound (as single sugar and starch), and this is good carbon source; In addition, manioc waste also contains close with cassava, a large amount of other nutritive ingredients, for example, comprise the multiple trace element useful to animal body.China's manioc waste resource storage is abundant, measures by present production level, can produce every year the manioc waste (dry-matter meter) of 9.25 ten thousand tons of left and right.
Both at home and abroad to manioc waste research, only with its manufacture order cell protein, alcohol, phytase, citric acid or cultured mushroom etc., very few to the utilization of manioc waste.In livestock industry is produced, the treatment process of manioc waste Fodder making is had to three kinds: the first is directly to add calorie-protein matter feed making with fresh wet condition to become mixed feed, the second is to be dried to after particle as energy feed or roughage, and the third is to add microorganism to make fermented feed.But the technique of these three kinds for the treatment of processs is all immature, and Disposal quality is uneven, feeding effect is not good.This has caused a large amount of manioc wastes to go out of use, and has not only caused the significant wastage of resource, and severe contamination environment.
Along with the fast development of aquaculture, the relative shortage of feed resource manifests day by day; In addition China has a large population and a few land, the basic condition that people subtracts with increasing, and people and animals strive the contradiction of grain; In the urgent need to developing the feed resource of non-grain class.Therefore, sufficiently and reasonably utilize agricultural byproducts tankage, can not only alleviate environmental pollution, and can alleviate to a certain extent the shortage problem of feed resource, reduce aquaculture cost.
In manioc waste, contain a large amount of starch, but protein content is low, crude fiber content is higher, and amino acid composition is extremely uneven, is of low nutritive value; Also contain a small amount of prussic acid, palatability is poor, is directly used for feeding fowl poultry weak effect.Therefore, improving protein content is the top priority of exploitation manioc waste as feedstuff raw material.
Existing research shows to add suitable nutritive substance in manioc waste, can be single-cell protein by part Substance Transformation through microorganism fermentation; Produce the micro-raw element of a large amount of B families and plurality of enzymes simultaneously, improve the nutritive value of manioc waste.Therefore, take manioc waste as raw material, utilize microorganism fermentation, be translated into single-cell protein feed, both can have solved China's feed resource shortage, improve cassava plantation and processing industry benefit; Can turn waste into wealth again, reduce environmental pollution, additional income.
Summary of the invention
One of object of the present invention is to provide a kind of composite fungus agent, is the special bacteria agent of fermentation manioc waste.
For achieving the above object, technical scheme of the present invention is:
Be applicable to the prebiotic bacterial classification of manioc waste solid state substrate fermentation, described probiotic bacterium is that preserve in laboratory, and is bacterium, yeast and the mould etc. of national regulation, and concrete probiotic bacterium kind refers to table 1 to be enumerated.
As preferred scheme, described composite bacteria is made up of aspergillus oryzae and geotrichum candidum; Or described composite bacteria is made up of with easily break wool is mould aspergillus oryzae; Or described composite bacteria is made up of yeast and aspergillus oryzae; Or described composite bacteria is made up of with easily break wool is mould yeast; Or described composite bacteria is by yeast, aspergillus oryzae with easily break wool is mould forms.
Further, described composite bacteria is by quantity ratio pichia spp: aspergillus oryzae: easily break wool mould=3～6:1～4:1～5.
Further, described composite bacteria, by quantity ratio pichia spp: aspergillus oryzae: easily break wool mould=6:2:1.
Two of object of the present invention is to provide a kind of biological fodder, and it is take the manioc waste discarded as fermentation main raw material, and production cost is low.
For achieving the above object, technical scheme of the present invention is:
The biological fodder that contains described composite bacteria, described biological fodder is obtained by described composite bacteria and manioc waste co-fermentation.
Further, described biological fodder, is added with wheat bran in manioc waste solid state substrate wherein.
Further, in described manioc waste solid state substrate, the addition of wheat bran is 15%～20% by weight.
Three of object of the present invention is to provide a kind of preparation method of biological fodder, is applicable to suitability for industrialized production.
For achieving the above object, technical scheme of the present invention is:
The preparation method of described biological fodder, specifically comprises the steps:
1) preparation of culture medium A and substratum B
According to glucose 2%, peptone 0.5%, yeast extract paste 0.2%, KH
2pO
40.1%, MgSO
47H
2o0.05%, simple distillation water 1000ml, is adjusted to 5.0 left and right by pH value, prepares liquid nutrient medium.Dry wheat bran and described liquid nutrient medium, according to the mass ratio of 1:1.0～1.1, are fully mixed, and high pressure steam sterilization, is cooled to 30 ℃, obtains culture medium A.
The potato 200g that weighs peeling, adds water boil 20～30min, by filtered through gauze, adds 20g glucose, stirs, to be dissolved completely after, add 15g agar.Supply afterwards moisture to 1000ml, packing Erlenmeyer flask.120 ℃ of sterilizing 20min, taking-up shakes up, and is down flat plate, stores for future use after cooling, obtains substratum B.
2) preparation of composite bacteria
The various daughter bacterias of composite bacteria described in claim 1 are carried out to coated plate inoculation with described substratum B, cultivate 2～3 days for 28-30 ℃, plant the preparation of daughter bacteria liquid with reference to " Fl á vera Camargo Prado; Luciana Porto de Souza Vandenberghe; Carlos Ricardo Soccol; Relation between citric acid production by solid-state fermentation from cassava bagasse and respiration of Aspergillus niger LPB21in semi-pilot scale[J], Brazilian Archives of Biology and Technology, 2005,48:29-36 " middle bacterial classification collection method execution.Use 0.01%Tween80 solution homogenate substratum B, suspension comprises 10
9cFU/ml microorganism, 4 ℃ of preservations.Obtain the bacterium liquid of planting separately daughter bacteria.
3) preparation of solid fungicide
By step 2) in the bacterium liquid of various daughter bacterias by 0.5%～2% inoculum size, be inoculated in culture medium A, mix, when humidity is 60%～65%, be placed under the condition of 28 ℃～30 ℃ of constant temperature, solid ventilation is cultivated 2～4 days, in culturing process, stir 2 every day, each 1 time in afternoon in the morning; Culture dehydrates as wheat bran song under 30 ℃～45 ℃ conditions, microbe population Ying≤10 in wheat bran song
9cfu/g; Obtain solid list microbial inoculum; , the solid list microbial inoculum of gained is mixed according to the ratio of 6:2:1 meanwhile, obtain composite bacteria.
4) preparation of manioc waste solid state substrate
Be manioc waste by manioc waste and wheat bran according to mass ratio: the ratio of wheat bran=80:10～20 is mixed, and after stirring, obtains the substratum of manioc waste solid state substrate.
5) preparation of biological fodder
Inoculum size by 0.5%～3.0% is seeded to step 4) gained composite fungus agent in described manioc waste solid substrate culture, carries out biological fermentation, and leavening temperature is 28 ℃～30 ℃, and fermentation time is 3～4 days, obtains the biological fodder of manioc waste; The content that makes probiotics in described biological fodder is>=10
9cfu/g.
Four of object of the present invention is to provide the new application of manioc waste, for the comprehensive utilization of manioc waste provides new thinking.
For achieving the above object, technical scheme of the present invention is:
Using the manioc waste biological fodder of fermentation as feedstuff raw material, coordinate and become complete feed with other feedstuff raw material, feeding animals; Reach and save food, reduce the object of aquaculture cost.
Beneficial effect of the present invention is: 1) select to be suitable for the bacterial classification of manioc waste growth, screen the microbial inoculum of best of breed according to consistency feature between bacterial classification.2) described combination microbial inoculum is prepared as the air-dry bacterial classification product of wheat bran song, can preserve for a long time.Microbial inoculum is with low cost, easy to use, and user only needs in conjunction with producing actual, adds auxiliary material wheat bran, and proportioning manioc waste is prepared solid state substrate raw material, adds compound ferment, then under proper temperature, cultivates and can obtain qualified leavened prod.3) described manioc waste solid medium, adopts wheat bran as auxiliary material, utilizes it as water suction and raising agent, and the addition of wheat bran is 15%～20%.4) adopt after this zymotechnique, tunning crude protein content can reach 10.27%, and 3.43% while fermentation improved more than 3 times, and protein content obviously improves.
Accompanying drawing explanation
Fig. 1 is that two bacterium are combined on manioc waste plate culture medium and contact growing state.
Fig. 2 is that three bacterium are combined in mixed growth situation on manioc waste plate culture medium.
Fig. 3 is aspergillus oryzae and pichia spp different ratios combined inoculation fermentation manioc waste product coarse albumen and aminoacids content mutation analysis figure.
Fig. 4 is pichia spp, aspergillus oryzae and the easy mould different ratios mixed fermentation of break wool manioc waste crude protein and aminoacids content mutation analysis figure.
Fig. 5 is the tunning crude protein content mutation analysis of optimizing microbial inoculum auxiliary material Different adding amount.
Fig. 6 is auxiliary material Different adding amount bottom fermentation product crude protein increasing amount mutation analysis figure.
Embodiment
Manioc waste waste refers to that cassava has been extracted the mixture of the later tankage of starch, and physical and chemical composition analysis shows, in art potato slag, the content of non-nitrogen compound is high, can reach 78.7%, and its main component is Zulkovsky starch compound (as single sugar and starch).Manioc waste in embodiment is to prepare alcohol manioc waste afterwards to use as test.
The inoculum size of mentioning in following embodiment is seed liquor or the quality of solid fungicide and the mass percent example of the rear substratum of inoculation referring to an access.
The prebiotic bacterial classification of embodiment 1 and manioc waste are cultivated altogether
Bacterial classification and manioc waste are cultivated altogether mainly and are carried out according to " dull and stereotyped dibbling " experimental technique, and screening adapts to the bacterial classification of manioc waste growth most." dull and stereotyped dibbling " test reference " Chen Song, Ding Lixiao, Zhang Li, Wang Tianlong, Zhu Yinglian, the screening [J] of the mixed strains of 2008. fermentation apple pomace to produce protein feed. food and fermentation industries, 2008,34(2): 94-96 " carry out.The reference " Liu Shuli, 2008.D increases the bacterial screening research of citrus peel residue fermented feed crude protein content " of preparing of manioc waste plate culture medium is carried out, and does suitably to improve; Be specially: manioc waste 3.0g, agar 2.0g, 100mL nutritive medium, 115 ℃ of sterilizing 20min, are down flat plate after mixing; Compounding (g/L) wherein: NaCl0.50g, MgSO
40.50g, (NH
4)
2sO
43.00g, KH
2pO
41.00g, CaCl
20.30g, FeSO
47H
2o0.01g, MnSO
47H
2o0.015g, ZnSO
47H
2o0.015g, CoCl
20.01g, distilled water 1L.
The bacterial classification of cultivating altogether comprises:
Probiotic bacteria: subtilis (Bacillus subtilis), Bacillus coagulans (Bacillus coagulans), lichens gemma (Bacillus licheniformis), bacillus cereus (Bacillus cereus), lactobacillus (Lactobacillus lactis), lactobacillus plantarum (Lactici plantarum), for preserving in laboratory.
Filamentous fungus: aspergillus niger (Aspergillus niger, numbering CICC2089), aspergillus oryzae (Aspergillus oryzae, numbering CICC2013), geotrichum candidum (Ceotrichum candidum, numbering CICC1315), Rhizopus oryzae (Rhiizopus oryzae, numbering CICC3037), viride (Trichoderma viride numbers CICC40502), easily mould (the Mucor fragilis of break wool, numbering CICC3093), Endomycopsis Fibnligera (Endomycopsis fibuliger, numbering CICC31258), Aspergillus usamii (Aspergillus usamii, numbering CICC2145), ball hair shell (Chaetomium globosum, numbering CICC41408), paecilomyces (Paecilomyces sp., numbering CICC41447), purchased from Chinese industrial microbial strains preservation center (China Center of Industrial Cuture Collection, be called for short CICC), Aspergillus nidulans (Aspergillus nidulans) is so kind as to give for Southwestern University.
Yeast: Candida utilis (Candida utilis, numbering CICC1422), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae, numbering CICC31362), Angel Yeast is (purchased from Angel Yeast company limited, numbering Saccharomyces angel), rice wine yeast (teacher of Southwestern University is so kind as to give, Rice wine yeast, numbering RWY SWU-1), pichia spp (Pichia sp. numbers CICC32708), preserve in laboratory.
Dull and stereotyped dibbling test demonstration, the growing state of strain screening on manioc waste flat board, the results are shown in Table .1.
The growth of table 1 probiotic bacterium on 3% the restricted plate culture medium of manioc waste agarose
Note :-,+, ++, +++ represent that respectively microbial strain culture is unchanged or grow hardly, thalline is grown on a small quantity or is grown slowly, and thalline is obviously visible, and thalli growth is good.
As seen from table:
(1) bacterial growth performance: nearly all prebiotic spore bacteria and milk-acid bacteria, a little less than nearly all can not growing or grow on the restricted plate culture medium of manioc waste.Analysis may be that what ammonium sulfate can be very fast is dissociated into SO because of in the manioc waste substratum for being made up of nutritive medium
4 2-and NH
4 +, NH
4 +for microorganism digests and assimilates, SO
4 2-relatively increase, cause pH to reduce, result has suppressed bacterial growth.
(2) fungal growth performance: five saccharomycete well-growns, show that these yeast can tolerate higher acidity.Although filamentous fungus also can tolerate the environment of low acidity, can be on the restricted substratum of manioc waste growth and breeding, the growth differences of filamentous fungus is obvious especially; Aspergillus nidulans growth is wherein poor; And aspergillus oryzae, Rhizopus oryzae and easily break wool is mould grows fine on solid medium are the bacterial classifications for the manioc waste that ferments preferably.
2 liang of bacterium combinations of embodiment are cultivated altogether with manioc waste
Consider that aspergillus oryzae is the bacterial strain that a class is produced prozyme, except producing proteolytic enzyme, also can produce amylase, saccharifying enzyme, cellulase, phytase etc.Under diastatic effect, the straight chain in manioc waste raw material, amylopectin are degraded to dextrin and various low molecular sugar class, as maltose, glucose etc.; Under the effect of proteolytic enzyme, stodgy macro-molecular protein is degraded to peptone, polypeptide and each seed amino acid; And can make the difficult mass degradation absorbing such as robust fibre, phytic acid in auxiliary material, improve nutritive value, health-care effect and the digestibility of manioc waste.Aspergillus oryzae is widely used in the fermentation industries such as food, feed, production kojic acid, wine brewing, and has been applied safely more than 1000 year.And, aspergillus oryzae is also (the TheAssociation of American Feed Control Officials of U.S. feed control official association, and (the Food and Drug Administration of food and drug administration AAFCO), FDA) be known as the microbial strains of safety (Generally Recognized as Safe, GRAS).Therefore, test and Selection aspergillus oryzae further investigation as combined sorting as indicator.
Using aspergillus oryzae as indicator, by equal-volume, same concentrations (10
7cfu/g) another strain bacterium liquid (filamentous fungus of mentioning in embodiment 1, adapt to manioc waste) dibbling is to equidistant position, symmetric line both sides of the restricted plate culture medium of manioc waste, cultivate 7 days for 28 ℃～30 ℃, continue the growing state of bacterium colony on observed and recorded flat board.Selection is grown fine, can be combined the culture experiment that carries out three bacterium combinations with yeast by microorganism compatible and collaborative symbiosis.
Result shows: since two bacterium point samples, until dull and stereotyped upper growth is to contacting with each other, Continuous Observation mixed culture microbial growth, can find: two strain filamentous fungus initial stages can both be on the restricted substratum of manioc waste well-grown; Different strain combination subsequently presents different growthhabits, is mainly divided into following three kinds of situations with the bacterial classification of aspergillus oryzae combination: combined this strain bacteria growing of (1) and aspergillus oryzae is slower, and the bacterium colony constantly being expanded by aspergillus oryzae gradually surrounds, covers.(2) fast with this strain growth of the combined combination of aspergillus oryzae, there is obvious space in the contact position of two bacterium colonies, around aspergillus oryzae growth, grow but cannot enter in aspergillus oryzae.(3) two strain bacterium of combination are in bacterium colony contact position, and mycelia intermeshes and is grown in together, and can enter field growth each other, and what all mixed culture were grown for 7 days the results are shown in Table 2.
A table 2 liang bacterium is combined on manioc waste plate culture medium and contacts growing state
As can be seen from the table, aspergillus oryzae and other the thread bacterial classification obvious difference of incubation growth that coexists.Generally speaking, in two bacterium combinations, the best filamentous fungus combination coexisting is mainly aspergillus oryzae (CICC2013)+geotrichum candidum (CICC1315), and aspergillus oryzae (CICC2013)+easily two groups of combinations of break wool mould (CICC3039).
The bacterium liquid of each primary yeast (embodiment 1) of that screen, same concentrations, equivalent is coated on to the restricted plate culture medium of manioc waste, aspergillus oryzae (the A.oryzae)+geotrichum candidum (C.candidum) screening with embodiment 2, and aspergillus oryzae (A.oryzae)+easily the combination bacterium of break wool mould (M.fragilis) is point sample bacterial classification, under same concentrations, dibbling is to equidistant position, symmetric line both sides of yeast plate culture medium, cultivate 7 days for 28 ℃～30 ℃, the growing state that continues bacterium colony on observed and recorded flat board, result shows:
Compare with the two bacterium assembled growths of aspergillus oryzae (A.oryzae)+geotrichum candidum (C.candidum) in control group (without yeast), the growing state of the filamentous fungus lawn in " three bacterium combinations " shows as: geotrichum candidum is grown poor or do not grown on all yeast culture bases, but the growth conditions of aspergillus oryzae bacterium colony is good.Therefore the combination that, can determine aspergillus oryzae (CICC2013)+geotrichum candidum (CICC1315)+yeast is not best strain combination.
In like manner, compare control group, filamentous fungus is combined as aspergillus oryzae (A.oryzae, CICC2013)+easily mould (M.Fragili of break wool, CICC3039) two bacterium can grow preferably on yeast flat board, but on different yeast flat boards, two filamentous fungus bacterium colony contact positions present different forms, in the time of mixed culture five days, colony growth records in Table .3.
Table 3 three bacterium are combined in mixed growth situation on the restricted flat board of manioc waste
Note :+, ++, +++ and ++++represent that respectively the effect of three bacterium combinations is poor, general, better and fine.
As seen from table: the flat board of various Yeast Growths is different to the growth effect of aspergillus oryzae and the mould combination of easy break wool bacterium.But it is known that observation and comparison is analyzed, aspergillus oryzae (CICC2013) wherein+easily the effect of mould (the CICC3039)+pichia spp of break wool (Pichia sp.) three bacterium combinations is best, and to be that filamentous fungus is collaborative during with yeast mixed culture promote the one of growing to react for this.Main manifestations is that the density of aspergillus oryzae is high, lawn self-sow, and size is little with contrast difference.In addition, easily the mould growth of break wool is rapid, covers whole cultivation face.
It is worth mentioning that, aspect the ductility of microorganism, not obvious compared with control group (two bacterium are cultivated) difference when three bacterium mixed culture, illustrate when this three bacterium grows jointly, when silk bacteria growing, secreting various enzymes and process nutritive substance (as starch) the formation monose in manioc waste, is yeast utilization; After monose is utilized, remove feedback inhibition, promoted the growth of thread strain combination.Therefore,, according to the result of three bacterium combinations, selecting aspergillus oryzae (CICC2013)+easy mould (CICC3039)+pichia spp of break wool (Pichia sp.) is best combination.
The best inoculation quantity of embodiment 4 ratio of mixture
1 method
The manioc waste 20g and the 20mL nutritive medium that in 250mL triangular flask, add pulverizing, 115 ℃ of sterilizing 20min make solid-state fermentation culture medium.Cooling rear interpolation mixed fermentation bacterial classification, bacteria suspension makes its bacterial concentration be controlled at 10 by adjustment
7individual/mL order of magnitude.When combined inoculation, the reference of best inoculation blending ratio " Xu Kangzhen; Song Jirong; Huang Jie; the research [J] of the best inoculation of Yin Tao single cell protein ratio of mixture. microbiology circular 2003,30(4): 36-39. " described method is carried out, and work suitably improves, cultivate after about 100 hours and measure protein content, crude protein adopts Micro-kjoldahl method (GB/T14771-93).
Determining of the 2 pairs of best inoculation of bacterium ratio of mixture
According to the formation bacterial classification of " three bacterium best of breed bacterium ".First,, by aspergillus oryzae and pichia spp combination, form " two bacterial classification " and ferment and be divided into one group.Under the consistent condition of bacterial classification concentration, according to pichia spp: aspergillus oryzae=5:1,4:1,3:1,2:1,1:1,1:2,1:3,1:4 and 1:5, when addition is identical, mixed culture manioc waste, cultivated through 4 days, dried tunning, measured gross protein value.Control group is the manioc waste solid fermentation that does not add bacterial classification.Utilize binary system of coordinates to analyze under different ratios gross protein value and corresponding amino acid change curve thereof in " solid state fermentation " manioc waste, determine the best inoculation blending ratio of the different adding proportions of two strain combinations, experimental result is as Fig. 3.
As seen from the figure: crude protein and aminoacids content variation present positive correlation, and when crude protein content is high, corresponding aminoacids content is also high.Just, in the time that bacterial classification does not add, the aminoacids content of the crude protein in manioc waste and correspondence is very low; Add after fermented bacterium, crude protein and amino acid whose amount increase obviously, yeast: aspergillus oryzae is near inoculation ratio of mixture is 3:1 time, and crude protein and amino acid whose content are the highest.Afterwards, along with mixing the reduction of ratio, crude protein quantity declines gradually.Therefore, selecting ratio is the better inoculation mixing ratio of 3:1 place for two bacterial classifications.
Determining of the best inoculation of 3 three bacterial classifications ratio of mixture
Two bacterial classifications (yeast and aspergillus oryzae) being added to " best of breed " (see above state be 3:1) of ratio does as a whole, again that this entirety is mould combined with easy break wool, under identical bacterial concentration, according to aforementioned identical method, be combined into different mixing proportion, add in fermentation manioc waste, through 4 days fermentative processing.Dry tunning, measure the gross protein value of tunning.The best inoculation ratio of mixture of analyzing three bacterial classifications under two-dimensional coordinate system, wherein combination is compared to X-coordinate, and crude protein content is as ordinate zou.Test-results is as Fig. 4.
Result shows: along with the kind of adding fermented bacterium increases, crude protein, aminoacids content increase thereupon; And the two bacteria fermentation increasing amounts of combination are than the height of single bacterium, but increase low than three bacterium, reflect that the species diversity in microflora can affect system function, the aggregate being combined into by different microorganisms is brought into play vital role more in the process of bio-transformation.Because filamentous fungus class can produce plurality of enzymes (as proteolytic enzyme, amylase, cellulase and phytase) etc., can well utilize the material such as Mierocrystalline cellulose, starch in manioc waste culture material to generate sugar, yeast class can utilize sugar to carry out growth metabolism, so both can alleviate product retarding effect, promote again the growth of yeast, thereby improved fermentation efficiency.In addition, in the result of three bacterial classification ratio of mixture fermentations manioc wastes, known ratio has the highest crude protein content and amount of amino acid in the time of 2:1, i.e. the best inoculation of three bacterial classifications quantity ratio of mixture are pichia spp: aspergillus oryzae: easily break wool mould=6:2:1.
The Analysis on amino acid components of embodiment 5 manioc waste leavened prods
Pichia spp by concentration that screen, unified: aspergillus oryzae: easily break wool mould=combination microbial inoculum that the ratio of 6:2:1 is mixed, mix with manioc waste, humidity reaches 50-70% left and right, and temperature is 28 ℃ of-30 ℃ of fermentations, fermentation time is about 100 hours, and leavened prod low temperature is dried.The Amino acid profile of measuring leavened prod, detected result is in table 4.
Contents of Amino Acids value in the product of table 4 three bacterium fermentation manioc wastes
As seen from table: compare with blank, nutritive medium group, single bacterium group and two bacterium group, in three bacterium combined fermentation products, crude protein, amino acid whose content increase fairly obvious.In 17 seed amino acids of measuring, indispensable amino acid has rising in various degree.Result proves that the protein increasing in tunning is that the single-cell protein being generated by thalline fermentation manioc waste causes.
Embodiment 6 auxiliary material mixed fermentation optimum ratios
For obtaining the easiest, best effect, most economical biological fermentation, select wheat bran for adding auxiliary material, as the raising agent of manioc waste water-retaining agent and fermentation.The addition of wheat bran is with eight gradients such as 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, is divided into sterilizing and unsterilised two kinds of modes are carried out.By aforementioned best three bacterium combinations bacterial classifications (pichia spp: aspergillus oryzae: easily break wool mould=6:2:1) add in the mixture of auxiliary material and manioc waste, keeping humidity is 50～70%, stir, at 28～30 ℃, cultivation 90h.Tunning, approximately drying in low temperature (approximately 40 ℃) baking oven, is measured the changing value of crude protein content as Fig. 5.
Result shows: along with the prolongation of fermentation time, in sterilizing and unsterilised sample, the content of crude protein increases gradually.But, under two kinds of modes the variation of crude protein quality not obvious, show that the effect of sterilizing or unsterilised fermentative processing is about the same; This ferments foundation is provided for producing the unsterilised mode of upper selection.
Analyse in depth under sterilizing or the different adding proportions of unsterilised two kinds of processing mode auxiliary materials the increment rate of protein in tunning.Overall observation discovery is along with wheat bran addition increases, and the crude protein in unit mass is first to increase, and reduces gradually afterwards, and the increment rate of protein presents the feature of normal distribution.Be under two kinds of processing modes, the wheat bran addition difference that the highest increased value of protein content is corresponding; Wherein unsterilised sample is in the time that auxiliary material wheat bran addition is 15%, and the increasing amount of protein is the most obvious; And sterilizing sample is in the time that auxiliary material wheat bran addition is 20%, the increasing amount of protein is the most obvious, refers to Fig. 6.
Comprehensive analytic explanation: biological fermentation manioc waste, improve its feed nutrition level, when exploitation manioc waste is used as feedstuff raw material, take unsterilised mode, when wheat bran is added to 15%-20%; The protein level of biological fermentation raising manioc waste is the most obvious; For the fermentation scheme of manioc waste the best.
The preparation of 7 kinds of daughter bacteria bacterium liquid of embodiment
Get aspergillus oryzae that lyophilized powder preserves, easily mould the and pichia spp PDA substratum of break wool carries out the inoculation of kind of daughter bacteria, cultivates 2～3 days for 28 ℃～30 ℃, observation growth also saves backup.Afterwards, collect the kind daughter bacteria on PDA flat board, obtain respectively aspergillus oryzae, the easy bacterium liquid of the mould and pichia spp three all daughter bacterias of break wool.
The preparation of 8 kinds of daughter bacteria solid fungicides of embodiment
Produce solid state fermentation agent by the strategy of making wheat bran song, method is: (1) selects the carrier of wheat bran song: select high-quality, dry, homogeneous wheat bran skin as the carrier of preparing wheat bran song, save backup; (2) obtaining liq substratum: glucose 2%, peptone 0.5%, yeast extract paste 0.2%, KH
2pO
40.1%, MgSO
47H
2o0.05%, simple distillation water 1000ml, is adjusted to 5.0 left and right by pH value.(3) prepare the bent solid medium A of wheat bran: the above-mentioned wheat bran carrier preparing and liquid nutrient medium are mixed according to the mass ratio of 1:1.0～1.1, stir, 115 ℃ of high pressure steam sterilizations 30 minutes, are cooled to 30 ℃, obtain solid medium.
By 0.5%～2% inoculum size inoculation aspergillus oryzae, the easy bacterium liquid of the mould and pichia spp three all daughter bacterias of break wool, carry out conventional solid ventilation fermentation (28 ℃ of-30 ℃ of 72h), in culturing process, stir 2 every day, normally each 1 time in afternoon in the morning.After cultivation finishes, carry out microorganism count, get tunning 20g, 150mL aqua sterilisa (containing 0.5ml Tween80), mixes by magnetic stir bar, filters the suspension obtaining containing spore, and spore concentration is added up with cell counter.Culture dehydrates under low temperature (30 ℃～45 ℃) condition, dries seed bacteria microorganism quantity Ying≤10 in product (solid-state microbial inoculum)
9cfu/g, the solid fermentation list microbial inoculum of acquisition manioc waste.
The preparation of embodiment 9 complex ferment microbial inoculums
The solid wheat bran aspergillus agent of kind daughter bacteria that described embodiment 8 is obtained (aspergillus oryzae, easily mould the and pichia spp of break wool), according to the ratio of 6:2:1, mixes, and carries out low temperature dewatering and dry, pulverize preservation, obtains complex ferment microbial inoculum.
With the biological fodder product of complex ferment microbial inoculum fermentation manioc waste, as feedstuff raw material, be combined into complete diet pellet, carry out the nursing experiment of growing and fattening pigs, the impact of the growth performance on it is in Table .5.Found that the complete feed that adds leavened prod and be combined into, can reduce aquaculture cost, save food.In addition, have suffered in experimentation growing and fattening pigs health anosis, search for food normal, food consumption is without obvious visible change.
In table 5 fattening pig basal diet, add the impact of manioc waste biological fermentation material on its growth performance
Note: colleague's shoulder motes complete different person's significant differences of lowercase (p < 0.05), colleague's shoulder motes lowercase has not remarkable (p > 0.05) of identical person's difference.
Finally explanation: above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (10)
1. the composite bacteria that is applicable to manioc waste solid fermentation, is characterized in that: described composite bacteria is by yeast and/or aspergillus oryzae and/or Rhizopus oryzae and/or easily break wool is mould forms.
2. composite bacteria according to claim 1, is characterized in that: described composite bacteria is made up of aspergillus oryzae and geotrichum candidum; Or described composite bacteria is made up of with easily break wool is mould aspergillus oryzae; Or described composite bacteria is made up of pichia spp and aspergillus oryzae; Or described composite bacteria is made up of with easily break wool is mould pichia spp; Or described composite bacteria is by pichia spp, aspergillus oryzae with easily break wool is mould forms.
3. composite bacteria according to claim 2, is characterized in that: by quantity ratio pichia spp: aspergillus oryzae: easily break wool mould=3～6:1～4:1～5.
4. composite bacteria according to claim 2, is characterized in that, by quantity ratio pichia spp: aspergillus oryzae: easily break wool mould=6:2:1.
5. contain biological fodder claimed in claim 1, composite bacteria, it is characterized in that: described biological fodder is obtained by described composite bacteria and manioc waste co-fermentation.
6. biological fodder according to claim 5, is characterized in that: in described biological fodder, be added with wheat bran.
7. biological fodder according to claim 6, is characterized in that: the addition of described wheat bran is 15%～20% by weight.
8. the preparation method of biological fodder claimed in claim 5, is characterized in that, specifically comprises the steps:
1) preparation of culture medium A and substratum B
According to glucose 2%, peptone 0.5%, yeast extract paste 0.2%, KH
2pO
40.1%, MgSO
47H
2o0.05%, surplus is simple distillation water, and pH value is adjusted to 5.0 left and right, prepares liquid nutrient medium, and dry wheat bran and described liquid nutrient medium, according to the mass ratio of 1:1.0～1.1, are fully mixed, high pressure steam sterilization, is cooled to 30 ℃, obtains culture medium A;
The potato 200g that weighs peeling, adds water boil 20～30min, by filtered through gauze, add 20g glucose, stir, to be dissolved completely after, add 15g agar, supply afterwards moisture to 1000ml, packing Erlenmeyer flask, 120 ℃ of sterilizing 20min, taking-up shakes up, be down flat plate, store for future use after cooling, obtain substratum B;
2) preparation of composite bacteria liquid
The various daughter bacterias of composite bacteria described in claim 1 are carried out to coated plate inoculation with described substratum B, cultivate 2～3 days for 28-30 ℃, use afterwards 0.01%Tween80 solution homogenate substratum B, suspension comprises 10
9cFU/ml microorganism, 4 ℃ of preservations;
3) preparation of solid fungicide
By step 2) in the bacterium liquid of various daughter bacterias by 0.5%～2% inoculum size, be inoculated in culture medium A, mix, when humidity is 60%～65%, be placed under the condition of 28 ℃～30 ℃ of constant temperature, solid ventilation is cultivated 2～4 days, and culture dehydrates as wheat bran song under 30 ℃～45 ℃ conditions, microbe population Ying≤10 in wheat bran song
9cfu/g; Obtain solid list microbial inoculum;
4) preparation of manioc waste solid state substrate
Be manioc waste by manioc waste and wheat bran according to mass ratio: the ratio of wheat bran=80:10～20 is mixed, and after stirring, obtains the solid state substrate substratum of manioc waste;
5) preparation of biological fodder
The solid fungicide of step 3), by 0.5%～3.0% inoculum size, is added to described in step 4) in manioc waste solid substrate substratum, carry out biological fermentation; Leavening temperature is 28 ℃～30 ℃, and fermentation time is 3～4 days, obtains biological fodder; The content that makes microorganism in described biological fodder is>=10
9cfu/g.
9. manioc waste is in the application of preparing in fungi culture medium, and described fungi comprises yeast and/or aspergillus oryzae and/or Rhizopus oryzae and/or easily break wool is mould.
10. application according to claim 9, is characterized in that: manioc waste is combined in the application of preparing in fungi culture medium with wheat bran.
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| CN106306361A (en) * | 2016-08-25 | 2017-01-11 | 宁夏泰瑞制药股份有限公司 | Method for preparing biological fermentation feed |
| CN107173556A (en) * | 2017-05-04 | 2017-09-19 | 凤台硕力畜禽养殖有限公司 | A kind of processing method for making an addition to manioc waste in sheep feed |
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| CN109329567A (en) * | 2018-12-05 | 2019-02-15 | 湖南赛福资源饲料科技有限公司 | It is a kind of using cassava and cassava cauline leaf as the fermented feed of raw material and preparation method |
| CN109805159A (en) * | 2019-03-01 | 2019-05-28 | 山西农业大学 | The production method of composite bacteria solid state fermentation linseed meal detoxification protein feed |
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