CN103098981B - Dewatered solid combined inoculant for increasing protein level of citrus pulp feed and preparation method of dewatered solid combined inoculant - Google Patents
Dewatered solid combined inoculant for increasing protein level of citrus pulp feed and preparation method of dewatered solid combined inoculant Download PDFInfo
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Abstract
The invention belongs to the technical field of biological fermentation, and mainly relates to the field of recycling of citrus pulp wastes and the field of biological feed. Technical points of a leavening agent are as follows: choosing aspergillus niger, geotrichum candidum and rice wine yeast which are appropriate for growing on the citrus pulp; resuscitating and respectively enlarging a solid culture which is used for producing seed bacteria, and dewatering and drying at low temperature; and mixing uniformly according to the content of microorganisms in the solid seed bacteria to form a composite solid biological leavening agent containing enough active rice wine saccharomycetes, aspergillus niger spores and geotrichum candidum spores. A product obtained by fermenting the citrus pulp by using the composite leavening agent is a good feed material; the inoculant is low in cost and convenient to use; enterprises just need to proportion solid matrix materials of the citrus pulp in combination of actual production, add a proper amount of the composite leavening agent and culture at proper temperature to obtain a qualified fermented product; and the use of the fermented product as the feed material can save the grain and increase the economic benefit of the breeding industry.
Description
Technical field
The invention belongs to biological fermentation field, particularly the recycling field of plant refuse and biological feedstuff field.
Background technology
Citrus, or writing both citrus, belong to Rutaceae, comprise tangerine, mandarin orange, shaddock, orange, lemon etc., be mainly distributed in the region on the south north latitude 35-degree in the world.Citrus plant tool unifoliate compound leaf, forms the mandarin orange fruit with keratin pericarp.Citrus fruits slag refers to that mandarin orange fruit processes the leftover bits and pieces of squeezing the juice or after can processed, accounts for the about 25-50% of weight of mandarin orange fruit.The main component of citrus fruits slag is the organic matters such as water-fast carbohydrate (comprising pectin, cellulose, hemicellulose), soluble sugar and bitter substance.It can provide carbon source and the energy of growth of microorganism.
China is the native place of citrus fruit, have every year a large amount of citrus fruits for the production of, processing.Particularly in recent years, along with the development of agricultural sector structure adjustment and agricultural modernization, its industry development impetus is swift and violent, and output significantly improves, citrus fruit is one of pillar industry becoming agricultural economy, for promoting rural economy, effectively improving the ecological environment and made positive contribution.Meanwhile, along with citrus fruit industry development, fruit is processed process in a large number, and cause the output of citrus fruits slag large especially, the storage of citrus fruits slag resource is enriched.
But the pericarp in citrus fruits slag, protein content is not high, and palatability is poor, and animal absorbs low, and a large amount of citrus pulps does not obtain comprehensive utilization, causes citrus pulp offal treatment cost day by day soaring.Current common processing method be by citrus pulp with land-fill method or enter rivers, cause serious environmental pollution.Therefore, the recycling of citrus pulp becomes the focus that people pay close attention to.
Along with the fast development of aquaculture, feed resource relative shortage, China has a large population and a few land in addition, and people and animals strive the contradiction of grain.Annual needs consumes a large amount of foreign exchange purchase grain, causes livestock feed cost constantly to increase.Sufficiently and reasonably utilize agricultural byproducts leftover bits and pieces to alleviate environmental pollution, and the shortage problem of feed resource can be alleviated to a certain extent, reduce feed cost.
In citrus pulp, add suitable nutriment, part inorganic matter can be converted into SCP through fermentable, produce the B race micro-element of large courage and multiple enzyme simultaneously, improve the nutritive value of citrus pulp.Therefore, be raw material with citrus pulp, utilize fermentable manufacture order cell protein feed, to the shortage of solution China feed resource, improve citrus pulp plantation and processing industry benefit, reduce all significant and using value of environmental pollution.
Summary of the invention
An object of the present invention is the new opplication providing aspergillus niger, and this albumen be applied as in supplementary citrus fruits and/or citrus fruits slag provides thinking.
For achieving the above object, technical scheme of the present invention is:
Aspergillus niger is preparing the application in biological feedstuff as microbial inoculum and citrus fruits and/or citrus fruits slag Dual culture.
Preferably, described aspergillus niger and/or geotrichum candidum and/or rice wine yeast are preparing the application in biological feedstuff as microbial inoculum and citrus fruits slag Dual culture.
Two of object of the present invention is to provide a kind of combination microbial inoculum, and it can work in coordination with the crude protein in afterfermentation thing in citrus fruits and/or the fermentation of citrus fruits slag.
For achieving the above object, technical scheme of the present invention is:
Combination microbial inoculum, by quantity by geotrichum candidum, rice wine yeast and aspergillus niger in 3-5: 0-4: 5-10 ratio mix.
Preferably, described combination microbial inoculum, by quantity by geotrichum candidum, rice wine yeast and aspergillus niger in 4: 1: 5 ratio mix.
Preferably, described combination microbial inoculum, mixes geotrichum candidum, rice wine yeast and aspergillus niger, obtains mixture, and mixture low temperature dewatering is dry, then pulverizes.
Three of object of the present invention is the preparation method providing this described combination microbial inoculum, and the method process stabilizing, cost is low.
For achieving the above object, technical scheme of the present invention is:
The preparation method of described combination microbial inoculum, specifically comprises the steps:
The preparation of the bacterium liquid of rice wine yeast bacterium: pulverize after citrus fruits and/or citrus fruits slag are dried, cross 40 mesh sieves, make powder; Often liter of citrusfruit waste extract, adds water to 1 liter by 300 grams, citrusfruit discarded object and stirs, and slagging-off, obtains citrusfruit waste extract; The fluid nutrient medium of rice wine yeast bacterium is prepared: brown sugar 30g, KH in following ratio and method
2pO
41.0g, MgSO
4.7H
2o0.5g, (NH
4)
2sO
42.0g, add citrusfruit waste extract leaching liquor 1L and form, high pressure steam sterilization, obtains the fluid nutrient medium of rice wine yeast bacterium; Press the inoculum concentration inoculation rice wine yeast bacterial classification of 5-10% after cooling, deep liquid aerobic fementation 72 hours at 28 DEG C, obtains the bacterium liquid of rice wine yeast bacterium, rice wine yeast bacterium quantity>=10 in the bacterium liquid of rice wine yeast bacterium
9cfu/mL;
Or wheat bran and water are mixed with the ratio that weight ratio is 1: 0.8-1, stir, high pressure steam sterilization, be cooled to 30 DEG C, obtain culture medium A; Rice wine yeast bacterium liquid is pressed the inoculum concentration inoculation of 0.5-2%, be placed in fermentation bed, thickness of feed layer 5-10cm, at 28 DEG C-30 DEG C, solid ventilation ferments 72 hours, obtains the microbial inoculum of rice wine yeast bacterium, and the saccharomycete quantity in the rice wine yeast microbial inoculum after drying answers>=10
9cfu/g;
The preparation of the microbial inoculum of black-koji mould: in the inoculum concentration inoculated aspergillus niger bacterium by 0.5% and described culture medium A, be placed in fermentation bed, thickness of feed layer 5-10cm, aerobic fementation 72 hours at 28 DEG C, obtain the microbial inoculum of black-koji mould, the black-koji mould quantity in the bacterium liquid of aspergillus niger answers>=10
9cfu/g;
The preparation of the microbial inoculum of Geotrichum: the inoculum concentration inoculated aspergillus niger bacterium by 0.5% is to described culture medium A, and be placed in fermentation bed, thickness of feed layer 5-10cm, at 28 DEG C, aerobic fementation 72 hours, obtains the microbial inoculum of Geotrichum, the spore quantity in the microbial inoculum of geotrichum candidum answers>=10
9cfu/g;
The microbial inoculum of the described microbial inoculum of rice wine yeast bacterium, the microbial inoculum of black-koji mould and black-koji mould is carried out low temperature dewatering drying separately, and mixes, obtain combination microbial inoculum.Inoculum concentration refers to the ratio of volume and the rear nutrient solution volume of inoculation moving into seed liquor.
Four of object of the present invention is the preparation method providing a kind of biological feedstuff, and feed cost prepared by the method is low, is of high nutritive value, and is applicable to industrial applications.
Citrus fruits slag prepares the method for biological feedstuff, specifically comprises the following steps:
The preparation of A solid medium
By the sterilizing of citrus fruits slag, obtain solid medium;
B fermentation
Described aspergillus niger and/or geotrichum candidum and/or rice wine yeast are made microbial inoculum, is seeded in described solid medium by the inoculum concentration of 4-6%, obtains tunning; Or described combination microbial inoculum is seeded in described solid medium by the inoculum concentration of 4-6%, obtain tunning; Described microbial inoculum or combination microbial inoculum>=10
9cfu/mL, fermentation temperature is 28-30 DEG C, and fermentation time is 48-72 hour.
Preferred described citrus fruits slag prepares the method for biological feedstuff, and after fully mixing with 80: 14-16 weight ratio after orange peel slag and the dry also pulverizing of wheat bran, sterilizing, obtains solid medium.
The product that five of object of the present invention is to provide said method to prepare and application thereof, it is high that this product is rich in protein, and this is applied as and reduces feed cost and provide new approaches.
The tunning of the reuse method gained of described orange peel slag.
Described tunning is preparing the application in feedstuff.
Beneficial effect of the present invention is: 1) select the bacterial classification being suitable for the growth of citrus fruits slag, screen the microbial inoculum of best of breed according to bacterium interspecific compatibility feature.Combine microbial inoculum described in 2 and be prepared as air-dry bacterial classification product, can preserve for a long time.Microbial inoculum is with low cost, easy to use, and user only need be actual in conjunction with production, and proportioning citrus pulp solid state substrate raw material, adds appropriate composite ferment, then carries out proper temperature cultivation and namely can obtain qualified fermented product.3) after adopting this zymotechnique, tunning crude protein content can reach 10.12%, and 6.47% when not fermenting improves 56.41%, and protein content significantly improves.
Accompanying drawing explanation
Fig. 1 is that geotrichum candidum and the combined inoculation of rice wine yeast different proportion are to the impact analysis figure of crude protein content.
Fig. 2 is geotrichum candidum, with rice wine yeast and the combined inoculation of aspergillus niger different proportion to the impact analysis figure of crude protein content.
Detailed description of the invention
Illustrated embodiment is to be described content of the present invention better, but is not that content of the present invention is only limitted to illustrated embodiment.So those of ordinary skill in the art carry out nonessential improvement and adjustment according to foregoing invention content to embodiment, still belong to protection scope of the present invention.
Citrusfruit discarded object: citrusfruit slag refers to that citrusfruit processes the leftover bits and pieces of squeezing the juice or after can processed, accounts for the about 25-50% of weight of mandarin orange fruit.The main component of citrusfruit slag is the organic matters such as water-fast carbohydrate (comprising pectin, cellulose, hemicellulose), soluble sugar and bitter substance.In embodiment, using the citrusfruit slag after preparing fruit juice as citrusfruit discarded object.
The inoculum concentration mentioned in following embodiment is the quality of seed liquor or the microbial inoculum referred to an access and the mass ratio of inoculation wild Oryza species.
Embodiment 1 bacterial classification and citrusfruit slag Dual culture
Bacterial classification and citrusfruit slag Dual culture mainly perform according to dull and stereotyped dibbling experimental technique, and screening adapts to the bacterial classification of citrus pulp growth most.Dull and stereotyped dibbling test bibliography " Chen Song, Ding Lixiao, Zhang Li, Wang Tianlong, Zhu Yinglian, the screening [J] of the mixed bacteria of 2008. fermentation apple pomace to produce protein feed. food and fermentation industries, 2008,34 (2): 94-96 " perform.The bibliography " Liu Shuli, 2008.D increase the bacterial screening research of citrus peel residue fermented feed crude protein content " of preparing of citrus pulp plating medium performs, and does suitably to improve.The restricted plating medium of citrus pulp is: citrus pulp 3g, agar 2g, 100mL nutrient solution, and 115 DEG C of sterilizing 20min, mix and be down flat plate; Compounding (g/L) wherein: NaCl0.50g, MgSO
40.50g, (NH
4)
28O
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 Dual culture has 1 respectively) probiotic bacteria: bacillus subtilis (Bacillus subtilis), bacillus coagulans (Bacillus coagulans), lichens gemma (Bacillus licheniformis), bacillus cereus (Bacillus cereus), animal Bacillus acidi lactici (Lactici lactis), lactobacillus plantarum (Lacticiplantarum).2) filamentous fungi: aspergillus oryzae (Aspergillus oryzae, numbering CICC2013), Rhizopus oryzae (Rhiizopus oryzae, numbering CICC3037), Trichoderma viride (Trichoderma viride, numbering CICC40502, purchased from Chinese industrial Culture Collection (CICC); Aspergillus nidulans (Aspergillusnidulans, numbering M4), Rhizopus oryzae preserves bacterial classification (numbering M1, M2, M3).3) saccharomycete: candida utili (Candida utilis, numbering CICC1422), saccharomyces cerevisiae (Saccharomyces cerevisiae, numbering CICC31362), commercially available song yeast, Angel Yeast (Saccharomyces angel).Condition of culture be bacterium 37 DEG C, fungi 28 DEG C-30 DEG C, the growth of the various bacterial classification of observed and recorded after 72 hours cultivate, dull and stereotyped dibbling result of the test is as shown in table 1.
The growing state of the dull and stereotyped dibbling test of table 1 microorganism
Note :-,+, ++, +++ represent that microbial strain culture is unchanged or grow hardly respectively, thalline grows on a small quantity or grows slowly, and thalline is obviously visible, and thalli growth is good.Result shows that wherein bacterium grows hardly, and the bacterium colony of fungi is all obviously visible.Aspergillus niger wherein, Rhizopus oryzae, geotrichum candidum and saccharomycete grow fine on the restricted plating medium of citrus pulp, illustrate that fungi bacterium all can tolerate the environment of Low acid, and bacterium can not.
Embodiment 2 liang of strain combinations and citrus fruits slag Dual culture
Aspergillus niger is that U.S. feed controls official association (Association of American Feed ControlOfficials AAFCO, 1991) thinking safe a kind of microorganism fungus kind, is production enzyme preparation (protease, amylase, pectase) and the bacterial classification being agriculturally used as to produce saccharified pig feed.Therefore aspergillus niger is selected to be used as combination research as indicator bacteria.Using aspergillus niger as indicator bacteria, bacterium liquid (filamentous fungi of the adaptation citrus pulp mentioned in the embodiment 1) dibbling of equivalent same concentrations is mentioned equidistant position, line of symmetry both sides of the restricted plating medium of citrus pulp to embodiment 1, cultivate 7 days for 28 DEG C-30 DEG C, the growing state of bacterium colony on continuous observation record flat board.Bacterial strain combination and the saccharomycete growing fine, can compatiblely coexist is selected to carry out the culture experiment of three bacterium combinations.
From the point sample inoculation of two bacterium, contact with each other to growing to, within continuous 7 days, observe Mixed culture microbial growth situation, can find two strain filamentous fungi initial stages can both on the restricted culture medium of citrus pulp well-grown, different strain combination subsequently presents different growthforms, the bacterial classification combined with aspergillus niger is mainly divided into following three kinds of situations: the strain growth that (1) combines is comparatively slow, and is surrounded covering by the bacterium colony that aspergillus niger constantly expands gradually.(2) strain growth combined is fast, has obvious space in the contact position of two bacterium colonies, around Aspergillus Niger Growth, but cannot enter in aspergillus niger and grows.(3) combine bacterial strain in bacterium colony contact position, mycelia intermeshes growth together, and can enter field growth each other, and what all Mixed culture grew for 7 days the results are shown in Table 2.
A table 2 liang bacterium is combined on citrus pulp plating medium and contacts growing state
As can be seen from the table, the filamentous fungi (M1, M2, M3) of aspergillus niger and preservation generally can not coexist growth very well, and aspergillus niger is obvious with the growth differences that coexists of remaining filamentous fungus strain.Aggregate analysis can determine filamentous fungi mainly A.niger+C.candidum and the A.niger+R.oryzae two kinds combination that the best coexists.
The combination of embodiment 3 three bacterium and citrusfruit slag Dual culture
The various yeast liquid that equivalent same concentrations embodiment 1 is mentioned coated plate on the restricted plating medium of citrus pulp, it is point sample bacterial classification that A.niger+C.candidum and A.niger+R.oryzae screened by embodiment 2 combines bacterium, dibbling is to equidistant position, line of symmetry both sides of yeast plating medium, cultivate 7 days for 28 DEG C-30 DEG C, the growing state of bacterium colony on continuous observation record flat board, selects the three bacterium combinations of growing fine, can compatiblely coexist.
Result shows: the Rhizopus oryzae in aspergillus niger+Rhizopus oryzae combination (A.niger+R.oryzae) and saccharomycete do not grow when Mixed culture, is three poor bacterium combinations, should not carries out deep research.But when aspergillus niger+geotrichum candidum combination (A.niger+C.candidum) combination and barms Mixed culture, saccharomycete is when the restricted grow on plates of citrus pulp is good, different impacts is produced on the growth of aspergillus niger and geotrichum candidum, present different growthforms, when Mixed culture seven days, colony growth situation was in table 3.
Table 3 three bacterium is combined in mixed growth situation on the restricted flat board of citrus pulp
Note :+, ++ with +++ represent that the effect of three bacterium combinations is poor, general and better respectively.
The relatively colony growth situation of control group (two bacterium Mixed culture), mold colony form growth when can find three bacterium Mixed culture is fine and close, and brightness is high, this be filamentous fungi and yeast Mixed culture time work in coordination with growth promoting effects one react.It is worth mentioning that, in the ductility of microorganism, the difference of comparatively control group (two bacterium cultivate) during three bacterium Mixed culture, when this three bacterium syntrophism is described, yeast may be first make use of in citrus pulp culture medium the nutrition being easy to absorb, make filamentous fungi secrete various ferment treatment citrus pulp or allow mycelia go deep into obtaining nutrition in citrus pulp culture medium, which results in filamentous fungi ductility and be subject to obvious impact.According to the result of three bacterium combinations, A.niger+C.candidum+S.cerevisiae SWU-1 is selected to be best combination.
Embodiment 4 the best inoculation quantity mixing ratio
1 method
In 250mL triangular flask, add citrus pulp 20g and the 20mL nutrient solution of pulverizing, 115 DEG C of sterilizing 20min make solid-state fermentation culture medium.Add mixed culture fermentation bacterial classification after cooling, bacteria suspension is by adjustment OD
450value makes its concentration control 10
9individual/mL order of magnitude.During combined inoculation, the reference of best inoculation mixed proportion " Xu Kangzhen; Song Jirong; Huang Jie; the research [J] of Yin Tao single cell protein the best inoculation mixing ratio. microbiology circular 2003,30 (4): 36-39. " describing method performs, and work suitably improves, cultivate and measure protein content after 62 hours, crude protein adopts Micro-kjoldahl method (GB/T14771-93).
The determination of 2 pairs of bacterium the best inoculation mixing ratios
According to the best of breed of bacterial classification, geotrichum candidum and rice wine yeast add in citrusfruit slag solid-state fermentation culture medium as microbial inoculum by test, the number ratio of geotrichum candidum and rice wine yeast is respectively 4: 1,3: 1,2: 1,1: 1,1: 2,1: 3 and 1: 4, and numerical value is by reading 0D
450value calculates; For reducing error, control group is the citrusfruit slag solid-state fermentation culture medium not adding bacterial classification.
Measure the crude protein of tunning.Utilize binary coordinate system to explore geotrichum candidum: the best inoculation mixed proportion of rice wine yeast, detect data as Fig. 1.When not adding bacterial classification, the crude protein content of citrus pulp is very low.After adding bacterial classification process, crude protein increases obviously, geotrichum candidum: rice wine yeast is when inoculation mixing ratio is near 4: 1, and the content of crude protein is the highest, and along with the reduction of mixing ratio, crude protein quantity declines gradually.Therefore, select ratio to be 4: 1 places be the better inoculation of two microorganisms to mix ratio.
The determination of 3 three bacterial classification the best inoculation mixing ratios
By geotrichum candidum: combination when rice wine yeast two bacterial classification mixing ratio is 4: 1 integrally, again compared with aspergillus niger, its ratio, as abscissa, comprises the citrus pulp of aseptic process, geotrichum candidum process, contrast (the two microbial inoculum of combination), three bacterium than fermentation.The content that crude protein detects, as ordinate, is selected the best inoculation mixing ratio of three bacterial classifications, is detected data as Fig. 2 under binary coordinate system.
The content of thick protein under more various treatment conditions, can find that the kind along with adding fermented bacterium increases, crude protein content increases thereupon; And the two bacteria fermentation of combination increases the height of crude protein quantity than single bacterium, but it is low to increase protein content than three bacterium, reflect that bio-diversity in microbiologic population can influential system function, in the process that the aggregate be combined into transforms at biology, play Main Function by different microorganisms.Because Mycophyta can produce multiple cellulase, amylase and pectase etc., well can utilize the cellulose in compost, the materials such as starch generate sugar, yeast class then can utilize sugar to carry out growth metabolism, so both can alleviate Product inhibiton effect, facilitate again the growth of yeast, thus improve fermentation efficiency.In addition, in the result of three bacterial classification mixing ratio fermentation citrus pulps, known ratio has the highest crude protein content 1: 1 time, and namely three bacterial classification the best inoculation quantity mixing ratios are geotrichum candidum: rice wine yeast: aspergillus niger=4: 1: 5.
The Analysis on amino acid components of embodiment 5 citrusfruit slag fermented product
By the combination microbial inoculum that the geotrichum candidum screened, rice wine yeast and aspergillus niger are mixed in the ratio that number ratio is 4: 1: 5, mix with citrus pulp, humidity reaches about 50-70%, and temperature is 28 DEG C-30 DEG C fermentations, and fermentation time is 62 hours.Fermented product low temperature is dried.Measure the Amino acid profile of fermented product, the results are shown in Table 4 with the comparative analysis of the citrus pulp Amino acid profile composition without any process simultaneously.
The analysis of amino acids of table 4 tunning
As seen from table: in tunning, gross protein value adds, compared with control group, increment rate reaches 56.41%.In the amino acid detected, the increment rate of cystine, threonine and methionine is obvious especially.These results prove that the protein increased in tunning is increased by bacterium protein and caused.
The preparation of embodiment 6 rice wine yeast microbial inoculum
Pulverize after 65 DEG C, citrusfruit discarded object is dried, cross 40 mesh sieves, make solid fermentation and want substrate.Often liter of citrusfruit waste extract, adds water to 1 liter by 300 grams, citrusfruit discarded object and stirs, and slagging-off, obtains citrusfruit waste extract.
The fluid nutrient medium of rice wine yeast bacterium is by eating brown sugar (granulated sugar) 30g, KH
2pO
41.0g, MgSO
47H
2o0.5g, (NH
4)
2sO
42.0g, citrusfruit waste extract leaching liquor 1L form, through high pressure steam sterilization (115 DEG C of 20min).Press the inoculum concentration inoculation rice wine yeast bacterial classification of 5%-10% after cooling, carry out the bacterium liquid that conventional deep liquid aerobic fementation (28 DEG C of-30 DEG C of 72h) prepares rice wine yeast bacterium, the rice wine yeast bacterium quantity in the bacterium liquid of rice wine yeast bacterium answers>=10
9cfu/mL.
Or produce solid state fermentation agent by the method making wheat bran song, culture medium with mass ratio range is: wheat bran: water=1: 1, stirs, and high pressure steam sterilization (115 DEG C of 20min), is cooled to 30 DEG C, obtains culture medium A.By the inoculum concentration inoculation rice wine yeast bacterium liquid of 0.5%-2%, carry out the solid fungicide that conventional solid ventilation fermentation (28 DEG C of-30 DEG C of 72h) prepares rice wine yeast bacterium, the rice wine yeast bacterium quantity in the microbial inoculum of the rice wine yeast bacterium after oven dry answers>=10
9cfu/g.
The preparation of embodiment 7 aspergillus niger
Produce solid state fermentation agent by the method making wheat bran song, culture medium take mass ratio range as wheat bran: water=1: 0.8-1, stirs, and high pressure steam sterilization (115 DEG C of 20min), is cooled to 30 DEG C, obtains culture medium A.By the inoculum concentration inoculated aspergillus niger bacterium (A.niger of 0.5%-2%, numbering CICC2089), carry out the microbial inoculum that conventional solid ventilation fermentation (28 DEG C of 72h) prepares black-koji mould, the black-koji mould quantity in the microbial inoculum of the aspergillus niger after oven dry answers>=10
9cfu/g.
The preparation of embodiment 8 geotrichum candidum
Produce solid state fermentation agent by the method making wheat bran song, culture medium take mass ratio range as wheat bran: water=1: 0.8-1, stirs, and high pressure steam sterilization (115 DEG C of 20min), is cooled to 30 DEG C, obtains culture medium A.By inoculum concentration inoculation Geotrichum (the Ceotrichum candidum of 0.5%, numbering CICC1315), carry out the microbial inoculum that conventional solid ventilation fermentation (28 DEG C of 72h) prepares Geotrichum, the black-koji mould quantity in the microbial inoculum of the geotrichum candidum after oven dry answers>=10
9cfu/g.
The preparation of embodiment 9 composite fermentation microbial inoculum
By the rice wine yeast bacteria agent (comprising bacterium liquid or solid fermentation microbial inoculum) of described embodiment 7 and the Geotrichum microbial inoculum of embodiment 8 and the black-koji mould microbial inoculum of embodiment 9, according to the ratio mixing of 4: 1: 5, carry out low temperature dewatering and dry, pulverize preservation, obtain composite fermentation microbial inoculum.
In embodiment 10 citrus pulp biological fermentation feed, bitter taste detects
Add 5% solid union bio-fermentation agent to citrus pulp solid medium (citrus pulp about 80%, wheat bran 15%, mixes; 115 DEG C of sterilizings 20 minutes, are cooled to 30 DEG C), move in suitable fermentation bed and spread out, thickness is about 30cm, is incubated to 25-28 DEG C, cultivates 4 days, dries and pulverizes.Measure the bitter principle of dry in fermented product, naringin content wherein detects bibliography " Davis Wb; Determination offlavanones in citrus fruits [J] .Analytical Chemistry, 1947,19 (7): 476-478. " and performs; Limonin content detects bibliography " Wilson KW; Crutchfield CA.Spectrophotometricdetermination of limonin in orange j uice [J] .Journal of Agricultural and food Chemistry; 1986; 16 (1): 118-124 " and performs, and the results are shown in Table 5.
Table 5 citrus pulp biological fermentation feed bitter principle is analyzed
As seen from table: along with the process of microorganism, in tunning, the content of bitter substance decreases, and aurantiin slip is wherein 42.55%; Limonin slip is 11.57%, reduces the most obvious.Because bitter substance is obvious especially on the impact of palatability, therefore this microbial composite starter has very significance to the raise competence improving citrus pulp.
Embodiment 11 measure of merit
With the citrus pulp fermented product that embodiment 10 obtains, as feedstuff, be combined into complete diet pellet, carry out the nursing experiment of growing and fattening pigs, on the impact of its growth performance in table 6, found that and add fermented product and the complete feed that is combined into, can aquaculture cost be reduced, save food.In addition, growing and fattening pigs pig searches for food normally, and feed intake is without obvious visible change.Citrus pulp biofermentation material is added on the impact of its growth performance in table 6 fattening pig basal diet
Note: the completely little same person's significant difference (p < 0.05) of colleague's shoulder note lowercase, colleague's shoulder note lowercase has identical person's difference little significantly (p > 0.05).
What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (8)
1. aspergillus niger and geotrichum candidum and rice wine yeast are preparing the application in biological feedstuff as microbial inoculum and citrus fruits slag Dual culture, it is characterized in that: the number ratio of described geotrichum candidum, rice wine yeast and aspergillus niger is 3-5:1-4:5-10.
2. combine microbial inoculum, it is characterized in that, mixed by geotrichum candidum, rice wine yeast and the aspergillus niger ratio in 4:1:5 by quantity.
3. combination microbial inoculum according to claim 2, is characterized in that, geotrichum candidum, rice wine yeast and aspergillus niger is mixed, obtains mixture, and mixture low temperature dewatering is dry, then pulverizes.
4. the preparation method of combination microbial inoculum according to claim 2, is characterized in that, specifically comprise the steps:
The preparation of the bacterium liquid of rice wine yeast bacterium: pulverize after citrus fruits and/or citrus fruits slag are dried, cross 40 mesh sieves, make powder; Often liter of citrus fruits and/or citrus fruits slag leaching liquor, add water to 1 liter by described citrus fruits and/or citrus fruits slag 300 grams and stir, and slagging-off, obtains described citrus fruits and/or citrus fruits slag leaching liquor; The fluid nutrient medium of rice wine yeast bacterium is prepared: brown sugar 30g, KH in following ratio and method
2pO
41.0g, MgSO
4.7H
2o 0.5g, (NH
4)
2sO
42.0g, adds described citrus fruits and/or citrus fruits slag leaching liquor 1L forms, and high pressure steam sterilization, obtains the fluid nutrient medium of rice wine yeast bacterium; Press the inoculum concentration inoculation rice wine yeast bacterial classification of 5-10% after cooling, deep liquid aerobic fementation 72 hours at 28 DEG C, obtains the bacterium liquid of rice wine yeast bacterium, rice wine yeast bacterium quantity>=10 in the bacterium liquid of rice wine yeast bacterium
9cfu/mL;
Or be the ratio mixing of 1:0.8-1 with weight ratio by wheat bran and water, stir, high pressure steam sterilization, be cooled to 30 DEG C, obtain culture medium A; Rice wine yeast bacterium liquid is pressed the inoculum concentration inoculation of 0.5%-2%, at 28 DEG C-30 DEG C, solid ventilation ferments 72 hours, obtains the microbial inoculum of rice wine yeast bacterium, and the saccharomycete quantity in the rice wine yeast microbial inoculum after drying answers>=10
9cfu/g;
The preparation of the microbial inoculum of black-koji mould: in the inoculum concentration inoculated aspergillus niger bacterium by 0.5% to described culture medium A, at 28 DEG C, solid ventilation ferments 72 hours, obtains the microbial inoculum of black-koji mould, the spore of Aspergillus niger quantity in the microbial inoculum of aspergillus niger answers>=10
9cfu/g;
The preparation of the microbial inoculum of Geotrichum: the inoculum concentration inoculated aspergillus niger bacterium by 0.5% is to described culture medium A, and at 28 DEG C, solid ventilation ferments 72 hours, obtains the microbial inoculum of Geotrichum, the mycotic spore quantity in the microbial inoculum of geotrichum candidum answers>=10
9cfu/g;
The microbial inoculum of the described microbial inoculum of rice wine yeast bacterium, the microbial inoculum of black-koji mould and black-koji mould is carried out low temperature dewatering drying separately, and mixes, obtain combination microbial inoculum.
5. citrus fruits slag prepares the method for biological feedstuff, it is characterized in that, specifically comprises the following steps:
The preparation of A solid medium
By the sterilizing of citrus fruits slag, obtain solid medium;
B fermentation
Combination microbial inoculum is seeded in described solid medium by the inoculum concentration of 4-6%, obtains tunning; Described combination microbial inoculum>=10
9cfu/g, fermentation temperature is 28-30 DEG C, and fermentation time is 48-72 hour; Described combination microbial inoculum is made up of geotrichum candidum, rice wine yeast and aspergillus niger, and the number ratio of geotrichum candidum, rice wine yeast and aspergillus niger is 3-5:1-4:5-10.
6. citrus fruits slag according to claim 5 prepares the method for biological feedstuff, it is characterized in that, after fully mixing with the weight ratio of 80:14-16 after orange peel slag and the dry also pulverizing of wheat bran, sterilizing, obtains solid medium.
7. the tunning of the method gained of biological feedstuff is prepared based on citrus fruits slag according to claim 6.
8. tunning according to claim 7 is preparing the application in feedstuff.
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| CN106035990B (en) * | 2016-07-15 | 2019-12-17 | 重庆市畜牧科学院 | Method for preparing biological feed by solid-state enzymolysis fermentation of citrus pulp, product and application thereof |
| CN107319173A (en) * | 2017-07-07 | 2017-11-07 | 合肥皖高特种家禽养殖科技有限公司 | A kind of utilization marc mycoprotein promotes the feed of young goose growth |
| CN110771722A (en) * | 2019-11-07 | 2020-02-11 | 贵州省水产研究所 | Preparation process of pomelo-derived fermented feed |
| CN111254079B (en) * | 2020-01-22 | 2023-07-25 | 华南理工大学 | Composite fermentation microbial inoculum and application thereof in preparation of citrus pulp bio-organic fertilizer |
| CN111557377B (en) * | 2020-05-12 | 2022-09-27 | 江南大学 | Method for preparing fruit and vegetable waste fermented feed |
| CN112868888B (en) * | 2021-02-03 | 2021-08-17 | 广东省农业科学院动物科学研究所 | Biological fermentation preparation and preparation method thereof |
| CN113773974A (en) * | 2021-10-12 | 2021-12-10 | 南宁市拜欧生物工程有限责任公司 | Feeding yeast and application thereof |
| CN114381376B (en) * | 2021-11-19 | 2023-06-30 | 中国热带农业科学院热带生物技术研究所 | Culture medium for co-culture of algae and bacteria and co-culture method thereof |
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