CN102943049B - Process for producing cryytococcus neoformans culture through solid fermentation - Google Patents
Process for producing cryytococcus neoformans culture through solid fermentation Download PDFInfo
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Abstract
The invention relates to the field of biology, particularly relates to a process for producing a yeast source feed additive and discloses a method for producing a cryytococcus neoformans culture through the solid fermentation which utilizes a sweet potato wet slag enzymic method and utilizes glucose by-product saccharified slag as a main material. According to the yeast source animal nutrition feed additive produced by the process, the mean value of crude protein content is 38.61%, the number of hydrolysis amino acids is eighteen, the varieties are complete, the essential amino acids for animal nutrition are provided, nutritional ingredients of an organic acid, alcohol, vitamins, microelements and the like are contained, and a mouse acute toxicity test shows that cryytococcus neoformans culture is non-toxic, safe and wide in application prospect.
Description
Technical field
The present invention relates to biological field, be specifically related to the solid fermenting producing method of the yeast source geotrichum candidum culture that uses for animal nutrition feed additives.
Background technology
At present, the high-accuracy machining equipment of the many employings of refining sweet potato starch production, carry out cleaning method production in clean room, process water is softening water, washes clean is carried by canalization without the potato piece of dirt, the automatization of sweet potato starch manufacturing procedure is controlled, and produces sweet potato residue and the enchylema soft water (being commonly called as " juice ", " waste water ") of a large amount of high health qualities.
Sweet potato residue (sweet potato residues) is a large amount of by products of producing in bright sweet potato starch producing process or as the accumulation of waste, accounts for 10% ~ 14% of raw material, and huge biomass renewable resource fail effectively to utilize, and serious environment pollution.The starch-containing average 50%(butt of potato slag),, by amylase, Glucoamylase hydrolysis, can produce dextrin, maltose and glucose.
Be attributed to Deuteromycotina on geotrichum candidum (Geotrichum Candidium Link) taxonomy, Moniliaceae, the mould family of avette spore, Geotrichum, be a kind yeast shape fungi (Bamett nineteen eighty-three).Geotrichum candidum is unicellular organism, distributes wide, and rich in proteins and multivitamin.Nourish and generate as fragmentation.Many weak glucose fermentations, decompose pectin and grease, the doubling time is 1.1 ~ 1.7h(30 ℃ or 25 ℃ in containing the glucose substrate).Geotrichum candidum assimilation nitrogenous source: peptone, ammonium sulfate, asparagine, urea, being of high nutritive value of geotrichum candidum, can be edible and make feed.
Geotrichum candidum is low to the substratum nutritional requirement, wide adaptability, be a kind of industrial microorganism that has development potentiality, the domestic and international multiplex alcohol waste water of culture material, tofu wastewater, bean curd wintercherry, qu-liquor losing grain the like waste carry out the fermentative production geotrichum candidum, as tropina, use.People often produce the Geotrichum body protein with the fermentation waste waters such as food wastewater and amino acid, organic acid, deep ventilation training method.
Utilize organic waste residues to make the bacterial classification of solid state fermentation with geotrichum candidum not single, the people such as Wu Suhuan are converted into feed with geotrichum candidum and the two strain solid fermentation changing food wastes of aspergillus oryzae, and its mycetome albumen 33.87%, increased by 6.85% than original.Also useful geotrichum candidum and aspergillus niger, moral formula lactobacillus, subtilis, cereuisiae fermentum miscegenation lactic acid-fermenting waste residue, improve protein content, makes simultaneously in product and have multiple digestive ferment and VITAMIN.
And take the secondary saccharification slag of sweet potato biomass sweet potato residue and sweet potato enchylema waste water as culture medium carbon source and other kind nutritive ingredient, utilize the purebred cultivation geotrichum candidum of sweet potato biomass enchylema waste water ventilation to make seed liquor, and with special fermentation mode scale operation Geotrichum body protein and meta-bolites that the shallow-layer solid state fermentation combines, make fodder additives,, with performance breed true advantage, easy, the product security of living contaminants, the bacterial classification single fermentation liquid-solid fermentation form preferably that reduces solid culture, have no report.
Summary of the invention
The saccharification slag that the present invention seeks to contain C, N nutritive ingredient is major ingredient, forms the required substratum of geotrichum candidum Growth and metabolism, the method for producing yeast source geotrichum candidum culture fodder additives by solid fermentation.
Solid fermentation (Solid fermentation) be take geotrichum candidum (Geotrichum candidum) and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) as purebred through liquid culture as Inoculant, utilize C, the N nutrition of saccharification slag, be aided with other medium components, by the solid culture technology, prepare yeast culture (yeast culture)
In the various techniques that prepare yeast, the substrate that is suitable for is sugary or the polysaccharide hydrolysis thing.Mainly with filter press technique or centrifuging, the filter residue residual sugar is high to the solid-liquid separation of sweet potato residue enzyme process converted mash, and 30% above hydrolyzation of glucose, soluble nitrogen, VB and trace mineral remain in the saccharification slag, and this is the high-quality nutrition source of culturing yeast and probiotic bacterium.Matrix does not need sterilizingly when medium pH 3.5 ~ 4.5, is that solid fermentation proposes a power-economizing method cheaply.
Although low with commercially available cellulase percent hydrolysis, potato slag fiber is through the processing in small, broken bits repeatedly of rasion, screening, cyclonic unit, particle diameter diminishes, and 80% less than 90 μ m; Again through High-temperature Liquefaction and Mashing process, the basic slaking of the fiber in the saccharification slag, proterties changes greatly, palatability is strong, suitable do to raise add the agent solid fermentation raw materials.
A kind of tray solid fermentation provided by the invention is produced the method for geotrichum candidum culture, comprises following steps:
Step 1: preparation test tube slant substratum: glucose 2%, yeast powder 1%, peptone 1%, agar 2%, carry out test tube slant at 30 ℃ of geotrichum candidum bacterial classifications and cultivate 24h;
Step 2: the inoculation liquid seeds carries out triangular flask to be cultivated, and culture condition is shaking table vibration 110rpm, 30 ℃, after cultivating 24h, carries out seed tank culture;
Step 3: after preparation solid fermentation substratum and sterilization, with step 2 gained geotrichum candidum seed liquor by 10% inoculation mix, sabot 3 ~ 5cm is thick, cultivate 30 ~ 48h at 30 ℃, relative humidity 75% left and right solid fermentation, stir 1-2 time gently, be covered with top layer to the white hypha body; Be dried to moisture 14% in 60 ℃ and namely obtain described geotrichum candidum culture.
The described geotrichum candidum bacterial classification of step 1, be preferably scientific research or department of colleges and universities through the classification evaluation and pass through the purebred of safety experiment.Repeatedly go down to posterity, for preventing spawn degeneration and variation, bacterial classification need to often carry out rejuvenation.
As preferably, the described strain inclined plane substratum of step 1 is glucose 2%, yeast powder 1%, peptone 1%, agar 2%.
As preferably, the described triangular flask liquid seed culture medium of step 2 is glucose 2%, yeast powder 1%, peptone 1%.
As preferably, the postvaccinal culture condition of the described triangular flask liquid seed culture medium of step 2: shaking table shakes to be full of and cultivates 110rpm, 30 ℃, 24h.
As preferably, step 2 seeding tank is aerobic bacteria fermentation universal machine stirred fermentor.
That tray is cultivated to one of common form of the solid fermentation (Solid fermentation) of aerobic bacteria, the principle of the described preparation solid fermentation of step 3 substratum: take the sweet potato residue enzyme process liquid sugar that contains Liquid Glucose, soluble nitrogen, VITAMIN and trace mineral from the saccharification slag as main.The standard that the described auxiliary material of step 3 is selected is safety non-toxic, cheap, the agricultural byproducts that contain C, N, P and fodder yeast applicable urea and ammonium sulfate usually, to meet geotrichum candidum growth, the requirement of metabolism desired nutritional.
As preferably, for antiforeign bacteria pollutes, the described sterilization of step 3 is 100 ℃, the sterilization of 60min solid medium and sterilization overnight totally 2 times; Be beneficial to ventilation, the described solid fermentation condition of step 3 is that tray or shallow-layer 3-5cm cultivate.
As preferably, the described solid fermentation condition of step 3 is 30 ℃ of left and right of temperature, humidity relative humidity 75% left and right, and the geotrichum candidum white hypha covers with and when caking is arranged on solid medium, turn, continue to cultivate with aseptic instrument, to reach white hypha, all cover with.
Explained hereafter geotrichum candidum culture yeast of the present invention source animal nutrition feed additives, its crude protein content average 38.61%, 18 kinds of hydrolysis amino acids, A wide selection of colours and designs, has the Animal nutrition indispensable amino acid, contain the nutritive ingredients such as organic acid, alcohol, VITAMIN and trace element,, through the anxious malicious test card of small white mouse nontoxic, the safety of mould culture expressly, have a extensive future.
Embodiment
The invention discloses a kind of solid fermentation and produce the technique of geotrichum candidum culture, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is, all similarly replace and change apparent to those skilled in the art, and they all are deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change methods and applications as herein described or suitably change and combination within not breaking away from content of the present invention, spirit and scope, realize and apply the technology of the present invention.
In order to make those skilled in the art understand better technical scheme of the present invention, the present invention is described in further detail below in conjunction with specific embodiment.
Embodiment 1: the composite solid medium take the saccharification slag as major ingredient is modulated with enchylema waste water.
(1) bacterial classification preparation:
Test tube slant bacterial classification (%):
Substratum: glucose 2%, yeast powder 1%, peptone 1%, agar 2%, surplus are water; 121 ℃ of sterilization 20min, be chilled to 30 ℃, 2.1084,30 ℃ of cultivations of the geotrichum candidum (Geotrichumcandidum) of the Chinese DSMZ of inoculation 24 hours.
Triangular flask liquid seeds (%):
Substratum: glucose 2%, yeast powder 1%, peptone 1%, surplus are water; 121 ℃ of sterilization 20min, be chilled to 30 ℃.
Inoculant: the inclined-plane seed, cultivated 24 hours for 30 ℃;
Seeding tank liquid seeds (%):
Substratum: glucose 2%, yeast powder 1%, peptone 1%, surplus are water; 121 ℃ of sterilization 20min, be chilled to 30 ℃.
Inoculant: inoculum size triangular flask seed liquor 10%, 30 ℃, cultivated 24 hours.
(2) solid medium preparation (%):
Saccharification slag 60%, sweet potato vine powder 20%, Semen Maydis powder 4%, cream yeast extract 2%, dregs of beans 4%, urea 2.2%, (NH
4)
2SO
43.5%, secondary calcium phosphate 0.5%, vitamin H 0.2%, wheat time powder 3.6%.I.e. loose (moisture determination material moisture 65% ~ 70% fast) the 100 ℃ of sterilization 60min that transfer to pinch agglomerating, lose with sweet potato enchylema; Sterilization and sterilization overnight totally 2 times; Be chilled to 30 ℃ and connect liquid seeds.
Inoculant: inoculum size seeding tank seed liquor 10%(W/W), shallow-layer 3 ~ 5cm, cultivated 48 hours for 30 ℃.
Aftertreatment:
The geotrichum candidum inoculum is at 60 ℃, and air stream drying, pack after the assay was approved.
Result: survey crude protein 38.25%; Its amino acid sees attached list full the decomposition.
Embodiment 2: the saccharification slag is that the composite solid medium of major ingredient is allocated with potato slag enzyme process liquid sugar.
(1) bacterial classification preparation:
Test tube slant bacterial classification (%):
Substratum: glucose 2%, yeast powder 1%, peptone 1%, agar 2%, surplus are water; 121 ℃ of sterilization 20min, be chilled to 30 ℃, and inoculation was cultivated 24 hours available from geotrichum candidum (Geotrichumcandidum) bacterial classification of Chinese DSMZ numbers 2.1084,30 ℃.
Triangular flask liquid seeds (%):
Substratum: glucose 2%, yeast powder 1%, peptone 1%, surplus are water; 121 ℃ of sterilization 20min, be chilled to 30 ℃.
Inoculant: the inclined-plane seed, cultivated 24 hours for 30 ℃
Seeding tank liquid seeds (%):
Substratum: glucose 2%, yeast powder 1%, peptone 1%, surplus are water; 121 ℃ of sterilization 20min, be chilled to 30 ℃.
Inoculant: inoculum size triangular flask seed liquor 10%, 30 ℃, cultivated 24 hours
(2) solid medium preparation (%):
Saccharification slag 60%, sweet potato vine powder 20%, Semen Maydis powder 4%, cream yeast extract 2%, dregs of beans 4%, urea 2.2%, (NH
4)
2SO
43.5%, secondary calcium phosphate 0.5%, vitamin H 0.2%, wheat time powder 3.6%, i.e. loose (moisture determination material moisture 65% ~ 70% fast) the 100 ℃ of 30min that sterilize that transfer to pinch agglomerating, lose with sweet potato enchylema; Sterilization and sterilization overnight totally 2 times; Be chilled to 30 ℃ and connect liquid seeds.
Inoculant: inoculum size seeding tank seed liquor 10%(W/W), shallow-layer 3 ~ 5cm, cultivated 48 hours for 30 ℃.
Aftertreatment:
The geotrichum candidum inoculum is at 60 ℃, and air stream drying, pack after the assay was approved.Result: survey crude protein 40.75%.
Embodiment 3: the saccharification slag is that the composite solid medium of major ingredient is allocated with tap water.
(1) bacterial classification preparation:
Test tube slant bacterial classification (%):
Substratum: glucose 2%, yeast powder 1%, peptone 1%, agar 2%, surplus are water; 121 ℃ of sterilization 20min, be chilled to 30 ℃, 2.1084,30 ℃ of cultivations of inoculation geotrichum candidum 24 hours.
Triangular flask liquid seeds (%):
Substratum: glucose 2%, yeast powder 1%, peptone 1%, surplus are water; 121 ℃ of sterilization 20min, be chilled to 30 ℃.
Inoculant: the inclined-plane seed, cultivated 24 hours for 30 ℃
Seeding tank liquid seeds (%):
Substratum: glucose 2%, yeast powder 1%, peptone 1%, surplus are water; 121 ℃ of sterilization 20min, be chilled to 30 ℃.
Inoculant: inoculum size triangular flask seed liquor 10%, 30 ℃, cultivated 24 hours.
(2) solid medium preparation (%):
Saccharification slag 60, sweet potato vine powder 20, Semen Maydis powder 4, cream yeast extract 2, dregs of beans 4, urea 2.2, (NH
4)
2SO
43.5, secondary calcium phosphate 0.5, vitamin H 0.2, wheat time powder 3.6.I.e. loose (moisture determination material moisture 65% ~ 70% fast) the 100 ℃ of sterilization 30min that transfer to pinch agglomerating, lose with sweet potato enchylema; Sterilization and sterilization overnight totally 2 times; Be chilled to 30 ℃ and connect liquid seeds.
Inoculant: inoculum size seeding tank seed liquor 10%(W/W), shallow-layer 3 ~ 5cm, cultivated 48 hours for 30 ℃.
Aftertreatment:
The geotrichum candidum inoculum is at 60 ℃, and air stream drying, pack after the assay was approved.Result: survey crude protein 36.84%; Its amino acid sees the following form full the decomposition.
| Sequence number | Interventions Requested | Technical requirements | Assay | Individual event is judged |
| 1 | Aspartic Acid, % | / | 1.15 | / |
| 2 | Threonine, % | / | 0.50 | / |
| 3 | Serine, % | / | 0.70 | / |
| 4 | L-glutamic acid, % | / | 2.00 | / |
| 5 | Proline(Pro), % | / | 0.37 | / |
| 6 | Glycine, % | / | 0.58 | / |
| 7 | L-Ala, % | / | 0.73 | / |
| 8 | Gelucystine, % | / | 0.03 | / |
| 9 | α-amino-isovaleric acid, % | / | 0.64 | / |
| 10 | Methionine(Met), % | / | 0.09 | / |
| 11 | Isoleucine, % | / | 0.54 | / |
| 12 | Leucine, % | / | 0.90 | / |
| 13 | Tyrosine, % | / | 0.37 | / |
| 14 | Phenylalanine, % | / | 0.55 | / |
| 15 | Methionin, % | / | 0.39 | / |
| 16 | Histidine, % | / | 0.23 | / |
| 17 | Arginine, % | / | 0.56 | / |
| 18 | Tryptophane, % | / | 0.02 | / |
The above is only the preferred embodiment of the present invention; should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. a solid fermentation is produced the method for geotrichum candidum culture, comprises following steps:
Step 1: preparation test tube slant substratum: glucose 2%, yeast powder 1%, peptone 1%, agar 2%, surplus are water, carry out test tube slant at 30 ℃ of geotrichum candidum bacterial classifications and cultivate 24h;
Step 2: the inoculation liquid seeds carries out triangular flask and cultivates, culture condition is shaking table vibration 110rpm, 30 ℃, after cultivating 24h, carries out seed tank culture, described triangular flask is cultivated and the liquid seed culture medium of seed tank culture is glucose 2%, yeast powder 1%, peptone 1%, and surplus is water;
Step 3: after preparation solid fermentation substratum and sterilization, with step 2 gained geotrichum candidum seed liquor by 10% inoculation mix, sabot 3～5cm is thick, cultivate 30～48h at 30 ℃, relative humidity 75% left and right solid fermentation, during stir 1-2 time gently, be covered with top layer to the white hypha body; Be dried to moisture 14% in 60 ℃ and namely obtain described geotrichum candidum culture; The formula of described solid fermentation substratum is: saccharification slag 60%, sweet potato vine powder 20%, Semen Maydis powder 4%, cream yeast extract 2%, dregs of beans 4%, urea 2.2%, (NH
4)
2SO
43.5%, secondary calcium phosphate 0.5%, vitamin H 0.2%, wheat time powder 3.6%, add sweet potato enchylema and mix, in mixture moisture 65%～70%.
2. method according to claim 1, is characterized in that, step 2 seeding tank is aerobic bacteria fermentation universal machine stirred fermentor.
3. method according to claim 1, is characterized in that, the described sterilization of step 3 is 100 ℃, the sterilization of 60min solid medium and sterilization overnight totally 2 times.
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| CN104273318A (en) * | 2013-07-10 | 2015-01-14 | 江苏谷硅新材料股份有限公司 | Method for producing bacterial protein based on succinic acid fermentation waste liquid |
| CN103798504A (en) * | 2014-01-03 | 2014-05-21 | 中国林业科学研究院林产化学工业研究所 | Method for preparing biological feed by solid fermenting olive leaves |
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