CN109527223A - A kind of functional feed and preparation method thereof - Google Patents
A kind of functional feed and preparation method thereof Download PDFInfo
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- CN109527223A CN109527223A CN201811653781.9A CN201811653781A CN109527223A CN 109527223 A CN109527223 A CN 109527223A CN 201811653781 A CN201811653781 A CN 201811653781A CN 109527223 A CN109527223 A CN 109527223A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
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- A23K20/10—Organic substances
- A23K20/174—Vitamins
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/163—Sugars; Polysaccharides
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- A—HUMAN NECESSITIES
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- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders
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Abstract
The invention discloses a kind of functional feeds and preparation method thereof, are related to technique for producing feed.Producing arachidonic acid microbial bacteria bacterium solution and production DHA microbial bacteria bacterium solution are seeded to mixed fermentation in same solid medium by the preparation method of functional feed disclosed by the invention, arachidonic acid and DHA are rich in functional feed obtained, arachidonic acid and DHA content are improved, and the preparation method step is simple, operation is convenient.
Description
Technical field
The present invention relates to technique for producing feed fields, in particular to a kind of functional feed and preparation method thereof.
Background technique
Polyunsaturated fatty acid (PUFA) refers to containing two or more double bonds, and a length of 18-22 carbon of carbochain is former
The straight chain fatty acid of son mainly includes ω -3 and ω -6 series.Wherein cause to be concerned with docosahexaenoic acid and flower extensively
Raw tetraenoic acid.DHA and AA and the mankind learn, memory and nervous system development are related, be adjustable the lipid-metabolism of human body, treatment and
Prevent cardiovascular and cerebrovascular disease, anticancer, confrontation obesity.In animal productiong, DHA and AA can promote the normal development of animal, improve
Growth performance, immune state and vigor.
In order to more efficient, economical production DHA and arachidonic acid feed need to be raw to microorganism is utilized in the prior art
It produces DHA and arachidonic method improves.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of functional feed, can strengthen feeding using the preparation method
DHA and arachidonic acid nutrient in material, effectively improve the nutritive value in feed.
Another object of the present invention is to provide a kind of functional feed, which is rich in DHA and arachidonic
Sour nutrient.
The present invention is implemented as follows:
The study found that algae, especially schizochytrium, dino flagellate, thraustochytriale can largely accumulate DHA, DHA in algae oil
The more other microalgaes of content are high.Mortierella alpina can accumulate the arachidonic acid of significant proportion, and arachidonic acid contains in mycelium
Amount is high, it is convenient to extract, and is generally acknowledged arachidonic acid production strain excellent.In addition, there are also it is other it is a variety of can be used to produce DHA and
Arachidonic microorganism fungus kind.Currently, DHA and arachidonic production are all by one of quasi-microorganism strain
Individually fermentation obtains and contains DHA or arachidonic single product.And neat liquid is mostly used to ferment, it is needed in fermentation process
Constantly ventilation and stirring are to guarantee dissolved oxygen, and liquid fermentation water content is up to 80% or more, and drying process energy consumption is higher, liquid hair
The DHA and arachidonic acid category polyunsaturated fatty acid of ferment output are oxidized easily.
Based on this, on the one hand, the present invention provides a kind of preparation methods of functional feed comprising following steps:
Fermentation step: producing arachidonic acid microbial bacteria bacterium solution and production DHA microbial bacteria bacterium solution are seeded to same solid
Mixed fermentation in culture medium；
Wherein, it is preferred that the inoculum concentration for producing DHA microbial bacteria is 0.1%-1% (wt)；The producing arachidonic acid
The inoculum concentration of microbial bacteria is 0.1%-1% (wt)；
The bacterium being wherein inoculated with is the thallus that will be collected after seed liquor centrifugal concentrating.
The condition of mixed fermentation: temperature is 20-30 DEG C, and the time is 100-140 hours.
Preferably, the temperature of mixed fermentation is 28 DEG C.
Further, in some embodiments of the present invention, in fermentation step, first by producing arachidonic acid microorganism
Bacterium bacterium solution, which is seeded in solid medium, cultivates 36-48h, then is seeded to same solid medium for DHA microbial bacteria bacterium solution is produced
Middle mixed culture is fermented 100-140 hours.
Further, in some embodiments of the present invention, produce DHA microbial bacteria be selected from schizochytrium limacinum, thraustochytriale,
Any one in dino flagellate and saccharomycete；
Further, in some embodiments of the present invention, the microbial bacteria of producing arachidonic acid is Mortierella alpina.
Producing arachidonic acid microbial bacteria is longer than producing DHA microbial bacteria growth cycle, and it is micro- to be first inoculated with producing arachidonic acid
Biological bacteria can form a more fluffy environment after producing arachidonic acid microbial bacteria mycelial growth gets up, and increase
Environment porosity is conducive to increase the dissolved oxygen for producing DHA microbial bacteria.
Further, in some embodiments of the present invention, the solid medium includes following component:
Protein matter, inorganic salts and vitamin.
Further, in some embodiments of the present invention, above-mentioned protein matter be corn, wheat, soya bean or
Its derivative or its processing byproduct (such as cornstarch, Dried Corn Steep Liquor Powder, cornstarch or Dried Corn Steep Liquor Powder, soyabean cake
Powder).
Further, in some embodiments of the present invention, above-mentioned inorganic salts are selected from potassium dihydrogen phosphate, magnesium sulfate, nitre
The combination of one or more of sour potassium, sodium chloride, potassium chloride and calcium chloride.
Further, in some embodiments of the present invention, vitamin combination includes thiamine, calcium pantothenate, biology
Element and cyanocobalamin.
Further, in some embodiments of the present invention, the vitamin combination contain 80% thiamine,
18.6% calcium pantothenate, 0.4% biotin and 1% cyanocobalamin.
Further, in some embodiments of the present invention, before fermentation step, the preparation method further includes bacterium
Liquid preparation step:
The bacterium solution preparation step includes the following steps (1) and/or step (2):
Step (1): production DHA microbial bacteria strain is seeded in DHA fluid nutrient medium and cultivates 36-48h, obtains the production
DHA microbial bacteria bacterium solution；
Step (2): producing arachidonic acid microbial bacteria strain is seeded in arachidonic acid fluid nutrient medium and cultivates 36-
48h obtains the producing arachidonic acid microbial bacteria bacterium solution.
Further, in some embodiments of the present invention, the DHA fluid nutrient medium includes: glucose, glutamic acid
Sodium, yeast extract and inorganic salts；
Preferably, the DHA fluid nutrient medium include: 5%-7% (wt) glucose, 2%-4% (wt) sodium glutamate,
0.8%-1% (wt) yeast extract and 3%-5% (wt) inorganic salts；
Preferably, the DHA fluid nutrient medium includes: 6% (wt) glucose, 3% (wt) sodium glutamate, 0.9% (wt)
Yeast extract and 4% (wt) inorganic salts；
Preferably, inorganic salts are selected from one of phosphate, sulfate, nitrate, sodium salt and calcium chloride or a variety of groups
It closes.
Further, in some embodiments of the present invention, the arachidonic acid fluid nutrient medium includes: glucose
And yeast powder；
Preferably, the arachidonic acid fluid nutrient medium includes: 3%-6% (wt) glucose and 1%-3% (wt) ferment
Female powder；
Preferably, the arachidonic acid fluid nutrient medium includes: 5% (wt) glucose and 2% (wt) yeast powder.
On the other hand, the present invention provides a kind of functional feeds, as obtained by preparation method as described above.
The present invention provides a kind of functional feeds comprising following ingredient:
DHA content 5.8%-30.5% (wt), arachidonic acid content 3.3%-28.5% (wt), protein content are
37%-65% (wt), crude fiber content are no more than 10% (wt) lower than 10% (wt), moisture content；
The granularity of the functional feed is 0.8-6mm, carbon-nitrogen ratio is (15-30): 1.
The beneficial effect comprise that the preparation method of functional feed of the invention, it will be micro- by producing arachidonic acid
Biological bacteria bacterium solution and production DHA microbial bacteria bacterium solution are seeded to mixed fermentation in same solid medium, and to fermentation parameter and item
Part carries out scientific optimization, to overcome the problems, such as that tradition separates DHA caused by liquid fermentation and arachidonic acid is easily oxidized, and improves
DHA and arachidonic acid content in product.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The preparation method of functional feed provided in this embodiment is as follows:
The thallus seed of 1 preparation fermentation:
The 1.1 production DHA microbial bacteria schizochytrium limacinums that will be frozen, after defrosting, are inoculated in DHA fluid nutrient medium, in 20 DEG C
Under conditions of cultivate 48h, be then centrifuged for being concentrated, collect concentration after schizochytrium limacinum thallus.
DHA fluid nutrient medium used contains the ferment of the glucose of 5% (wt), the sodium glutamate of 2% (wt), 0.8% (wt)
The inorganic salts of female medicinal extract and 3% (wt), surplus are water；
Wherein, inorganic salts contain the nitric acid of the potassium dihydrogen phosphate of 0.5% (wt), the magnesium sulfate of 0.7% (wt), 0.6wt%
Potassium, the sodium chloride of 1.5% (wt), the calcium chloride of 0.02% (wt) and 0.40% (wt) sodium sulphate, surplus is water, pH from
So.
1.2, by Mortierella alpina strain inoculated to potato dextrose agar (PDA) culture medium flat plate, train at 25 DEG C
It supports 9 days and generates simultaneously maturation to spore, remove the mycelia on culture medium and spore, be configured to spore suspension with sterile water.By spore
Sub- inoculation of suspension liquid is cultivated into arachidonic acid fluid nutrient medium, and inoculum concentration is 10% (volume ratio), cultivation temperature 25
DEG C, it is 120 revs/min, incubation time 48h that shaking table, which shakes revolving speed,, it is then centrifuged for being concentrated, the Mortierella alpina after collecting concentration
Bacterium thallus.
Wherein, arachidonic acid fluid nutrient medium used contains the glucose of 3% (wt) and the yeast powder of 1% (wt), remaining
Amount is water, pH7.0.
2 prepare functional feed
2.1 preparation fermentation solid mediums
By mass percentage be 20%:38%:18%:16%:5%:3% ratio, extracting corn starch, wheat, soya bean,
Dried Corn Steep Liquor Powder, inorganic salts and vitamin combination mixing, then be added in the ratio that the weight ratio of water and cornstarch is 10:18
Water, the boiling 20min under conditions of 95 DEG C, sterilize 20min, the solid medium of obtained fermentation.
Wherein, inorganic salts ingredients used in this step are the same as the inorganic salts in above-mentioned steps 1.1.
Vitamin combination used be dry powder, contain 80% thiamine, 18.6% calcium pantothenate, 0.4% biology
Element and 1% cyanocobalamin.
2.2 inoculation fermentation
First the Mortierella alpina thallus after concentration is transferred in solid medium for 0.1% (wt) by inoculum concentration, in 20
Fermented and cultured, fermentation time 48h are carried out under conditions of DEG C；It is primary to stir solid medium every 10h, stirs example every time period
5min can such as be continued.Sterile water is supplemented into solid medium by 5% (wt) of solid medium during stirring.
After fermentation, then in the ratio that inoculum concentration is 0.1% (wt) the schizochytrium limacinum thallus after concentration is transferred in same
In one solid medium, co-fermentation time 100h is further continued under conditions of 26 DEG C.Period stirs solid culture every 10h
Base is primary, and 5min can for example be continued every time by stirring.5% (wt) during stirring by solid medium is into solid medium
Supplement sterile water.
After fermentation, resulting culture medium and thallus will be fermented dry power is 5kw, drying temperature is 90 DEG C micro-
Then drying and crushing 2h in wave drying machine is granulated to get the functional feed of arachidonic acid and DHA is rich in.
3 detections
The functional feed obtained for being rich in arachidonic acid and DHA is taken, its index of correlation is detected, the results are shown in Table 1.
Embodiment 2
The preparation method of functional feed provided in this embodiment is as follows:
The thallus seed of 1 preparation fermentation:
The 1.1 production DHA microbial bacteria schizochytrium limacinums that will be frozen, after defrosting, are inoculated in DHA fluid nutrient medium, in 30 DEG C
Under conditions of cultivate 36h, be then centrifuged for being concentrated, collect concentration after schizochytrium limacinum thallus.
DHA fluid nutrient medium used contains the yeast of the glucose of 7% (wt), the sodium glutamate of 4% (wt), 1% (wt)
The inorganic salts of medicinal extract and 5% (wt), surplus are water, and pH is natural；
Wherein, inorganic salts ingredients used are the same as embodiment 1.
1.2, by Mortierella alpina strain inoculated to potato dextrose agar (PDA) culture medium flat plate, train at 28 DEG C
It supports 6 days and generates simultaneously maturation to spore, remove the mycelia on culture medium and spore, be configured to spore suspension with sterile water.By spore
Sub- inoculation of suspension liquid is cultivated into arachidonic acid fluid nutrient medium, and inoculum concentration is 20% (volume ratio), cultivation temperature 25
DEG C, it is 120 revs/min that shaking table, which shakes revolving speed, and incubation time 48h is then centrifuged for being concentrated, the Mortierella alpine mould after collecting concentration
Thallus.
Wherein, arachidonic acid fluid nutrient medium used contains the glucose of 6% (wt) and the yeast powder of 3% (wt), remaining
Amount is water, pH6.8.
2 prepare functional feed
2.1 preparation fermentation solid mediums
By mass percentage be 14%:42%:12%:12%:5%:5% ratio, extracting corn starch, wheat, soya bean,
Dried Corn Steep Liquor Powder, inorganic salts and vitamin combination mixing, then water is added in the ratio that the weight ratio of water and starch is 10:18,
The boiling 20min under conditions of 95 DEG C, sterilize 20min, the solid medium of obtained fermentation.
Wherein, inorganic salts ingredients used in this step are the same as embodiment 1.
Vitamin combination ingredient used is the same as embodiment 1.
2.2 inoculation fermentation
First the Mortierella alpina thallus after concentration is transferred in solid medium for 1% (wt) by inoculum concentration, in 30 DEG C
Under conditions of carry out fermented and cultured, fermentation time 36h；It is primary to stir solid medium every 14h, stirs every time for example period
It can continue 10min.Sterile water is supplemented into solid medium by 1% (wt) of solid medium during stirring.
After fermentation, then in the ratio that inoculum concentration is 1% (wt) the schizochytrium limacinum thallus after concentration is transferred in same
In solid medium, co-fermentation time 100h is further continued under conditions of 30 DEG C.Period stirs solid medium every 14h
Once, 10min can for example be continued by stirring every time.10% (wt) during stirring by solid medium is into solid medium
Supplement sterile water.
After fermentation, resulting culture medium and thallus will be fermented dry power is 10kw, drying temperature is 110 DEG C
Then drying and crushing 0.5h in microwave dryer is granulated to get the functional feed of arachidonic acid and DHA is rich in.
3 detections
The functional feed obtained for being rich in arachidonic acid and DHA is taken, its index of correlation is detected, the results are shown in Table 1.
Embodiment 3
The preparation method of functional feed provided in this embodiment is as follows:
The thallus seed of 1 preparation fermentation:
The 1.1 production DHA microbial bacteria schizochytrium limacinums that will be frozen, after defrosting, are inoculated in DHA fluid nutrient medium, in 24 DEG C
Under conditions of cultivate 44h, be then centrifuged for being concentrated, collect concentration after schizochytrium limacinum thallus.
DHA fluid nutrient medium used contains the ferment of the glucose of 6% (wt), the sodium glutamate of 3% (wt), 0.9% (wt)
The inorganic salts of female medicinal extract and 4% (wt), surplus are water, and pH is natural；
Wherein, inorganic salts ingredients used are the same as embodiment 1.
1.2, by Mortierella alpina strain inoculated to potato dextrose agar (PDA) culture medium flat plate, train at 26 DEG C
It supports 8 days and generates simultaneously maturation to spore, remove the mycelia on culture medium and spore, be configured to spore suspension with sterile water.By spore
Sub- inoculation of suspension liquid is cultivated into arachidonic acid fluid nutrient medium, and inoculum concentration is 15% (volume ratio), cultivation temperature 25
DEG C, it is 120 revs/min that shaking table, which shakes revolving speed, and incubation time 48h is then centrifuged for being concentrated, the Mortierella alpine mould after collecting concentration
Thallus.
Wherein, arachidonic acid fluid nutrient medium used contains the glucose of 5% (wt) and the yeast powder of 2t%, and surplus is
Water, pH7.5.
2 prepare functional feed
2.1 preparation fermentation solid mediums
By mass percentage be 12%:42%:10%:10%:1%:1% ratio, extracting corn starch, wheat, soya bean,
Dried Corn Steep Liquor Powder, inorganic salts and vitamin combination mixing, then water is added for the ratio of 10:18 in the weight ratio of starch, in
Boiling 20min under conditions of 95 DEG C, sterilize 20min, the solid medium of obtained fermentation.
Wherein, inorganic salts ingredients used in this step are the same as embodiment 1.
Vitamin combination ingredient used is the same as embodiment 1.
2.2 inoculation fermentation
First the Mortierella alpina thallus after concentration is transferred in solid medium for 0.5% (wt) by inoculum concentration, in 28
Fermented and cultured, fermentation time 44h are carried out under conditions of DEG C；It is primary to stir solid medium every 12h, stirs example every time period
7.5min can such as be continued.Sterile water is supplemented into solid medium by 7% (wt) of solid medium during stirring.
After fermentation, then in the ratio that inoculum concentration is 0.5% (wt) the schizochytrium limacinum thallus after concentration is transferred in same
In one solid medium, co-fermentation time 130h is further continued under conditions of 24 DEG C.Period stirs solid culture every 12h
Base is primary, and 7.5min can for example be continued every time by stirring.By 7% (wt) of solid medium to solid medium during stirring
Middle supplement sterile water.
After fermentation, resulting culture medium and thallus will be fermented dry power is 12kw, drying temperature is 100 DEG C
Then drying and crushing 1h in microwave dryer is granulated to get the functional feed of arachidonic acid and DHA is rich in.
3 detections
The functional feed obtained for being rich in arachidonic acid and DHA is taken, its index of correlation is detected, the results are shown in Table 1.
Embodiment 4
The preparation method of functional feed provided in this embodiment is as follows:
The thallus seed of 1 preparation fermentation:
The 1.1 production DHA microbial bacteria schizochytrium limacinums that will be frozen, after defrosting, are inoculated in DHA fluid nutrient medium, in 29 DEG C
Under conditions of cultivate 40h, be then centrifuged for being concentrated, collect concentration after schizochytrium limacinum thallus.
DHA fluid nutrient medium used contains the ferment of the glucose of 6% (wt), the sodium glutamate of 3% (wt), 0.9% (wt)
The inorganic salts of female medicinal extract and 4% (wt), surplus are water, and pH is natural；
Wherein, inorganic salts ingredients used are the same as embodiment 1.
1.2, by Mortierella alpina strain inoculated to potato dextrose agar (PDA) culture medium flat plate, train at 26 DEG C
It supports 9 days and generates simultaneously maturation to spore, remove the mycelia on culture medium and spore, be configured to spore suspension with sterile water.By spore
Sub- inoculation of suspension liquid is cultivated into arachidonic acid fluid nutrient medium, and inoculum concentration is 18% (volume ratio), cultivation temperature 25
DEG C, it is 120 revs/min that shaking table, which shakes revolving speed, and incubation time 48h is then centrifuged for being concentrated, the Mortierella alpine mould after collecting concentration
Thallus.
Wherein, arachidonic acid fluid nutrient medium used contains the glucose of 5% (wt) and the yeast powder of 2t%, and surplus is
Water, pH7.2.
2 prepare functional feed
2.1 preparation fermentation solid mediums
By mass percentage be 12%:42%:10%:10%:1%:1% ratio, extracting corn starch, wheat, soya bean,
Dried Corn Steep Liquor Powder, inorganic salts and vitamin combination mixing, then be added in the ratio that the weight ratio of water and cornstarch is 10:18
Water, the boiling 20min under conditions of 95 DEG C, sterilize 20min, the solid medium of obtained fermentation.
Wherein, inorganic salts ingredients used in this step are the same as embodiment 1.
Vitamin combination ingredient used is the same as embodiment 1.
2.2 inoculation fermentation
First the Mortierella alpina thallus after concentration is transferred in solid medium by inoculum concentration 0.1% (wt), in 29 DEG C
Under conditions of carry out fermented and cultured, fermentation time 40h；It is primary to stir solid medium every 12h, stirs every time for example period
It can continue 7.5min.Sterile water is supplemented into solid medium by 8% (wt) of solid medium during stirring.
After fermentation, then in the ratio that inoculum concentration is 0.3% (wt) the schizochytrium limacinum thallus after concentration is transferred in same
In one solid medium, co-fermentation time 110h is further continued under conditions of 24 DEG C.Period stirs solid culture every 12h
Base is primary, and 7.5min can for example be continued every time by stirring.By 8% (wt) of solid medium to solid medium during stirring
Middle supplement sterile water.
After fermentation, resulting culture medium and thallus will be fermented dry power is 15kw, drying temperature is 105 DEG C
Then drying and crushing 1h in microwave dryer is granulated to get the functional feed of arachidonic acid and DHA is rich in.
3 detections
The functional feed obtained for being rich in arachidonic acid and DHA is taken, its index of correlation is detected, the results are shown in Table 1.
Embodiment 5
The preparation method of functional feed provided in this embodiment is as follows:
The thallus seed of 1 preparation fermentation:
The 1.1 production DHA microbial bacteria schizochytrium limacinums that will be frozen, after defrosting, are inoculated in DHA fluid nutrient medium, in 28 DEG C
Under conditions of cultivate 42h, be then centrifuged for being concentrated, collect concentration after schizochytrium limacinum thallus.
DHA fluid nutrient medium used contains the ferment of the glucose of 6% (wt), the sodium glutamate of 3% (wt), 0.9% (wt)
The inorganic salts of female medicinal extract and 4% (wt), surplus are water, and pH is natural；
Wherein, inorganic salts ingredients used are the same as embodiment 1.
1.2, by Mortierella alpina strain inoculated to potato dextrose agar (PDA) culture medium flat plate, train at 27 DEG C
It supports 8 days and generates simultaneously maturation to spore, remove the mycelia on culture medium and spore, be configured to spore suspension with sterile water.By spore
Sub- inoculation of suspension liquid is cultivated into arachidonic acid fluid nutrient medium, and inoculum concentration is 17% (volume ratio), cultivation temperature 25
DEG C, it is 120 revs/min that shaking table, which shakes revolving speed, and incubation time 48h is then centrifuged for being concentrated, the Mortierella alpine mould after collecting concentration
Thallus.
Wherein, arachidonic acid fluid nutrient medium used contains the glucose of 5% (wt) and the yeast powder of 2t%, and surplus is
Water, pH6.8.
2 prepare functional feed
2.1 preparation fermentation solid mediums
By mass percentage be 12%:42%:10%:10%:1%:1% ratio, extracting corn starch, wheat, soya bean,
Dried Corn Steep Liquor Powder, inorganic salts and vitamin combination mixing, then be added in the ratio that the weight ratio of water and cornstarch is 10:18
Water, the boiling 20min under conditions of 95 DEG C, sterilize 20min, the solid medium of obtained fermentation.
Wherein, inorganic salts ingredients used in this step are the same as embodiment 1.
Vitamin combination ingredient used is the same as embodiment 1.
2.2 inoculation fermentation
First the Mortierella alpina thallus after concentration is transferred in solid medium for 0.1% (wt) by inoculum concentration, in 28
Fermented and cultured, fermentation time 36h are carried out under conditions of DEG C；It is primary to stir solid medium every 12h, stirs example every time period
7.5min can such as be continued.Sterile water is supplemented into solid medium by 7.5% (wt) of solid medium during stirring.
After fermentation, then in the ratio that inoculum concentration is 0.1% (wt) the schizochytrium limacinum thallus after concentration is transferred in same
In one solid medium, co-fermentation time 120h is further continued under conditions of 28 DEG C.Period stirs solid culture every 12h
Base is primary, and 7.5min can for example be continued every time by stirring.By 7.5% (wt) of solid medium to solid culture during stirring
Sterile water is supplemented in base.
After fermentation, resulting culture medium and thallus will be fermented dry power is 8kw, drying temperature is 95 DEG C micro-
Then drying and crushing 1.5h in wave drying machine is granulated to get the functional feed of arachidonic acid and DHA is rich in.
3 detections
The functional feed obtained for being rich in arachidonic acid and DHA is taken, its index of correlation is detected, the results are shown in Table 1.
Embodiment 6
The preparation method of functional feed provided in this embodiment is as follows:
The thallus seed of 1 preparation fermentation:
The 1.1 production DHA microbial bacteria thraustochytriales that will be frozen, after defrosting, are inoculated in DHA fluid nutrient medium, in 28 DEG C
Under conditions of cultivate 42h, be then centrifuged for being concentrated, collect concentration after thraustochytriale thallus.
DHA fluid nutrient medium used contains the ferment of the glucose of 6% (wt), the sodium glutamate of 3% (wt), 0.9% (wt)
The inorganic salts of female medicinal extract and 4% (wt), surplus are water, and pH is natural；
Wherein, inorganic salts ingredients used are the same as embodiment 1.
1.2, by Mortierella alpina strain inoculated to potato dextrose agar (PDA) culture medium flat plate, train at 27 DEG C
It supports 8 days and generates simultaneously maturation to spore, remove the mycelia on culture medium and spore, be configured to spore suspension with sterile water.By spore
Sub- inoculation of suspension liquid is cultivated into arachidonic acid fluid nutrient medium, and inoculum concentration is 17% (volume ratio), cultivation temperature 25
DEG C, it is 120 revs/min that shaking table, which shakes revolving speed, and incubation time 48h is then centrifuged for being concentrated, the Mortierella alpine mould after collecting concentration
Thallus.
Wherein, arachidonic acid fluid nutrient medium used contains the glucose of 5% (wt) and the yeast powder of 2t%, and surplus is
Water, pH7.3.
2 prepare functional feed
2.1 preparation fermentation solid mediums
By mass percentage be 12%:42%:10%:10%:1%:1% ratio, extracting corn starch, wheat, soya bean,
Dried Corn Steep Liquor Powder, inorganic salts and vitamin combination mixing, then be added in the ratio that the weight ratio of water and cornstarch is 10:18
Water, the boiling 20min under conditions of 95 DEG C, sterilize 20min, the solid medium of obtained fermentation.
Wherein, inorganic salts ingredients used in this step are the same as embodiment 1.
Vitamin combination ingredient used is the same as embodiment 1.
2.2 inoculation fermentation
First the Mortierella alpina thallus after concentration is transferred in solid medium for 0.4% (wt) by inoculum concentration, in 28
Fermented and cultured, fermentation time 40h are carried out under conditions of DEG C；It is primary to stir solid medium every 12h, stirs example every time period
7.5min can such as be continued.Sterile water is supplemented into solid medium by 7.5% (wt) of solid medium during stirring.
After fermentation, then in the ratio that inoculum concentration is 0.4% (wt) the thraustochytriale thallus after concentration is transferred in same
In one solid medium, co-fermentation time 120h is further continued under conditions of 28 DEG C.Period stirs solid culture every 12h
Base is primary, and 7.5min can for example be continued every time by stirring.By 7.5% (wt) of solid medium to solid culture during stirring
Sterile water is supplemented in base.
After fermentation, resulting culture medium and thallus will be fermented dry power is 8kw, drying temperature is 95 DEG C micro-
Then drying and crushing 1.5h in wave drying machine is granulated to get the functional feed of arachidonic acid and DHA is rich in.
3 detections
The functional feed obtained for being rich in arachidonic acid and DHA is taken, its index of correlation is detected, the results are shown in Table 1.
Embodiment 7
The preparation method of functional feed provided in this embodiment is as follows:
The thallus seed of 1 preparation fermentation:
The 1.1 production DHA microbial bacteria dino flagellates that will be frozen, after defrosting, are inoculated in DHA fluid nutrient medium, in 28 DEG C
Under conditions of cultivate 42h, be then centrifuged for being concentrated, collect concentration after dino flagellate thallus.
DHA fluid nutrient medium used contains the ferment of the glucose of 6% (wt), the sodium glutamate of 3% (wt), 0.9% (wt)
The inorganic salts of female medicinal extract and 4% (wt), surplus are water, and pH is natural；
Wherein, inorganic salts ingredients used are the same as embodiment 1.
1.2, by Mortierella alpina strain inoculated to potato dextrose agar (PDA) culture medium flat plate, train at 25 DEG C
It supports 9 days and generates simultaneously maturation to spore, remove the mycelia on culture medium and spore, be configured to spore suspension with sterile water.By spore
Sub- inoculation of suspension liquid is cultivated into arachidonic acid fluid nutrient medium, and inoculum concentration is 20% (volume ratio), cultivation temperature 25
DEG C, it is 120 revs/min that shaking table, which shakes revolving speed, and incubation time 48h is then centrifuged for being concentrated, the Mortierella alpine mould after collecting concentration
Thallus.
Wherein, arachidonic acid fluid nutrient medium used contains the glucose of 5% (wt) and the yeast powder of 2t%, and surplus is
Water, pH6.6.
2 prepare functional feed
2.1 preparation fermentation solid mediums
By mass percentage be 12%:42%:10%:10%:1%:1% ratio, extracting corn starch, wheat, soya bean,
Dried Corn Steep Liquor Powder, inorganic salts and vitamin mixing, then water is added in the ratio that the weight ratio of water and starch is 10:18, in 95 DEG C
Under conditions of boiling 20min, sterilize 20min, the solid medium of obtained fermentation.
Wherein, inorganic salts ingredients used in this step are the same as embodiment 1.
Vitamin combination ingredient used is the same as embodiment 1.
2.2 inoculation fermentation
First the Mortierella alpina thallus after concentration is transferred in solid medium for 0.1% (wt) by inoculum concentration, in 28
Fermented and cultured, fermentation time 40h are carried out under conditions of DEG C；It is primary to stir solid medium every 12h, stirs example every time period
7.5min can such as be continued.Sterile water is supplemented into solid medium by 7.5% (wt) of solid medium during stirring.
After fermentation, then in the ratio that inoculum concentration is 0.4% (wt) the dino flagellate thallus after concentration is transferred in same
In one solid medium, co-fermentation time 120h is further continued under conditions of 28 DEG C.Period stirs solid culture every 12h
Base is primary, and 7.5min can for example be continued every time by stirring.By 7.5% (wt) of solid medium to solid culture during stirring
Sterile water is supplemented in base.
After fermentation, resulting culture medium and thallus will be fermented dry power is 8kw, drying temperature is 95 DEG C micro-
Then drying and crushing 1.5h in wave drying machine is granulated to get the functional feed of arachidonic acid and DHA is rich in.
3 detections
The functional feed obtained for being rich in arachidonic acid and DHA is taken, its index of correlation is detected, the results are shown in Table 1.
Embodiment 8
Method is substantially the same manner as Example 3, the difference is that: after being inoculated with Mortierella alpina, after fermentation 20 hours again
It is inoculated with schizochytrium limacinum.
Embodiment 9
Method is substantially the same manner as Example 3, the difference is that: after being inoculated with Mortierella alpina, after fermentation 60 hours again
It is inoculated with schizochytrium limacinum.
Embodiment 10
Method is substantially the same manner as Example 3, the difference is that: it is first inoculated with schizochytrium limacinum, then accesses Mortierella alpina.Two
Kind bacterium inoculation interval time is same as Example 3.
Embodiment example 11
Method is substantially the same manner as Example 3, the difference is that: Mortierella alpina and schizochytrium limacinum are inoculated with simultaneously.
Comparative example 1
After this comparative example is using liquid fermentation is separated, remixes and be prepared into feed.
The fermentation of DHA
(1) seed activation culture: take deposit number of the present invention: the schizochytrium limacinum of CCTCC NO:M2012494 is prominent
Mutant is inoculated into activation medium and cultivates, and 28 DEG C of cultivation temperature, it is 150-250r/min, incubation time that shaking table, which shakes revolving speed,
48h, the activation medium are as follows: glucose 10g/L, sodium glutamate 25g/L, yeast extract 10g/L, sodium chloride 20g/L, sulfuric acid
Magnesium 0.5g/L, pH are natural.
Wherein, which is preserved in China typical culture collection center on December 3rd, 2012, address: Wuhan, China is military
Chinese university, deposit number: CCTCC NO:M2012494, taxology name: Schizochytrium sp..
(2) seed expands culture: by the activated seed culture solution of above-mentioned steps (1) shaking culture according to 10% (volume ratio)
Inoculum concentration is inoculated into expand and be cultivated in culture medium, and seed tank volume used is 10L, and culture medium loading amount is 60% in seeding tank
(volume ratio), incubation technology controlling and process are as follows: 28 DEG C of cultivation temperature, 150 revs/min of mixing speed, ventilatory capacity 1vvm (L/
L.min), incubation time 44h, the expansion culture medium are as follows: glucose 40g/L, sodium glutamate 25g/L, yeast extract 10g/L,
Sodium chloride 10g/L, magnesium sulfate 5g/L, potassium dihydrogen phosphate 1g/L, calcium chloride 0.5g/L, pH are natural.
(3) fermentation tank culture: the expansion seed culture fluid of above-mentioned steps (2) shaking culture is connect according to 10% (volume ratio)
Kind amount is inoculated into fermented and cultured in the 50L fermentation flask equipped with fermentation medium, incubation technology controlling and process are as follows: cultivation temperature 28
DEG C, 150 revs/min of mixing speed, ventilatory capacity 1.5vvm (L/L.min), tank presses 0.1MPa to support time 90h, leads in fermentation process
Overcurrent adds glucose to control in fermentation liquid concentration of glucose in 10-20g/L,
(4) it puts the direct microwave drying of DHA fermentation liquid after tank, carries out microwave drying 8h at 95 DEG C, obtain DHA bacterium powder；
Arachidonic fermentation:
(1) seed culture: by Mortierella alpina strain inoculated to potato dextrose agar (PDA) culture medium flat plate,
It is cultivated at 27 DEG C 8 days and generates simultaneously maturation to spore, remove the mycelia on culture medium and spore, it is outstanding to be configured to spore with sterile water
Supernatant liquid.Spore suspension is inoculated into arachidonic acid fluid nutrient medium and is cultivated, inoculum concentration is 17% (volume ratio), training
25 DEG C of temperature is supported, it is 120 revs/min that shaking table, which shakes revolving speed, incubation time 48h.
Wherein, arachidonic acid fluid nutrient medium used contains the glucose of 5% (wt) and the yeast powder of 2t%, and surplus is
Water, pH6.8.
(2) seed expands culture: final fermentation tank culture uses the volume of 50L, therefore seeding tank selects 10L seed to expand
Fermentor.The Mortierella alpina shake-flask seed fermentation liquid of above-mentioned steps is inoculated into seeding tank and carries out seed expansion culture,
In seed culture medium carbon source sucrose 35g/l；Nitrogen source yeast extract 12g/l controls pH 7, and fermentation temperature is 28 DEG C, stirring speed
220 revs/min, ventilatory capacity 1vvm (L/L.min) of degree, tank press 0.1MPa, cultivate 42h.
(3) fermented and cultured: bacterium is dense in seeding tank reach 20% after, be linked by culture transferring pipeline equipped with 30L fermented and cultured
It is cultivated in the 50L fermentor of base, inoculum concentration 15% (volume ratio), 28 DEG C of temperature of fermentor control, 220 turns of mixing speed/
Minute, ventilatory capacity 1vvm (L/L.min), tank presses 0.1MPa, cultivates 170h.Fermentation is controlled in fermentation process by stream plus carbon source
Carbon source concentration is in 10g/L in liquid.
(4) arachidonic acid fermentation liquid Direct spraying is dry, it is carried out under 200 degree of inlet air temperature, 80 degree of leaving air temp
Microwave drying 8h obtains arachidonic acid bacterium powder；
By after two kinds of bacterium powder mixing granulations up to feed.
Comparative example 2
Access Mortierella alpina and schizochytrium limacinum simultaneously in liquid fermentation medium.Tank is put after fermentation 170h.Drying is made
Grain is up to feed.
The testing result that feed indices are made in the above embodiments 1-7 is shown in Table 1, the above embodiments 8-11 and comparison
The testing result of feed indices made from example 1 and comparative example 2 is shown in Table 2.
Feed testing result made from 1 embodiment 1-7 of table
Feed testing result made from table 2 embodiment 8-11, comparative example 1-2
Wherein, protein detection method: GB/T 6432-1994 is referred to, crude fibre detection method: refers to GB/T 6434-
1994, the measurement of moisture in feed: GB/T 6435-2014 is referred to, particle size detection method: referring to GB/T 5917.1-2008.
According to the result of Tables 1 and 2 it can be seen that
It can be seen that the feed of separated liquid fermentation in fermentation and drying by comparing the result of comparative example 1, comparative example 2
It is easy to cause the oxidation (POV value 9.6,8.8) of polyunsaturated fatty acid in the process, and embodiment 1-11 is all made of in solid culture
Base fermentation, obtained feed POV value are only up to 5, are lower than comparative example and 5, meet inner quality standard, thus illustrate, the present invention will
Two kinds of bacterium, which are placed in co-incubation on solid medium, effectively to overcome DHA caused by separated liquid fermentation and arachidonic acid easy
It is oxidized problem；
Mortierella alpine mould and schizochytrium limacinum are placed in same liquid in addition, can be seen that by comparing the result of comparative example 2
It ferments in body fermentation medium, due to competitive relation, Mortierella alpina is in a disadvantageous position, and can not also obtain desired result, feed flower
Raw tetraene acid content is very low, illustrates that two kinds of bacterium are placed in co-incubation on solid medium and can greatly improve peanut by the present invention
Tetraene acid content.
Compared by the result of embodiment 8-9, it can be seen that the inoculation interval time of Mortierella alpine mould and schizochytrium limacinum
Have an impact to the yield of arachidonic acid and DHA in product, using interval time range inoculating two kinds bacterial strain of the invention,
The total output of arachidonic acid and DHA in product is improved significantly；
In addition, can be seen that Mortierella alpine mould and schizochytrium limacinum inoculation successively by comparing the result of embodiment 10,11
Sequence has an impact to the yield of arachidonic acid and DHA, is first inoculated with Mortierella alpine mould and is followed by kind of a schizochytrium limacinum, hence it is evident that improves
The total output of arachidonic acid and DHA in product.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of preparation method of functional feed, which is characterized in that it includes the following steps:
Fermentation step: producing arachidonic acid microbial bacteria bacterium solution and production DHA microbial bacteria bacterium solution are seeded to same solid culture
Mixed fermentation in base；
Preferably, the inoculum concentration for producing DHA microbial bacteria is 0.1%-1% (wt)；
The inoculum concentration of the producing arachidonic acid microbial bacteria bacterium solution is 0.1%-1% (wt)；
The condition of mixed fermentation: temperature is 20-30 DEG C, and the time is 100-140 hours.
2. preparation method according to claim 1, which is characterized in that in fermentation step, first by the yield peanut tetraene
Sour microbial bacteria bacterium solution, which is seeded in the solid medium, cultivates 36-48h, then the production DHA microbial bacteria bacterium solution is inoculated with
To mixed culture fermentation 100-140 hours in the solid medium.
3. preparation method according to claim 2, which is characterized in that produce DHA microbial bacteria and be selected from schizochytrium limacinum, broken capsule pot
Any one in bacterium, dino flagellate and saccharomycete.
4. preparation method according to claim 3, which is characterized in that the microbial bacteria of producing arachidonic acid is Mortierella alpine
It is mould.
5. preparation method according to claim 1-4, which is characterized in that the solid medium includes such as the following group
Point:
Protein matter, inorganic salts and vitamin.
6. preparation method according to claim 1-4, which is characterized in that before fermentation step, the preparation
Method further includes bacterium solution preparation step:
The bacterium solution preparation step includes the following steps (1) and/or step (2):
Step (1): production DHA microbial bacteria strain is seeded in DHA fluid nutrient medium and cultivates 36-48h, obtains the production DHA
Microbial bacteria bacterium solution；
Step (2): producing arachidonic acid microbial bacteria strain being seeded in arachidonic acid fluid nutrient medium and cultivates 36-48h,
Obtain the producing arachidonic acid microbial bacteria bacterium solution.
7. preparation method according to claim 6, which is characterized in that the DHA fluid nutrient medium includes: glucose, paddy
Propylhomoserin sodium, yeast extract and inorganic salts；
Preferably, the DHA fluid nutrient medium includes: 5%-7% (wt) glucose, 2%-4% (wt) sodium glutamate, 0.8%-
1% (wt) yeast extract and 3%-5% (wt) inorganic salts；
Preferably, the DHA fluid nutrient medium includes: 6% (wt) glucose, 3% (wt) sodium glutamate, 0.9% (wt) yeast
Medicinal extract and 4% (wt) inorganic salts；
Preferably, inorganic salts are selected from one of phosphate, sulfate, nitrate, sodium salt and calcium chloride or a variety of combinations.
8. preparation method according to claim 6, which is characterized in that the arachidonic acid fluid nutrient medium includes: Portugal
Grape sugar and yeast powder；
Preferably, the arachidonic acid fluid nutrient medium includes: 3%-6% (wt) glucose and 1%-3% (wt) yeast powder；
Preferably, the arachidonic acid fluid nutrient medium includes: 5% (wt) glucose and 2% (wt) yeast powder.
9. a kind of functional feed, which is characterized in that it is as obtained by the described in any item preparation methods of claim 1-8.
10. a kind of functional feed, which is characterized in that it includes following ingredient:
DHA content 5.8%-30.5% (wt), arachidonic acid content 3.3%-28.5% (wt), protein content 37%-
65% (wt), crude fiber content are no more than 10% (wt) lower than 10% (wt), moisture content；
The granularity of the functional feed is 0.8-6mm, carbon-nitrogen ratio is (15-30): 1.
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| CN102987096A (en) * | 2012-12-31 | 2013-03-27 | 厦门金达威集团股份有限公司 | Microbiological feed additive and preparation method and application thereof |
| CN103882071A (en) * | 2014-03-14 | 2014-06-25 | 嘉必优生物工程（武汉）有限公司 | Microbial oil and preparation method thereof |
| CN104630077A (en) * | 2015-02-05 | 2015-05-20 | 江南大学 | Mortierella alpine CCFM442 strain and use thereof |
| CN106260547A (en) * | 2016-08-05 | 2017-01-04 | 湖北凌卓生物工程有限公司 | A kind of fermenting organism feedstuff and preparation method thereof |
| 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 |
| CN108065037A (en) * | 2017-12-28 | 2018-05-25 | 嘉必优生物技术（武汉）股份有限公司 | Microbial fermentation prepares the method and functional feed of functional feed |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102987096A (en) * | 2012-12-31 | 2013-03-27 | 厦门金达威集团股份有限公司 | Microbiological feed additive and preparation method and application thereof |
| CN103882071A (en) * | 2014-03-14 | 2014-06-25 | 嘉必优生物工程（武汉）有限公司 | Microbial oil and preparation method thereof |
| CN104630077A (en) * | 2015-02-05 | 2015-05-20 | 江南大学 | Mortierella alpine CCFM442 strain and use thereof |
| CN106260547A (en) * | 2016-08-05 | 2017-01-04 | 湖北凌卓生物工程有限公司 | A kind of fermenting organism feedstuff and preparation method thereof |
| 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 |
| CN108065037A (en) * | 2017-12-28 | 2018-05-25 | 嘉必优生物技术（武汉）股份有限公司 | Microbial fermentation prepares the method and functional feed of functional feed |
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