CN104630077B - A kind of Mortierella alpina CCFM442 bacterial strains and application thereof - Google Patents
A kind of Mortierella alpina CCFM442 bacterial strains and application thereof Download PDFInfo
- Publication number
- CN104630077B CN104630077B CN201510062057.9A CN201510062057A CN104630077B CN 104630077 B CN104630077 B CN 104630077B CN 201510062057 A CN201510062057 A CN 201510062057A CN 104630077 B CN104630077 B CN 104630077B
- Authority
- CN
- China
- Prior art keywords
- feed additive
- mortierella alpina
- ccfm442
- production
- temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Fodder In General (AREA)
Abstract
The present invention relates to a kind of Mortierella alpina CCFM442 bacterial strains and application thereof.The present invention includes inclined-plane culture, seed culture, fermented and cultured and post-processing step using Mortierella alpina CCFM442 bacterial strains production thalli feed additive, the method for the production thalli feed additive.The thalli feed additive is safe.Aliphatic acid composition in the thalli feed additive is reasonable, and content of the polyunsaturated fatty acid in total fatty acids reaches 42.4 45.8%, and the ω 3 of ω 6/ meet WHO and FAO proposed standard than being 4.5 5.3.The thalli feed Additive Production used medium is cheap glucose and dregs of beans, and the two market price is relatively low and is readily available, therefore production cost is low.
Description
【Technical field】
The invention belongs to technical field of animal.A kind of more particularly it relates to Mortierella alpina CCFM442 bacterium
Strain, further relate to the purposes of the Mortierella alpina CCFM442 bacterial strains.
【Background technology】
Polyunsaturated fatty acid (PUFAs) can be divided into ω -6 and ω -3 grade series.ω -6 Series Ps UFAs mainly has linoleic acid
(LA), gamma-Linolenic acid (GLA), arachidonic acid (AA) etc..AA is the important precursor of human prostate's element synthesis, and it has
There is increase blood vessel elasticity and improve the physiological functions such as immunity, be important milk powder nutritious supplementary pharmaceutical.ω -3 Series Ps UFAs is main
There are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), clupanodonic acid (DPA) and docosahexaenoic acid (DHA).
EPA can reduce blood cholesterol levels and atherosclerosis occurrence risk.DHA has in raising infant's intelligence etc.
Important function.WHO and FAO proposes ω -6PUFAs and ω -3PUFAs ratio in meals (hereinafter referred to as ω -6/ ω -3 compare)
Most just when for (5-10)：1, in China's ratio serious unbalance, up to (10-30)：1.Epidemiologic data shows, ω in diet-
6/ ω -3 is occurred frequently more closely related with some diseases than too high, such as diabetes, coronary heart disease, breast cancer.Therefore, improve in diet
ω -3PUFAs ratios simultaneously maintain ω -6/ ω -3 more significant than in optimum range.
ω -3 Series Ps UFAs comes from the materials such as animal's liver and fish oil more, because of factors such as environmental pollutions, its produce by
Limitation.Mortierella alpina (Mortierella alpina, M.alpina) is unique by formal peace in current production PUFAs fungies
The strain that full property is assessed, its fermenting and producing ω -3PUFAs have been confirmed in experiment feasible.Bacterium feed is made using M.alpina thalline
Laying hen is fed, can increase the ω -3PUFAs contents in egg, this is favorably improved the ratio of ω -3PUFAs in diet.
【The content of the invention】
[technical problems to be solved]
It is an object of the invention to provide a kind of Mortierella alpina CCFM442 bacterial strains.
It is a further object to provide the purposes of the Mortierella alpina CCFM442 bacterial strains.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to a kind of Mortierella alpina Mortierella alpina CCFM442, the bacterial strain is in 2014 12
The moon 25 is in the Chinese microorganism strain preservation of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Administration committee's common micro-organisms center preservation, its preserving number are CGMCC No 10295.
Mortierella alpine trichoderma strain CCFM442 is the engineering to being obtained after Mortierella alpina wild-type strain progress genetic modification
Bacterial strain.The biological property of specific genetic modification method and obtained strains is referring to Chinese patent application CN
201410087487.1 record.The specific condition of culture of Mortierella alpina of gained is as follows：It first in temperature 28 DEG C be with rotating speed
Cultivate 2 days, then cultivated 8 days under conditions of 12 DEG C of temperature and rotating speed 200r/min, Mortierella alpine under conditions of 200r/min
Compared with wild-type strain significant change occurs for the rule of trichoderma strain CCFM442 accumulation fat, and the ratio that wherein EPA accounts for total fatty acids is bright
Aobvious increase, about the 7 of wild-type strain times.Just because of the change of this property, turn into for Mortierella alpine trichoderma strain CCFM442
Industrial strain with respect to high yield ω -3PUFAs is laid a good foundation.
The invention further relates to purposes of the described Mortierella alpina CCFM442 bacterial strains in thalli feed additive is produced.
A preferred embodiment of the invention, the step of producing thalli feed additive of the present invention, are as follows：
A, inclined-plane culture
The ring of Mortierella alpina CCFM442 bacterial strains picking one of freezen protective is inoculated on PDA slant mediums, Ran Hou
Incubated 14 days under conditions of 25 DEG C of temperature, the bacterial strain activation culture thing is obtained.
The PDA slant mediums that the present invention uses are prepared according to its conventional formulation.PDA inclined-plane cultures based component is such as
Under：200g potatos, 20g glucose, 15g agar and 1L running water, are adjusted its pH using sodium hydroxide and hydrochloride aqueous solution
Save as 6.0-6.2.
In this step, equipment used in inclined-plane culture is the gloomy reliable water proof for testing Instrument Ltd.'s sale in Shanghai
Formula constant incubator (model：GRP-9080).
B, seed culture
The ring of activation culture thing picking one that step A is obtained is inoculated into seed culture medium, in 25 DEG C of temperature and 200rpm
Under conditions of constant-temperature shaking culture 3 days, then claim to obtain wet thallus weight, by wet thallus and sterilized water according to wet thallus and sterile water quality
Than 1：4 are mixed, and scattered 10min are then carried out to the mixed system with dispersion machine to prepare bacteria suspension, according still further to every liter of institute
The inoculum concentration that bacteria suspension is 10mL is stated to be inoculated in the seed culture medium, it is permanent under conditions of 25 DEG C of temperature and rotating speed 200rpm
Warm shaken cultivation 36h, obtain Mortierella alpina CCFM442 seed liquors.
The seed culture medium that the present invention uses is prepared according to following formulas：20g/L glucose, 10g/L potassium nitrate,
3g/L potassium dihydrogen phosphates, 5g/L yeast extracts and 0.25g/L magnesium sulfate, natural pH.
In this seed culture step, the culture device used is the vibration training of Shanghai Zhi Chu Instrument Ltd. sale
Support case (model：ZQZY-70B).
The dispersion machine that the present invention uses is the dispersion machine (model that card experimental instruments and equipment limited is sold that ends：T10B).
C, fermented and cultured
According to fermentation medium stereometer 1%-5% inoculum concentrations, the Mortierella alpina CCFM442 kinds that step B is obtained
Sub- liquid is inoculated into fermentation medium, cultivates 2 days under conditions of 25 DEG C of temperature first, is then trained under conditions of 6 DEG C of temperature
Support 5 days, obtain the zymotic fluid containing Mortierella alpina CCFM442.
According to the present invention, fermentation uses the main purpose of Fluctuation temperature culture to be 25 DEG C of temperature for Mortierella alpina CCFM442
Optimum growth temperature, cultivating 2 days can make Fungal biodiversity increase to maximum at such a temperature；6 DEG C of temperature is Mortierella alpina
CCFM442 fermenting and producings EPA preference temperature, cultivating can make thalline accumulate a large amount of EPA for 5 days at this temperature.
In this step, judge whether Fungal biodiversity increases to maximum according to zymotic fluid dissolved oxygen amount, due to Initial stage of culture
Thalline fast-growth need to consume a large amount of oxygen, so zymotic fluid dissolved oxygen amount progressively declines, there is slowly dissolved oxygen after dropping to minimum point
Rise, Fungal biodiversity maximum during time point (cultivate 2 days at 25 DEG C of temperature).
In this fermented and cultured step, the culture device used is the 4L fermentation tanks of New Brunswick companies sale
(model：BioFlo 115).
The fermentation medium preparation method is as follows：Prepare containing 50g/L glucose carbon sources, 20g/L dregs of beans, 13.34g/L
The mixing of potassium nitrate, 3g/L dipotassium hydrogen phosphates, 1g/L sodium sulphate, 0.5g/L CALCIUM CHLORIDE DIHYDRATEs and 0.5g/L Magnesium dichloride hexahydrates
The thing aqueous solution, the pH of its aqueous solution is adjusted to 6.0, then the 20min that sterilized at 121 DEG C of temperature, obtain described fermented and cultured
Base.
The pH of the aqueous solution is adjusted using sulfuric acid and potassium hydroxide aqueous solution.
D, post-process
The zymotic fluid containing Mortierella alpina CCFM442 for allowing step C to obtain is centrifuged, and obtained wet thallus is used
Distilled water is washed, then is centrifuged, and washing wet thallus is then dried, and then obtains a kind of dry thalline, i.e. described bacterium
Body feedstuff additive.
In this step, described washing is that use distills water washing 2-4 times for 3-5 times with wet thallus stereometer.
In this step, first time and second of centrifugation are centrifuged under conditions of rotating speed 6000rpm
Separate 10min.Equipment used in centrifugation is the centrifuge (model of Thermo sale：LYNX 4000).
In this step, described drying is that 85- is dried under conditions of 48-52 DEG C of temperature and wind speed 2.1-2.5m/s
95min。
The drying equipment that the present invention uses is the gloomy reliable electric heating constant temperature forced air drying for testing Instrument Ltd.'s sale in Shanghai
Case (model：DGG-9123A).
The drying thalline obtained using the inventive method, the i.e. moisture content of described thalli feed additive are to dry bacterium
Body gross weight meter 25.4-26.4%, total fatty acid content 7.6-10.7%, wherein polyunsaturated fatty acid is in total fatty acids
Content be 42.4-45.8%.
Described measurement of water-content coefficient refers to GB/T 5009.3-2003.
Described crude fat measure refers to GB/T 14772-2008.
Described determination of fatty acid bibliography Wang L, Chen W, Feng Y, et al.Genome
characterization of the oleaginous fungus Mortierella alpina.Plos One6:
e28319.。
After total fatty acids measure, described polyunsaturated fatty acid can be obtained after being summed up to corresponding content of fatty acid
Content.
It can be calculated with the serial content of polyunsaturated fatty acid of ω -3 by ω -6 series in the polyunsaturated fatty acid
Go out, ω -6/ ω -3 ratios are 4.5-5.3.
According to the present invention, the dosage of the thalli feed additive is the 5-10% of basal feed gross weight.
The aliphatic acid composition of thalli feed additive of the present invention is reasonable, can be provided with the chicken beneficial to eater's health
Egg.The additive has the advantages that safe to use, PUFAs contents are high, aliphatic acid composition is reasonable, is not reducing egg laying performance
The content of AA, DHA in egg are improved in the case of parameter, and maintains ω -6/ ω -3 ratios in the reasonable scope.
[beneficial effect]
The beneficial effects of the invention are as follows：
The thalli feed additive is safe.By formal safety evaluation, it is produced the strain that the present invention uses
Security is guaranteed；The present invention is not introduced into the material safe to the human body impacted in process of production.
Aliphatic acid composition in the thalli feed additive is reasonable, and content of the polyunsaturated fatty acid in total fatty acids reaches
To 42.4-45.8%, ω -6/ ω -3 ratios are 4.5-5.3, meet WHO and FAO proposed standard.
The thalli feed Additive Production used medium is cheap glucose and dregs of beans, and the two market price is relatively low and holds
Easily obtain, therefore production cost is low.
Mortierella alpina Mortierella alpina CCFM442 of the present invention exist on December 25th, 2014
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, its preserving number is CGMCC No.10295.
【Embodiment】
The present invention is will be better understood that by following embodiments.
Embodiment 1：Produce the thalli feed additive of the present invention
The implementation steps of the embodiment are as follows：
A, inclined-plane culture
The ring of Mortierella alpina CCFM442 bacterial strains picking one of freezen protective is inoculated on described PDA slant mediums,
Then by the gloomy reliable water isolation type constant incubator (model for testing Instrument Ltd.'s sale in Shanghai：GRP-9080 in temperature in)
Incubated 14 days under conditions of 25 DEG C, the bacterial strain activation culture thing is obtained.
B, seed culture
The ring of activation culture thing picking one that step A is obtained is inoculated into the seed that described method is prepared to specifications to train
Support in base, then in the shaken cultivation case (model sold by Shanghai Zhi Chu Instrument Ltd.：ZQZY-70B in temperature 25 in)
DEG C with constant-temperature shaking culture 3 days under conditions of rotating speed 200rpm, then claim to obtain wet thallus weight, by wet thallus with sterilized water according to wet
Thalline and sterilized water mass ratio 1：4 are mixed, then with the dispersion machine (model sold by Chinese mugwort card experimental instruments and equipment limited：
T10B scattered 10min) is carried out to the mixed system to prepare bacteria suspension, according still further to the inoculum concentration that bacteria suspension every liter described is 10mL
It is inoculated in described seed culture medium, it is permanent under conditions of 25 DEG C of temperature and rotating speed 200rpm in same seed culture device
Warm shaken cultivation 36h, obtain Mortierella alpina CCFM442 seed liquors.
C, fermented and cultured
Prepare containing 50g/L glucose carbon sources, 20g/L dregs of beans, 13.34g/L potassium nitrate, 3g/L dipotassium hydrogen phosphates, 1g/L
The mixture aqueous solution of sodium sulphate, 0.5g/L CALCIUM CHLORIDE DIHYDRATEs and 0.5g/L Magnesium dichloride hexahydrates, uses 1M sulfuric acid or hydrogen-oxygen
Change aqueous solutions of potassium to adjust the pH of its aqueous solution to 6.0, then the 20min that sterilized at 121 DEG C of temperature, obtain described fermented and cultured
Base；
According to the inoculum concentration of fermentation medium stereometer 2%, the Mortierella alpina CCFM442 seed liquors that step B is obtained
It is inoculated into fermentation medium, in the 4L fermentation tank (models sold by New Brunswick companies：BioFlo 115) in carry out
Fermented and cultured.2 angel's Fungal biodiversities are cultivated under conditions of 25 DEG C of temperature first and increase to maximum, basis for estimation is that zymotic fluid is molten
Oxygen amount, because Initial stage of culture thalline fast-growth need to consume a large amount of oxygen, so zymotic fluid dissolved oxygen amount progressively declines, drop to most
Dissolved oxygen amount has slow rise after low spot, and Fungal biodiversity is maximum during time point (being cultivated 2 days at 25 DEG C of temperature), then in temperature
5 days are cultivated under conditions of 6 DEG C of degree to promote thalline to accumulate EPA.
D, post-process
Use the centrifuge (model sold by Thermo：LYNX 4000), allow what step C obtained to contain Mortierella alpina
CCFM442 zymotic fluid is centrifuged under conditions of rotating speed 6000rpm, and obtained wet thallus use is with wet thallus volume
4 times of meter distillation water washing 3 times, is centrifuged using same centrifuge under conditions of rotating speed 6000rpm, obtained washing precipitation
Thing is then in the gloomy reliable electric heating constant-temperature blowing drying box (model for testing Instrument Ltd.'s sale in Shanghai：DGG-9123A in)
Temperature 50 C obtains drying thalline, i.e. described thalli feed additive with drying 90min under conditions of wind speed 2.3m/s.
The measurement of water-content coefficient described using this specification refers to GB/T 5009.3-2003.Crude fat measure refers to GB/T
14772-2008.Determination of fatty acid bibliography Wang L, Chen W, Feng Y, et al.Genome
characterization of the oleaginous fungus Mortierella alpina.Plos One6:
e28319.。
The moisture content for the drying thalline (thalli feed additive) that the present embodiment is prepared is to dry thalline gross weight
Meter 26.38%, the serial polyunsaturated fatty acid of total fatty acid content 10.56%, wherein polyunsaturated fatty acid and ω -6
It is listed in the table below with the serial content of polyunsaturated fatty acid of ω -3 in 1.
Table 1：The aliphatic acid composition and content of thalli feed additive
Note：Data are represented with average ± standard deviation in table；ω -6/ ω -3 are than being 4.47 ± 0.76.
This thalline additive fatty acid species compared with horn of plenty, PUFAs contents account for total fatty acid content 45.76% ±
0.81%, wherein the AA contents as important baby milk powder additive account for total fatty acid content 24.43% ± 0.75%, tool
The EPA content for having important immunologic function accounts for the 6.09% ± 0.15% of total fatty acid content；ω -3PUFAs in common egg
Predominantly DHA, 0.04%, the AA for accounting for shell egg gross weight account for the 0.11% of shell egg gross weight.China regulation baby in 2000
AA addition is 0.16%-0.26% in strengthening model, and DHA addition is 0.04%-0.18%.Thus from AA and DHA
Seen in the angle of content, common egg fatty acid composition is improved especially increase the content this respect of the two especially must
Will.Therefore, this thalline additive has the superiority in production, and the DHA in improving egg aliphatic acid composition, improving egg
With the importance in AA content.
Embodiment 2：Produce the thalli feed additive of the present invention
The embodiment of the embodiment is same as Example 1, and simply seed liquor inoculum concentration is with fermented and cultured in step C
Base stereometer 5%.
Precipitated using the method for this specification description, the drying thalline (thalli feed additive) that the present embodiment is prepared
Moisture content be to dry thalline gross weight meter 25.37%, total fatty acid content 9.07%, wherein polyunsaturated fatty acid exist
Content in total fatty acids is 42.37%.The ratio of the serial polyunsaturated fatty acids of ω -6 and the serial polyunsaturated fatty acids of ω -3
For 4.95.
Embodiment 3：Produce the thalli feed additive of the present invention
The embodiment of the embodiment is same as Example 1, and simply seed liquor inoculum concentration is with fermented and cultured in step C
Base stereometer 1%.
Precipitated using the method for this specification description, the drying thalline (thalli feed additive) that the present embodiment is prepared
Moisture content be to dry thalline gross weight meter 25.99%, total fatty acid content 7.64%, wherein polyunsaturated fatty acid exist
Content in total fatty acids is 43.76%.The ratio of the serial polyunsaturated fatty acids of ω -6 and the serial polyunsaturated fatty acids of ω -3
For 5.28.
The result of the test of above example 1,2,3 is summarized, is shown in Table 2.
Table 2：Drive member index in each embodiment
Note：Data are represented with average ± standard deviation in 1 table.
The female different expression significant differences (P ＜ 0.05) of 2 colleague's shoulder marking-ups.
Comparative example 1：Produce the thalli feed additive of the present invention
The embodiment of the comparative example is same as Example 1, simply in step C seed cultures, before fermented and cultured
Phase and cultivated under conditions of 25 DEG C of temperature in the fermented and cultured later stage.
Precipitated using the method for this specification description, the drying thalline (thalli feed additive) that the present embodiment is prepared
Moisture content be to dry thalline gross weight meter 25.11%, wherein total fatty acid content 15.56%, polyunsaturated fatty acid
Content in total fatty acids is 39.15%.The serial polyunsaturated fatty acids of ω -6 and the serial polyunsaturated fatty acids of ω -3
Than for 23.72..
Comparative example 2：Produce the thalli feed additive of the present invention
The embodiment of the comparative example is same as Example 1, simply in step C seed cultures, before fermented and cultured
Phase and cultivated under conditions of 6 DEG C of temperature in the fermented and cultured later stage.
The method described using this specification, because whole process all carries out fermented and cultured, thalline under conditions of 6 DEG C
Hardly grow, Fungal biodiversity is less than 1g/L, and less than the 50g/L under Fluctuation temperature culture, only very small amount EPA is detected in thalline.
By embodiment 1 compared with the result of implementation of comparative example 1 and 2 it is recognised that according to Mortierella alpina
CCFM442 production fat rule, using Fluctuation temperature culture strategy thalline can be promoted to accumulate substantial amounts of EPA, and incubated (6 DEG C of temperature
Or 25 DEG C) be difficult to meet actual requirement.
Test example 1
In order to more effectively illustrate the practicality of the thalli feed additive, first by thalli feed additive respectively with basis
Feed gross weight meter 5% or 10% and basal feed mixing granulation, then take a series of animal experiments and are verified.Below
Embodiment and interpretation of result for animal experiment：
1st, the preparation of thalli feed.
Basal feed is bought from Lv Kemao animal husbandry Co., Ltd, the product Contents of Main Components such as table 3 below.
Table 3：Basal feed forms and fatty acid profile table
Note：Data are represented with average ± standard deviation.
The present invention is dried into thalline additive to crush, then respectively according to the 5% of basal feed gross weight and 10% addition
Cool Room 4 DEG C preservation is placed in into described basal feed, after mixing granulation.
2nd, experiment packet and design
25 weeks chicken age laying hens are chosen, totally 45, are randomly divided into 3 groups, are designated as I, II, III group respectively.Wherein I group is control
Group, II, III group is experimental group.
Whole experiment periods are divided into laundering period and experimental period.All fed in tri- groups of laundering period 14d with basal feed.Trying
Test the phase, I, II, III group is raised by basal feed, low dosage (5% in terms of basal feed weight, hereinafter referred to as 5% group) thalline respectively
Feed additives feed, high dose (10% in terms of basal feed weight, hereinafter referred to as 10% group) thalli feed addition agent feedstuff are raised
Hello.Every 5d measure egg index (see 4, testing index), to yolk fatty acid levels it is constant when change feed basal feed.
3rd, feeding and management method
Whole feeding experiment is carried out in the south of the River institute of driving school of Wuxi City Lake District Jin Cheng roads, takes laying hen free-ranging, illumination
14h/ days (during sunshine deficiency, using artificially feed), feed, water are enough.Free choice feeding and drinking-water, periodically beat hen house
Sweep.Daily 8:00、16:00 feeding twice, per day entry on the day of inventory and feed surplus.Daily 16:00 picks up egg and marks
Number, numeration.
4th, testing index
4.1 average egg weights, laying rate, feedstuff-egg ratio, food-intake production performance index.
Computational methods are as follows：
Average egg weight (g/ pieces)=daily egg production/total piece of number of daily egg
Laying rate=(a piece number of laying eggs daily/livestock on hand chicken number) × 100%
Feedstuff-egg ratio=feed consumption rate daily/weight of laying eggs daily
Food-intake (g)=M-m
In its formula：
M is daily every group of input feeding quality, unit g；
M is that the residual feed amount in trough, unit g are collected before feeding intake next day
4.2 egg quality indexs, including egg shape index, yolk color, yolk are relatively heavy, shell thickness, Hough unit.
Egg shape index assay method：
With the vertical footpath of vernier caliper measurement egg and maximum transverse diameter, measurement is accurate to 0.02mm.
Egg shape index=(transverse diameter/vertical footpath) × 100%
Yolk color assay method：
Fanned using Roche colorimetric, take 5 pieces of eggs at random from each test group respectively, using black as background, under available light
Estimated.
The relatively heavy assay method of yolk：
Egg white and yolk are kept completely separate using Yolk separator, yolk is collected and weighs weight.
Yolk is relatively heavy=(yolk weight/shell egg weight) × 100%
Shell thickness assay method：
Measure blunt end, the thickness at three positions in centre and sharp end of eggshell respectively with slide measure, average, be accurate to
0.01mm。
Hough unit assay method：
Egg is broken and is on horizontal positioned flat glass plate, with the height of vernier caliper measurement dense albumen.
Hu=100 × log10(H-1.7×W0.37+7.57)
In formula：
Hu is Hough unit；
H is dense albumen height (mm)；
W is egg size (g).
4.3rd, egg fatty acid analysis.
Egg fatty acid analysis method bibliography Wang L, Chen W, Feng Y, et al.Genome
characterization of the oleaginous fungus Mortierella alpina.Plos One6:
e28319.。
4.4th, egg fatty acid profile mutation analysis.
Carried out with feeding process, until each aliphatic acid accounts for total fatty acids percentage and no longer changed in egg, as feed
Middle aliphatic acid is transformed into the saturation point in egg.
5th, data calculating and statistical method
The otherness of single factor analysis each group index is used by the softwares of SPSS 17.0.
6th, result and analysis
Experiment finds that each aliphatic acid accounts for total fatty acids percentage no longer in egg after thalline additive supplement is fed 10 days
Change, thus index determining is carried out to the egg sample before supplement 10 days phases and 10 days.
6.1 production performance indexs
Each experimental group performance in layers index is listed in Table 4 below.
Table 4：Each experimental group performance in layers index
Note：1 data are represented with average ± standard deviation；
The female different expression significant differences (P ＜ 0.05) of 2 colleague's shoulder marking-ups.
Result of the test is found：Compared with control group, 5% and 10% thalline additive of addition is to laying hen body weight, food ration, production
Egg rate, average egg weight Index Influence difference is not notable.Due to taking the environmental conditions such as outdoor free-ranging, temperature to be difficult to keep laying hen
Control, thus laying rate of laying hen and average egg weight are below the data of common batterylaying；Compared with batterylaying, free-ranging laying hen
Activity is big, and according to energy Distribution dynamics, thus free-ranging laying hen feedstuff-egg ratio is higher, and the above results are consistent with document report.
6.2nd, egg quality index
Each experimental group laying hen production egg quality index is listed in Table 5 below.
Each experimental group egg quality index of table 5
Note：1 data are represented with average ± standard deviation；
The female different expression significant differences (P ＜ 0.05) of 2 colleague's shoulder marking-ups.
Result of the test shows：Control group is compared, and 5% and 10% thalli feed additive of addition is to laying hen egg shape index, egg
It is not notable that yellow chromaticity, yolk are relatively heavy, shell thickness, Hough bit indicator influence difference.Therefore, the thalli feed additive is not
Egg quality index can be reduced.
6.3 egg aliphatic acid forms
Main fatty acid content results are listed in Table 6 below in each experimental group laying hen production egg yolk.
Table 6：Main fatty acid assay result in yolk
Note：
Ith, II, III ω -6/ ω -3 ratios respectively 29.63 ± 0.55a、15.91±1.93b、12.89±0.52b；
Ith, II, III total fatty acids amount is 22.37 ± 0.54 respectively for the percentage of yolk gross weighta、21.91±
0.83a、23.51±0.93a；
Data are represented with average ± standard deviation in table；The female different expression significant differences (P ＜ 0.05) of shoulder marking-up of going together.
It can be seen that by above-mentioned result of the test：
For adding 5% Mortierella alpina thalli feed additive, DHA accounts for total fatty acids ratio and increased to by 0.45%
1.06% (P ＜ 0.05), AA account for total fatty acids ratio and increase to 3.26% (P ＜ 0.05), no EPA detections, ω -6/ by 2.69%
ω -3 is reduced to 15.91 (P ＜ 0.05) than by 29.63；
For adding 10% Mortierella alpina thalli feed additive, DHA accounts for total fatty acids ratio and is further increased to
1.27% (P ＜ 0.05), AA account for total fatty acids ratio and are 2 times (P ＜ 0.05) of control group, while have very small amount (0.07%)
EPA is detected, and ω -6/ ω -3 ratios are further reduced to 12.89 (P ＞ 0.05).
Above-mentioned as shown by data, 5% Mortierella alpina thalline additive of addition can significantly improve AA and DHA in free-ranging chicken egg
Content；After the addition of thalli feed additive increases to 10%, DHA content significantly increases compared with 5% additive group in egg
Add (P ＜ 0.05), while AA accounts for total fatty acids ratio and increased compared with 5% additive group in egg, but the two is poor without conspicuousness
Different (P ＞ 0.05).Above result of the test shows, after the addition certain proportion thalli feed additive, ω in thalline fat-
3PUFAs can deposit conversion in egg yolk and mainly exist in the form of DHA, and AA accounts for total fatty acids ratio can compared with control group
1 times of increase, total ω -3PUFAs account for total fatty acids ratio can increase by 2 times compared with control group, while maintain ω -6/ ω -3 in yolk
Than in the range of suitable absorption of human body.It is computed, after adding the 5% thalline additive in feed, AA contents account for shell egg
0.32%, DHA content accounts for shell egg 0.11%, AA the and DHA proportions closer in strengthening model.
More than experiment shows, Mortierella alpina thalline fermentation using more cheap glucose, dregs of beans as carbon and nitrogen sources,
Not adding allogenic material in fermentation process promotes thalline to produce fat, and fermentation time is short, thus can be obtained by high-volume fermentation
Thalline.Manufactured thalline additive is added in feed with certain proportion and carries out animal feeding trials, to laying hen production target,
Found after egg quality index and egg Analysis of Fatty Acids Composition, the thalline additive is not reducing laying hen production target and chicken
While Egg Quality index, the ratio of DHA and AA in egg fat can be significantly improved, while maintains ω -6/ ω -3 rational
In the range of.Therefore, the present invention has important production meaning, available for the aliphatic acid composition for improving egg.
Claims (8)
1. a kind of method of Mortierella alpina (Mortierella alpina) CCFM442 productions thalli feed additive, its
The step of being characterised by the production method is as follows：
A, inclined-plane culture
The ring of Mortierella alpina CCFM442 bacterial strains picking one of freezen protective is inoculated on PDA slant mediums, then in temperature
Incubated 14 days under conditions of 25 DEG C, the bacterial strain activation culture thing is obtained；
B, seed culture
The ring of activation culture thing picking one that step A is obtained is inoculated into seed culture medium, then in 25 DEG C of temperature and 200rpm
Under conditions of constant-temperature shaking culture 3 days, then claim to obtain wet thallus weight, the matter by wet thallus and sterilized water according to wet thallus and sterilized water
Measure ratio 1：4 are mixed, and scattered 10min are then carried out to the mixed system with dispersion machine to prepare bacteria suspension, according still further to every liter
The bacteria suspension is that 10mL inoculum concentrations are inoculated in the seed culture medium, permanent under conditions of 25 DEG C of temperature and rotating speed 200rpm
Warm shaken cultivation 36h, obtain Mortierella alpina CCFM442 seed liquors；
C, fermented and cultured
According to fermentation medium stereometer 1%-5% inoculum concentrations, the Mortierella alpina CCFM442 seed liquors that step B is obtained
It is inoculated into fermentation medium；Cultivate 2 days under conditions of 25 DEG C of temperature first, then cultivate 5 under conditions of 6 DEG C of temperature
My god, obtain the zymotic fluid containing Mortierella alpina CCFM442；
The fermentation medium preparation method is as follows：Prepare containing 50g/L glucose carbon sources, 20g/L dregs of beans, 13.34g/L nitric acid
Potassium, 3g/L dipotassium hydrogen phosphates, 1g/L sodium sulphate, the mixture water of 0.5g/L CALCIUM CHLORIDE DIHYDRATEs and 0.5g/L Magnesium dichloride hexahydrates
Solution, the pH of its aqueous solution is adjusted to 6.0, then the 20min that sterilized at 121 DEG C of temperature, obtain described fermentation medium；
D, post-process
The zymotic fluid containing Mortierella alpina CCFM442 for allowing step C to obtain is centrifuged, obtained wet thallus distillation
Water is washed, then is centrifuged, and washing wet thallus is then dried, and then obtains a kind of dry thalline, i.e. described thalline is raised
Feed additives；
Mortierella alpina (Mortierella alpina) CCFM442, the bacterial strain is on December 25th, 2014 in court of Beijing
China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of the positive institute 3 of area's North Star West Road 1 is commonly micro-
Bio-Centers preservation, its preserving number are CGMCC No.10295.
2. the method for production thalli feed additive according to claim 1, it is characterised in that in step C, the water
The pH of solution is adjusted using sulfuric acid and potassium hydroxide aqueous solution.
3. the method for production thalli feed additive according to claim 1, it is characterised in that in step D, described
Centrifugation is that 10min is centrifuged under conditions of rotating speed 6000rpm.
4. the method for production thalli feed additive according to claim 1, it is characterised in that in step D, described
Washing is that use distills water washing 2-4 times for 3-5 times with wet thallus stereometer.
5. the method for production thalli feed additive according to claim 1, it is characterised in that in step D, described
Drying is to dry 85-95min under the conditions of 48-52 DEG C of temperature and wind speed 2.1-2.5m/s are.
6. the method for production thalli feed additive according to claim 1, it is characterised in that in step D, described dry
The moisture content of dry thalline is to dry thalline gross weight meter 25.4-26.4%, total fatty acid content 7.6-10.7%, wherein more
Content of the unrighted acid in total fatty acids is 42.4-45.8%.
7. the method for production thalli feed additive according to claim 6, it is characterised in that in described how unsaturated
In aliphatic acid, the ratio of the serial polyunsaturated fatty acids of ω -6 and the serial polyunsaturated fatty acids of ω -3 is 4.5-5.3.
8. the method for the production thalli feed additive according to any one of claim 1-7 claim, its feature exist
In the dosage of the thalli feed additive be the 5-10% of basal feed gross weight.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510062057.9A CN104630077B (en) | 2015-02-05 | 2015-02-05 | A kind of Mortierella alpina CCFM442 bacterial strains and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510062057.9A CN104630077B (en) | 2015-02-05 | 2015-02-05 | A kind of Mortierella alpina CCFM442 bacterial strains and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104630077A CN104630077A (en) | 2015-05-20 |
| CN104630077B true CN104630077B (en) | 2017-12-12 |
Family
ID=53209329
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510062057.9A Active CN104630077B (en) | 2015-02-05 | 2015-02-05 | A kind of Mortierella alpina CCFM442 bacterial strains and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104630077B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104855689A (en) * | 2015-05-28 | 2015-08-26 | 河南双成生物科技有限公司 | Method for preparing fatty acid-rich protein feed raw material from amino acid fermentation residual liquor |
| CN106636297B (en) * | 2016-12-29 | 2020-05-08 | 江南大学 | Method for screening novel fatty acid synthesis inhibitor |
| CN106754436B (en) | 2017-01-13 | 2023-07-04 | 江南大学 | Preparation of Mortierella alpina CCFM698 thallus and application of Mortierella alpina CCFM698 thallus in feed additives |
| CN108651755A (en) * | 2018-05-08 | 2018-10-16 | 江南大学 | A kind of application of high yield EPA Mortierella alpinas in egg feedstuff |
| CN109527223A (en) * | 2018-12-29 | 2019-03-29 | 嘉必优生物技术（武汉）股份有限公司 | A kind of functional feed and preparation method thereof |
| CN112625912B (en) * | 2020-09-09 | 2023-06-06 | 中国科学院微生物研究所 | Mortierella alpina strain XY05201 and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102058018A (en) * | 2009-11-18 | 2011-05-18 | 中国科学院大连化学物理研究所 | Animal feed additive and application thereof |
| CN102776213B (en) * | 2012-08-28 | 2014-07-30 | 江南大学 | Desaturase omega 3 Des for biosynthesis of polyunsaturated fatty acids |
| CN102987096B (en) * | 2012-12-31 | 2014-12-10 | 厦门金达威集团股份有限公司 | Microbiological feed additive and preparation method and application thereof |
| CN103820335B (en) * | 2014-03-11 | 2017-02-08 | 江南大学 | Mortierella alpina, M. alpina genetic engineering strain of overexpression omega 3 desaturase gene and construction method of strain |
-
2015
- 2015-02-05 CN CN201510062057.9A patent/CN104630077B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104630077A (en) | 2015-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104630077B (en) | A kind of Mortierella alpina CCFM442 bacterial strains and application thereof | |
| CN101892160B (en) | Schizochytrium LX0809 (marine fungus) and industrial application thereof | |
| CN103917636A (en) | Algal lipid compositions and methods of preparing and utilizing the same | |
| CN103519035B (en) | Preparation method of chlorella fermented jelly | |
| US11447803B2 (en) | Anaplerotic oil production in microbials | |
| CN104872384B (en) | Needle mushroom foot feed addictive | |
| WO2021104165A1 (en) | Schizochytrium and application thereof, and sn-2 dha-rich microbial oil, preparation method therefor, and application thereof | |
| US20210051988A1 (en) | Human and non-human animal use of microbial anaplerotic oil | |
| CN105018544A (en) | Carrageenan oligosaccharide with trepang growth promotion and adverse resistance functions and preparation method and application thereof | |
| CN107586742A (en) | The bacillus amyloliquefaciens of one plant height production levulan and its application | |
| CN102911872B (en) | Scenedesmus sp. strain and application thereof | |
| CN106754436A (en) | The preparation of Mortierella alpina CCFM698 thalline and its application in feed addictive | |
| CN103468575B (en) | Crypthecodinium cohnii mutant strain with high yield of DHA (docosahexaenoic acid), as well as fermentation method and application thereof | |
| CN104004661B (en) | A kind of layer dietses for improving egg quality | |
| CN103815150A (en) | Lactic acid bacterium nutrient enhancer for holothurian culture | |
| CN103205377B (en) | Quality bacillus coagulans for breeding broilers and application of excellent bacillus coagulans | |
| CN109988674A (en) | A kind of fat or oil composition and preparation method thereof | |
| CN102399832A (en) | Forage grease producing method by solid state fermentation of maize starch and wheat bran by mortierella isabellina | |
| WO2019192182A1 (en) | Application of schizochytrium limacinum and preparation thereof in improvement of quality and yield of animal product | |
| US11758934B2 (en) | Human and non-human animal use of microbial anaplerotic oil | |
| CN105331670B (en) | Split the method for pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi | |
| CN104498557B (en) | The toxin producing medium of AFG1 a kind of and the AFG1 of aspergillus parasiticus produce malicious fermentation process | |
| CN104946691A (en) | Method for producing DHA-containing oil through fermentation by utilizing schizochytrium limacinum | |
| EP3897185A1 (en) | Human and non-human animal use of microbial anaplerotic oil | |
| CN117016671A (en) | Gamma-polyglutamic acid-rich fermented feed additive for laying hens in later egg laying period and laying hen later egg laying period feed prepared by same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |