CN105441344B - One plant of candida utili and its application with anti-oxidation function - Google Patents
One plant of candida utili and its application with anti-oxidation function Download PDFInfo
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Abstract
The invention discloses one plant of candida utili with anti-oxidation function, it is candida utili (Candida utilis) BLCR07, China typical culture collection center is preserved on November 23rd, 2015, deposit number is CCTCC NO:M 2015694.The invention also discloses application of the candida utili in preparing the antioxidant for removing interior free yl.The present invention candida utili can be used as a kind of novel, green safe antioxidant for alleviate including heat stress due to various extraneous factors caused by animal body stress reaction, ensure the health of animal body, harmful effect caused by considerably reducing heat stress makes the function of saccharomycete further be deeply developed.
Description
Technical field
The candida utili for having anti-oxidation function the present invention relates to one plant and its application belong to microbial technique neck
Domain.
Background technology
With the acceleration and reinforcement of the intensive process of animal productiong, people in order to make the production performance of animal maximize, with
This obtains more animal products, and the intervention and pressure apply to animal is increasing.Therefore, modern cultivation means and side
Method produces tremendous influence to animal, make animal be constantly in stress state, stress be in animal body by foreign objects
After reason or chemical stimulation, a series of physiological reaction can occur in vivo to resist extraneous stimulation, wherein most important be exactly
Excessive free radical can be formed in vivo.Under normal physiological conditions oxygen radical can in vivo spontaneously by enzymatic reaction or
Non-enzyme reaction and generate, due to needing O in certain physiological actions or biochemical reaction-And OH-The participation of free radical.If free radical
Generation with eliminate disequilibrium will cause to lead to pathological state to the damage of body.Excessive free radical will make body device
The film fat of official is damaged, and is most embodied directly in the rising of malonaldehyde in blood (MDA) content.Therefore, body will also be by certainly
Remain gaining everything and lose nothing by generation and the elimination of base, physiological is low-level, stable equilibrium number of free radical.In vivo freely
This balance of base is completed by some anti-oxidant defense systems.These systems of defense include the enzyme of some specificity to activity
The scavenging effect of oxygen, if catalase is to H2O2Decomposition, to O-Decomposition and glutathione peroxidase to H2O2And fat
Decomposition of class Peroxidation Product etc. and some nonspecific antioxidants, such as vitamin E, vitamin C, transferrins.
However, no one of these systems of defense antioxidant is most strong for reactivity, damage force is maximum
OH-Free radical.OH-Free radical is drawn by attacking biomembrane polyunsaturated fatty acid, DNA, protein and other biological macromolecular
The oxidational losses of cell and tissue is sent out, cell tissue cracking causes animal body lesion, productivity to decline even dead.Due to certainly
By the oxidation of base, by oxidative damage, the myoglobins in muscle is oxidized to time for cell membrane and Subcellular membrane in muscle
Myoglobins, while film is destroyed, intracellular fluid exudation, animal carcasses color becomes brown from red, reduces fresh meat product product
Matter influences mouthfeel.So stress can it is serious damage animal health, lead to the decline of production performance, reduce productivity effect,
Modern aquaculture is set to be in a kind of situation of vicious circle.
The content of candida utili also known as edible torula, protein and vitamin B is all higher than brewer's yeast, is training
Supporting in base can grow it is not necessary that any growth factor is added.Currently, focusing primarily upon its richness for the research of candida utili
Selenium is rich in active peptide and ethionine resistance etc., and there is not been reported for the anti-oxidation function of candida utili.Cause
This, screens the new candida utili bacterial strain with removing interior free yl function with highly important application value.
Invention content
For the above-mentioned prior art, the object of the present invention is to provide one plant of candida utili bacterium with anti-oxidation function
Strain and its application.
To achieve the above object, the present invention uses following technical proposals：
One plant of candida utili with anti-oxidation function is candida utili (Candida utilis)
BLCR07 is preserved on November 23rd, 2015 in the China typical culture collection of Wuhan University of Wuhan, China city
The heart, deposit number are CCTCC NO:M 2015694.
Applications of the above-mentioned candida utili BLCR07 in preparing the antioxidant for removing interior free yl is also this hair
The range of bright protection.
In above application, it is preferred that the free radical is OH-Free radical.
Have it is a further object of the present invention to provide one kind and removing OH-The antioxidant of free radical function, active constituent
For above-mentioned candida utili BLCR07 or its tunning.
Above-mentioned antioxidant, which removes to have, removes OH-Outside the function of free radical, moreover it is possible to the significant work for improving SOD and GSH-PX
Property.
It is above-mentioned that there is removing OH-In the antioxidant of free radical function, the fermentation of the candida utili BLCR07 is produced
Object is prepared as follows：The seed liquor of candida utili BLCR07 is seeded in the fermentation medium containing derivant,
Fermented and cultured obtains tunning.
The group of the fermentation medium containing derivant becomes：Yeast extract 5g, peptone 10g, glucose 20g,
KH2PO42g, distilled water 1000mL；A concentration of 2.0-3.0mmol/L of derivant；Preferably 2.5mmol/L.
The derivant is CuCl2、CuSO4Or ZnSO4；Preferably CuCl2。
The condition of fermented and cultured is：Fermentation temperature is 25-30 DEG C, fermentation time 20-28h.
The preparation method of the seed liquor of candida utili BLCR07 is：By candida utili (Candida utilis)
The slant strains of BLCR07 are seeded in fluid nutrient medium, and shaking table culture 18-24 hours is to get candida utili BLCR07 kinds
Sub- liquid.
The specific preparation method of the seed liquor of candida utili BLCR07 is：1/5 volume is packed into triangular flask
Fluid nutrient medium, sterilizing are inoculated with a ring solid slope candida utili (Candida utilis) after cooling with oese
BLCR07,30 DEG C of shaking table 200rpm are cultivated 20 hours, obtain candida utili BLCR07 seed liquors.
The group of the fluid nutrient medium becomes：Yeast extract 5g, peptone 10g, glucose 20g, KH2PO42g, distilled water
1000mL。
Above-mentioned antioxidant is being also the protection of the present invention as the application in feed addictive and/or food additives
Range.
Third object of the present invention is to provide a kind of feeds for alleviating animal stress reaction, which is by base
The above-mentioned antioxidant for adding 0.1%-0.2% in plinth feed by mass fraction is prepared.
Beneficial effects of the present invention：
(1) candida utili with anti-oxidation function of the invention can remove internal excessive free radical, can be with
Directly remove OH-Free radical can also significantly decrease the activity of MDA contents and raising SOD in blood in addition.
(2) candida utili with anti-oxidation function of the invention can be as a kind of novel, green safe
Antioxidant be used for alleviate including heat stress due to various extraneous factors caused by animal body stress reaction,
Ensure the health of animal body.Harmful effect caused by significantly reducing heat stress makes the function of saccharomycete obtain further deeply
Exploitation, it is often more important that anti-oxidant yeast can be widely used as feed addictive in aquaculture.
Description of the drawings
Fig. 1：Candida utili ultraviolet mutagenesis destruction curve；
Fig. 2：Influence of the fermentation time to biomass and free radical scavenging activity.
Specific implementation mode
The present invention is further illustrated in conjunction with the embodiments, it should which explanation, following the description is merely to explain this
Invention, is not defined its content.
Embodiment 1：The screening of bacterial strain
1 materials and methods
1.1 material
1.1.1 bacterial strain：Candida utili.
1.1.2 culture medium
Solid slope culture medium：Yeast extract 5g, peptone 10g, glucose 20g, KH2PO42g, agar 15g, distilled water
1000mL。
Liquid seed culture medium：Yeast extract 5g, peptone 10g, glucose 20g, KH2PO42g, distilled water 1000mL.
Mutagenesis culture medium：Yeast extract 5g, peptone 10g, glucose 20g, KH2PO42g, distilled water 1000mL.
Fermentation medium：Same liquid seed culture medium.
1.1.3 test reagent
Ultrasonic disruption buffer solution：0.01mol/L Tris-HCl, pH 8.6.
Determining free radicals reagent：1mol/L HCl, Tris, TEMED, ammonium persulfate, FeSO4, H2O2, crystal violet, α-naphthalene
Amine, p-aminobenzene sulfonic acid.
Derivant：CuCl2、CuSO4And ZnSO4。
1.1.4 testing equipment
Sonicator, water-bath, high-pressure sterilizing pot, superclean bench, supercentrifuge, UV spectrophotometers and perseverance
Warm shaking table etc..
1.2 detection method
1.2.1 anti-oxidant saccharomycete oxidation resistance --- the detection of free radical scavenging activity
Bacterium solution 5000r/min after the fermentation medium culture containing derivant is centrifuged into 10min, uses 0.01mol/
The precipitation (yeast cells) that LTris-HCl cleaning centrifugations obtain, and repeat 2~3 times, with the 0.01mol/ of precipitation two volumes
The yeast cells cleaned is resuspended in LTris-HCl.Broken wall, and 30 DEG C of constant temperature oscillations are carried out to yeast cells with sonicator
Be placed within 30 minutes 70 DEG C of water-baths 5 minutes, then 5000r/min centrifuges 10min, centrifugation twice, take supernatant detect its to from
By the clearance rate of base.
The assay method of free radical scavenging activity：It is separately added into 1.5mL crystal violets in a series of 50mL color-comparison tubes
(0.4mmol/L)、2.0mL FeSO4Solution (1mmol/L), 1.0mL H2O2Solution (2.0mmol/L) adjusts pH value to 4.0, fixed
Hold 50mL and shake up, stand 30min, surveys the absorbance A at 580nmb, while measuring and being not added with H2O2When 580nm at absorbance
A0.Then the yield of hydroxy radical can use △ A=A0-AbTo calculate.The measurement of apparent anti-hydroxy radical oxygenation efficiency：Above-mentioned
Add H in reaction system2O2A certain amount of anti-oxidant reagent is added before, measures its absorbance A, free radical scavenging activity S can be pressed
Formula calculates：S (%)=(A-Ab)/(A0-Ab) × 100%.
1.2.2 the detection of biomass
100mL zymotic fluid mixings, centrifugation removal supernatant is taken to be put into baking oven and dry 48h, weigh the weight after drying, unit：g/
100mL。
1.3 test method
Screening anti-oxidation function yeast is induced using ultraviolet mutagenesis zygotic induction agent.
1.3.1 primary dcreening operation
By candida utili strain in slant activation culture to logarithmic phase, a ring is chosen in equipped with 100mL sterile physiological salt
In the 250mL triangular flasks of water and appropriate bead, oscillation triangular flask is to break up cell, and using a times dilution process is passed, to adjust this bacterium outstanding
The concentration of liquid is about 107cfu/mL.It takes this bacteria suspension of 5mL to be added in the sterilized petri dishes of a diameter of 9cm, sets apart from ultraviolet lamp 20cm
Place is irradiated, and is sampled respectively in 10s, 20s, 30s, 40s, 50s, 60s, 90s, 120s, with l0 times of dilution method by irradiating
Bacteria suspension debita spissitudo (about 60 bacterium colonies can be grown on tablet) is diluted in sterile water, take 1mL be added tablet,
Enter culture medium, shake up, wrapped with black cloth after solidification, be placed in 30 DEG C of incubators and cultivate 36h, each period do three it is parallel.
Cultured tablet is taken out, bacterium colony counting is carried out.According to control tablet and the clump count on tablet is handled, calculates each period
Lethality and draw destruction curve.Ultraviolet light, which irradiates later operation, to carry out under red light, to avoid photoreactivation phenomenon
Occur.
Lethality calculation formula is as follows：
Lethality=[(0.1mL viable counts after control 0.1mL viable counts-processing)/control 0.1mL viable counts] × 100%.
Irradiation tablet of the lethality between 70%-80% is chosen, primary dcreening operation, picking are carried out by index of colonial morphology size
The more consistent single bacterium colony of fast, neat in edge, microscopy thalline is grown, is seeded in solid slope culture medium and cultivates.Experiment is chosen altogether
140 single strains, inclined-plane culture are taken for 24 hours, after passage 2 times, then to access fermentation medium CuCl2Induction, 30 DEG C of cultures
36h surveys its biomass and free radical scavenging activity.15 plants of qualified single strains are picked out, after measured biomass and free radical
After clearance rate, 5 plants of biomass and equal the higher person of free radical scavenging activity are filtered out.
1.3.2 secondary screening
The mutant strain for the Strong oxdiative ability that primary dcreening operation is filtered out accesses CuCl containing 2.5mmol/L2Fermentation medium
In, 30 DEG C of shaking table culture 36h, triangular flask liquid amount 100mL/500mL (v/v) they are to be trained on 200r/min shaking tables in 30 DEG C, rotating speed
36h is supported, measures biomass and free radical scavenging activity, while setting the contrast test of starting strain.
1.3.3 genetic stability is tested
The bacterial strain that secondary screening is obtained carries out 5 passages in the fermentation medium, measures its biomass and free radical scavenging activity.
The contrast test of the preceding bacterial strain of passage is set simultaneously.
2 results and discussion
2.1 primary dcreening operation results
Mutagenesis is carried out to the candida utili bacterial strain that sets out using ultraviolet irradiation method, with the increase of irradiation time, bacterial strain
Survival rate decline, show that starting strain is sensitive to ultraviolet irradiation, thus can be used the method carry out mutagenesis.It will be each flat after culture
Plate carries out bacterium colony counting, calculates bacterium colony lethality, and each time of ultraviolet irradiation count results are shown in Table 1 with lethality.
Count results and lethality after the irradiation of 1 ultraviolet light of table
By table 1 as it can be seen that the UV treatment time is within 10s, lethality is relatively low, illustrates at the ultraviolet light in the short time
Reason, it is little to the growth effect of the bacterial strain；And processing time, when increasing to 20s, lethality has apparent increase, reaches
67.30%, illustrate that the bacterium is more sensitive to ultraviolet light.Often increasing 10s later, lethality grows steadily, when processing time is 120s,
Lethality is 99.66%, therefore tentatively concludes that the bacterial strain is 120s to the longest tolerance time of ultraviolet light.
Using the time as abscissa, destruction curve, the result is shown in Figure 1 are drawn by ordinate of lethality.It can be seen from figure 1 that 10s it
Interior curve is shallower, and lethality variation is little；Hereafter slope of curve abruptly increase, display lethality this period are changed significantly；60s
Later curve tends towards stability again, illustrates this phase lethality variation also unobvious, close to maximum value.According to above-mentioned destruction curve, choosing
Appropriate lethality (70%~80%) the i.e. tablet of mutagenesis 30s~40s is selected, the qualified single bacterium colony of picking 140 carries out excellent
The selection and breeding of good strain.
It is screened by the shaking flask to 140 plants of bacterial strains, obtains biomass and higher 5 plants of the bacterial strain of free radical scavenging activity, point
Not Wei No. BLCR05, No. BLCR07, No. BLCR37, No. BLCR96 and BLCR121 bacterial strains, using this 5 plants of mutagenic strains as multiple
Object is sieved, picking single bacterium colony is saved in solid slope culture medium from tablet.
2.2 secondary screening results
5 plants of biomass and the higher bacterial strain of free radical scavenging activity that primary dcreening operation obtains are respectively connected to contain with starting strain
2.5mmol/L CuCl2Fermentation medium in, set 30 DEG C of shaking table culture 36h and carry out secondary screenings, measurement result is shown in Table 2, as a result shows
Show that the yeast strain biomass after primary dcreening operation and starting strain are almost the same, but free radical scavenging activity improves,
Middle BLCR07 bacterial strains free radical scavenging activity improves 78.27% than starting strain.
2 ultraviolet mutagenesis secondary screening result of table
2.3 genetic stability test results
BLCR07 bacterial strains are carried out to 5 passages in the fermentation medium added with derivant, measure its biomass and oneself
Illustrate it with good genetic stability, still without reducing (the results are shown in Table 3) compared with before passage by base clearance rate
BLCR07 bacterial strains are set to the yeast strain having compared with strong anti-oxidation ability that ultraviolet mutagenesis screens.
3 mutant strain inheritance stability Journal of Sex Research of table
Candida utili (Candida utilis) BLCR07 of above-mentioned acquisition was preserved on November 23rd, 2015
Positioned at the China typical culture collection center of Wuhan University of Wuhan, China city, deposit number is CCTCC NO:M
2015694。
Embodiment 2：The optimization of bacterial strain inducing and fermentation condition
1 materials and methods
1.1 material
Strain：Embodiment 1 screens obtained candida utili (Candida utilis) BLCR07.
Other reagents and culture medium are the same as embodiment 1.
1.2 method
1.2.1 the determination of derivant
Derivant selects CuCl2、CuSO4、ZnSO4, concentration is 2.5mmol/L；The liquid amount of culture medium is 250mL's
The bottled 50mL fermentation mediums of triangle；Inoculum concentration is 3%；Inoculation strain is to have anti-oxidation function through what ultraviolet mutagenesis obtained
Candida utili BLCR07.30 DEG C of shaking table culture 36h measure biomass and free radical scavenging activity.
1.2.2 the determination of inducer concentrations
Derivant selects CuCl2；Liquid amount is the bottled 50mL fermentation mediums of triangle of 250mL；Inoculum concentration 3%, induction
Agent concentration is adjusted to tetra- gradients of 2.0mmol/L, 2.5mmol/L, 3.0mmol/L and 3.5mmol/L, is inoculated with candida utili
BLCR07.30 DEG C of shaking table culture 36h measure biomass and free radical scavenging activity.
1.2.3 the determination of initial pH value of medium
Saccharomycete needs could be grown under the conditions of certain acid-base value, and pH value is excessively high or too low can all inhibit saccharomycete
Growth.Derivant selects CuCl2, a concentration of 2.5mmol/L；Liquid amount is the bottled 50mL fermentation mediums of triangle of 250mL；It connects
Kind amount is 3%；Medium's PH Value is adjusted to 4.0,4.5,5.0,5.5,6.0,6.5 and 7.0 respectively, is inoculated with candida utili
BLCR07.30 DEG C of shaking table culture 36h measure biomass and free radical scavenging activity.
1.2.4 the screening of liquid amount (ventilatory capacity)
Derivant selects CuCl2, a concentration of 2.5mmol/L；Inoculum concentration is 3%；PH value is natural；Liquid amount：The three of 500mL
The bottled liquid measure in angle is respectively tetra- gradients of 50mL, 100mL, 150mL and 200mL, inoculation candida utili BLCR07.30 DEG C are shaken
Bed culture 36h, measures biomass and free radical scavenging activity.
1.2.5 the determination of inoculum concentration
Derivant selects CuCl2, a concentration of 2.5mmol/L；PH value is natural；Liquid amount is the bottled 50mL of triangle of 500mL
Fermentation medium；It is inoculated with candida utili BLCR07, inoculum concentration is set to 1%, 2%, 3%, 4% and 5%.30 DEG C of shaking tables
36h is cultivated, biomass and free radical scavenging activity are measured.
1.2.6 the determination of fermentation time
Derivant selects CuCl2, a concentration of 2.5mmol/L carries out fermentation life in the 5L mechanical agitating fermentation tanks of laboratory
Production.30 DEG C of fermentation temperature, every 4 hours sampling is primary during experiment, measures biomass and free radical scavenging activity, draws biology
Amount and free radical scavenging activity curve, determine best fermentation time.
2 results and discussion
The screening of 2.1 derivants
The selection result of derivant is shown in Table 4.
4 derivant the selection result of table
As can be seen from Table 4, after adding three kinds of different derivants, the biomass after candida utili BLCR07 fermentations
It is relatively high with free radical scavenging activity, but difference is not notable, the highest CuCl of clearance rate is chosen in follow-up test2As induction
Agent.
The screening of 2.2 inducer concentrations
The selection result of inducer concentrations is shown in Table 5.
5 inducer concentrations the selection result of table
As can be seen from Table 5, with the increase of inducer concentrations, biomass is in downward trend, but in 0-2.5mmol/L
Decline unobvious in concentration range, and free radical scavenging activity is in downward trend after first rising, in CuCl2A concentration of 2.5mmol/
Clearance rate highest when L.So selecting anti-oxidant saccharomycete derivant CuCl2A concentration of 2.5mmol/L.
The screening of 2.3 initial pH value of medium
The selection result of initial pH value of medium is shown in Table 6.
The selection result of 6 initial pH value of medium of table
As can be seen from Table 6, with the raising of fermentation medium pH value, biomass is in downward trend, and free radical is clear
Except rate is in downward trend after first rising.So considering, to select the pH value of anti-oxidant microzyme culture medium be 5 or so, i.e.,
Culture medium natural ph.
The screening of 2.4 liquid amounts (ventilatory capacity)
The selection result of liquid amount (ventilatory capacity) is shown in Table 7.
The selection result of 7 liquid amount of table (ventilatory capacity)
As can be seen from Table 7, with the increase of liquid amount, biomass and free radical scavenging activity are all in downward trend,
Middle biomass declines apparent.So the liquid amount of zymotic fluid selects 50mL/500mL triangular flasks, ferment tank to be built when producing
View increases ventilatory capacity.
The screening of 2.5 inoculum concentrations
The selection result of inoculum concentration is shown in Table 8.
The selection result of 8 inoculum concentration of table
As can be seen from Table 8, with the increase of inoculum concentration, biomass and free radical scavenging activity are in first to rise to decline afterwards
Trend, biomass and free radical scavenging activity reach maximum when 3% inoculum concentration, so inoculum concentration selection 3% is advisable.
The screening of 2.6 fermentation times
The selection result of fermentation time is shown in Fig. 2.Figure it is seen that as time increases, free radical scavenging activity is 16
It is in rising trend before hour, reach maximum when by 16 hours, tends towards stability later.Biomass is in rising trend before 20 hours,
It is declined slightly later.In general, the ferment tank time is to be advisable for 20 hours.
3 brief summaries
The candida utili bacterial strain BLCR07 that ultraviolet mutagenesis screens, derivant select CuCl2, a concentration of
2.5mmol/L, fermentation optimum process condition are the triangular flask liquid amount 50mL that liquid amount is 500mL, inoculum concentration 3%, pH
Value naturally, fermentation temperature be 30 DEG C, fermentation time 20h.
Embodiment 3：Anti-oxidation function candida utili BLCR07 safety testings
1.1 experimental animal
Experimental animal is Kunming mouse, weight 18-20g, half male and half female, purchased from Tai'an Tai Bang biotech firms.
1.2 experimental design
Mouse is raised be grouped after a week in advance, and control group and three test groups are randomly divided into；Control group mice feeds basal diet,
Test group mouse diet is the candida utili bacterial strain BLCR07 bacterium that 0.5%, 2%, 5% is separately added into basal diet
Powder；Mouse is freely eaten during experiment, and feeding environment is identical.Test group is continuously fed 30 days.On-test, is distinguished at the end of
It weighs, calculates mouse weight variation；Mouse is cutd open when off-test and is killed, each internal organs weight is weighed.
1.3 test result
Mouse average daily gain result and internal organs it is averagely heavy result difference it is as shown in Table 9 and Table 10.
9 mouse average daily gain test result of table (g/d is only)
Note：Colleague's data shoulder is designated as different lowercase letter indication differences significantly (P<0.05), there is identical lowercase letter
Not notable (the P of difference>0.05), similarly hereinafter.
10 each test group internal organs of table are averagely heavy (g)
It can be seen that the daily gain of test group mouse is dramatically increased compared with control group from table 9, table 10, illustrate that anti-oxidant production protein is false
Silk yeast has certain growth promoting function.Dissection the case where finding no organ disease when off-test, three test groups with
Heart, liver, spleen, lungs, kidney between control group all do not differ significantly, it is seen that the production protein with anti-oxidation function
Candida is safe within the scope of experimental concentration.
Embodiment 4：Candida utili with anti-oxidation function alleviates the application test of heat stress of laying hens reaction
1 test site
Experiment is carried out in Shandong Baolai-leelai Bio-engineering Co., Ltd.'s breeding layer chicken Demonstration Base, test period peace
Come August 24 days to 2015 July in 2015 8, mean temperature is (32.0-33.0) DEG C, relative humidity 50%- in henhouse
60%.
2 experimental designs
Experiment is the blue grey laying hen in sea of peak period of laying eggs with chicken, is basic daily ration with chicken house egg feedstuff, before pre-feeding period, to each
The chicken of group is adjusted, and rejects the chicken for having apparent morbid state, makes the number of elements of each group unanimously and production performance is close.Choose 120
The laying hen for peak period of only laying eggs is randomly divided into 3 groups using cage mode, each group 40, wherein control group fed basic day
Grain, other each test groups add the candida utili BLCR07 bacterium powders of various concentration in basal diet.Pre-feeding period one week, examination
During testing, chicken group health status, each group egg number, egg size, soft broken egg number and feed consumption are observed and recorded daily.Every group of feeding
10 chickens of grab sample during supporting take a blood sample in feeding, take a blood sample 2 times altogether for the 9th day and the 16th day, venous blood collection under each every chicken wings
Sample 2-3mL makees anticoagulation and handles and save backup according to a conventional method after blood sampling.
3 testing indexs
The production performances index such as feedstuff-egg ratio, laying rate；SOD and MDA contents in Plasma of Laying Hens
4 assay methods
SOD assays use xanthine oxidase compound enzyme process, MDA assays to use thiobarbituricacidα- method (TBA), survey
Determine reagent and is all made of the kit that Bioengineering Research Institute's production is built up in Nanjing.
5 test results
A groups are control group, feed basal diet；B groups and C groups are test group, add 0.1% He respectively in basal diet
0.2% candida utili BLCR07 bacterium powders.
The influence of 5.1 pairs of production performances
Table 11 adds influence of the anti-oxidant yeast to performance in layers
As shown in Table 11, anti-oxidant saccharomycete (candida utili BLCR07) is added in daily ration significantly improves heat stress
The laying rate of laying hen, reduces feedstuff-egg ratio under state, and illustrating that anti-oxidant saccharomycete has improves heat stress performance in layers
Effect, and 0.2% addition group is better than 0.1% addition group.
5.2 influence laying hen antioxidant levels under heat stress state
12 anti-oxidation function saccharomycete of table is to SOD in Plasma of Laying Hens (μm ol/L) activity and MDA (mmol/mL) content
It influences
Heat stress can induce more in the interior generation excess LPO, main component MDA, MDA of cell membrane and oxidable cell membrane
Unsaturated fatty acid destroys the integrality of cell membrane, leads to cell membrane major injury, so influence the immune function of animal body with
Normal metabolic function.Therefore MDA is the important indicator for weighing body totality antioxidant levels.Test result shows in daily ration
The test group content of MDA in chicken blood of laying eggs declines and significant difference compared with control group after addition anti-oxidation function saccharomycete, SOD
Content significantly rises, and illustrates that adding anti-oxidation function saccharomycete can significantly reduce the MDA contents generated due to heat stress, alleviate
Destruction of the heat stress to animal cell membrane increases the content of internal SOD, improves the activity of the antioxidases such as SOD, and then protects it
Internal organs and internal bioactive substance such as enzyme, nucleic acid are not destroyed, and ensure normal physiological function.
It can be seen that：Anti-oxidant saccharomycete BLCR07 can be very good to alleviate and be brought not to laying hen due to heat stress
Profit influences, and ensures the health of the laying hen body under high temperature season, while improving the production performance of laying hen at high temperature, increases cultivation
Economic benefit.So this plant of anti-oxidant saccharomycete can be used for as a kind of novel, green safe antioxidant
Alleviate including heat stress including due to various extraneous factors caused by animal body stress reaction, ensure animal body be good for
Health.
Claims (9)
1. one plant of candida utili with anti-oxidation function is candida utili (Candida utilis)
BLCR07, is preserved in China typical culture collection center on November 23rd, 2015, and deposit number is CCTCC NO:M
2015694。
2. one kind, which has, removes OH-The antioxidant of free radical function, which is characterized in that its active constituent is candida utili
BLCR07 or its tunning；Candida utili (Candida utilis) BLCR07, on November 23rd, 2015
It is preserved in China typical culture collection center, deposit number is CCTCC NO:M 2015694.
3. antioxidant according to claim 2, which is characterized in that the tunning of the candida utili BLCR07
Preparation method be：The seed liquor of candida utili BLCR07 is seeded in the fermentation medium containing derivant, fermentation training
It supports, obtains tunning.
4. antioxidant according to claim 3, which is characterized in that the preparation of the seed liquor of candida utili BLCR07
Method is：The slant strains of candida utili BLCR07 are seeded in fluid nutrient medium, shaking table culture 18-24 hours, i.e.,
Obtain candida utili BLCR07 seed liquors.
5. antioxidant according to claim 3, which is characterized in that the composition of the fermentation medium containing derivant
For：Yeast extract 5g, peptone 10g, glucose 20g, KH2PO42g, distilled water 1000mL；A concentration of 2.0- of derivant
3.0mmol/L。
6. antioxidant according to claim 3, which is characterized in that the derivant is CuCl2、CuSO4Or ZnSO4。
7. antioxidant according to claim 3, which is characterized in that the condition of fermented and cultured is：Fermentation temperature is 25-30
DEG C, fermentation time 20-28h.
8. claim 2-7 any one of them antioxidant is as the application in feed addictive.
9. it is a kind of for alleviate heat stress of laying hens reaction feed, which is characterized in that the feed be by basal feed by
Claim 2-7 any one of them antioxidants of mass fraction addition 0.1%-0.2% are prepared.
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| CN103289907A (en) * | 2013-03-20 | 2013-09-11 | 广州格拉姆生物科技有限公司 | A candida utilis protein product and applications thereof in breeding industry |
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Non-Patent Citations (5)
| Title |
|---|
| Accumulation and Storage of Zn2+ by Candida utilis;M. L. FAILLA et al.;《Journal of General Microbiology》;19761231;第94卷;第23-36页 * |
| 假丝酵母菌筛选及金属硫蛋白制备工艺研究;李靖元;《中国优秀硕士学位论文全文数据库 基础科学辑》;20130815(第08期);摘要、第19页第3.1.2节、第21页第3.2.2节、第33页第5.1.5节、第36页讨论 * |
| 李靖元.假丝酵母菌筛选及金属硫蛋白制备工艺研究.《中国优秀硕士学位论文全文数据库 基础科学辑》.2013,(第08期), * |
| 益生菌诱导合成金属硫蛋白及其对自由基清除率和安全性的实验;朱丽梅等;《饲料广角》;20131231(第10期);摘要、第18页右栏第2段 * |
| 高产类金属硫蛋白假丝酵母菌株的筛选;李靖元等;《中国生物制品学杂志》;20131130;第26卷(第11期);第1585-1587,1592页 * |
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