CN105331546B - A kind of selenium-rich Candida glabrata strain FXY-4 and its cultural method and the application as fish feed additive - Google Patents
A kind of selenium-rich Candida glabrata strain FXY-4 and its cultural method and the application as fish feed additive Download PDFInfo
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Abstract
The present invention relates to a kind of selenium-rich Candida glabrata strain FXY 4 and its cultural method and as the application of fish feed additive, belong to microorganisms technical field.The present invention is to be applied on YEPD culture mediums to cultivate after diluting pickle juice sample, and picking goes out single bacterium colony, then with containing Na2SeO3The YEPD culture mediums of (final concentration of 0.5g/L) carry out yeast rich in selenium screening, then are obtained by mutagenesis method, and Classification And Nomenclature is：Candida glabrata (Candida glabrata) FXY 4, has been preserved in China typical culture collection center (CCTCC), deposit number is：CCTCC NO：M 2015664.Candida glabrata (Candida glabrata) FXY 4 of the present invention, which has, significantly increases immune, somatotrophic function, and it is verified to aquatic livestock growth and health with facilitation by animal experiment, it can be directly applied in fish meal as additive.
Description
Technical field
The invention belongs to microorganisms technical fields, specifically, the present invention relates to a kind of selenium-rich Candida glabrata strains
FXY-4 and its cultural method and application as fish feed additive.
Background technology
Selenium (Se) is micro- necessary to animal body, is played an important role in terms of safeguarding animal health, but excessive
Selenium can generate toxic action to body, therefore selenium most suitable addition quantifier elimination in animal feed has obtained extensive development.Fish
The Nutritional studies of selenium show that Se content too high or too low in feed can influence healthy fish in class feed.Aquatic products is intensive
Change in breeding process, some pathogenic bacteria, especially enteron aisle conditionity pathogenic bacteria can breed rapidly, and then cause corresponding disease.
Currently, mainly stress to control and weaken by adding the method for antibiotic in feed.It can inhibit after antibiotic supply
The growth and breeding of fish pathogenic entero becteria, the growth for being conducive to beneficial bacterium, to maintain the microflora group structure of enteron aisle.So
And antibiotic is a large amount of using there is serious drawbacks, such as antibiotic to cause autogenous infection and two double infections in feed
Residual etc. in dye, the generation of antibody-resistant bacterium, immunity degradation and aquatic products and environment.Probiotics have it is safe and pollution-free, have
The advantages that immune enhancing function, becomes the first choice of Substitutes For Antibiotic, and yeast is even more most popular one kind in probiotics
Probiotics.
It gives full play to Se-enriched yeast enhancing and is immunized and promotes the activity of growth, screening obtains the strong yeast of selenium rich ability, right
Culture fishery has important practical significance.This patent screening has enhancing immune and the selenium rich ability of antistress function is strong
Candida glabrata strain；Meanwhile after adding the bacterial strain in feed, additive amount of the antibiotic in feed can be reduced, to
Effectively reduce Feed Manufacturing cost.Currently, there has been no reports for application of the high-yield selenium-rich Candida glabrata in aquaculture feed
Road.
Invention content
For the deficiencies in the prior art, of the invention first is designed to provide a kind of smooth false silk ferment of selenium-rich
Mother obtains 1 plant height production Se-enriched yeast FXY-4 then by mutagenesis method.By identification, which not only has light
The physio-biochemical characteristics of sliding Candida (Candida glabrata), and it is provided simultaneously with selenium-enriched ability and prebiotic attribute.
Another object of the present invention is the provision of Candida glabrata described above (Candida glabrata) FXY-4's
Cultural method.
A further purpose of the present invention is to provide selenium-rich Candida glabrata described above (Candida glabrata)
Applications of the FXY-4 as fish feed additive.
In order to achieve the above object, the present invention takes following technical scheme：A kind of selenium-rich Candida glabrata FXY-4,
Acquisition pattern is as follows：
Will pickle juice sample dilution after be applied to YEPD (10g yeast powders, 20g peptones, 20g glucose, 1L water,
PH6.0 it) on the tablet of culture medium, is cultivated at 28 DEG C, observes bacterium colony growth conditions at regular intervals, and picking single bacterium colony is transferred
It is preserved on to the inclined-planes YEPD, then with containing Na2SeO3Yeast rich in selenium is carried out in the YEPD culture mediums of (final concentration of 0.5g/L)
Screening, then cultivates the saccharomycete that primary dcreening operation obtains with YEPD culture mediums, and every plant of bacterium is divided into blank group and experimental group carries out
Culture, is added Na in experimental group2SeO3(final concentration of 0.5g/L) is added without in blank group, after culture for 24 hours, is marked using country
Quasi- method (GB 5009.93-2010)-atomic fluorescence spectrophotometer method measures selenium element content, and it is strongest to obtain 1 plant of selenium rich ability
Yeast original strain determines that its Classification And Nomenclature is after being identified with Physiology and biochemistry after mutagenesis：Candida glabrata
(Candida glabrata) FXY-4, the bacterium have been preserved in China typical culture collection center (CCTCC), address：China
Wuhan Wuhan University, deposit number are：CCTCC NO：M 2015664, the deposit date is on November 6th, 2015.
Another object of the present invention is the provision of selenium-rich Candida glabrata described above (Candida glabrata)
The cultural method of FXY-4, described method includes following steps：
(1) Candida glabrata (Candida glabrata) FXY-4 is subjected to fermented and cultured, fermentation temperature in shaking flask
It it is 28~30 DEG C, pH value is 6.0~6.8, and rotating speed is 180~200r/min, and fermentation time is 24~32h, shake flask fermentation culture
Base is YEPD fluid nutrient mediums：Glucose 2%, peptone 2%, yeast powder 1%；
(2) pilot scale fermentation is carried out after shake flask fermentation in fermentation tank, shake flask fermentation seed liquor is taken to be inoculated into 10L fermentations
In tank, inoculum concentration is 6~8%, and the culture medium of 4~7L is filled in fermentation tank, and fermentation temperature is 28~30 DEG C, pH value is 6.3~
6.8, mixing speed 300r/min, ferment 28~32h, ventilation ratio 1:1, fermentation culture is placed in 4 DEG C after fermentation
It is spare, wherein fermentation medium is：Lactose 15g, peptone 20g, yeast powder 10g, potassium nitrate 2g, Na2SeO30.5g, go from
Sub- water 1000ml, pH 6.5~6.8.
A kind of application of selenium-rich Candida glabrata FXY-4 as fish feed additive, application process are：It will be of the invention
Selenium-rich Candida glabrata (Candida glabrata) FXY-4 zymotic fluids directly added in business fish meal, additive amount
It is 5 × 106~1010CFU/Kg fish meals.
The fish can be carp, Tilapia mossambica, side fish, blunt snout bream etc..
Compared with prior art, the present invention has the following advantages：
1, Candida glabrata (Candida glabrata) FXY-4 of the invention, which has, significantly increases immune, growth promotion
Function, and by animal experiment verify its to aquatic livestock growth and health have facilitation, to save aquaculture cost,
It increases economic efficiency, has a good application prospect；
2, Candida glabrata (Candida glabrata) FXY-4 of the invention can during growth and breeding selenium-rich,
To realize the supply of Organic Selenium in feed；
3, after Candida glabrata (Candida glabrata) FXY-4 of the invention is added in feed, can increase
Organic selenium content in aquatic products increases its added value, additive amount of the Organic Selenium in feed is saved, to effectively reduce feed
Production cost.
Specific implementation mode
Following embodiment is not used in and limits the scope of the invention for illustrating the present invention, if not otherwise specified, the present invention
Method therefor is routine techniques, and agents useful for same is purchased from biochemical shop.
Embodiment 1：
Candida glabrata FXY-4, acquisition pattern are as follows：
The separation of original strain：
It is applied on the tablet of YEPD culture mediums after pickle juice sample is diluted, cultivates at 28 DEG C, see at regular intervals
Bacterium colony growth conditions are examined, and picking single bacterium colony is transferred on the inclined-planes YEPD and preserves, and following yeast strain is screened by primary dcreening operation：
FXY-1、FXY-3、FXY-4、FXY-5、FXY-7、FXY-10、FXY-11、FXY-19、FXY-21、FXY-23、FXY-24、FXY-
25、FXY-38、FXY-42、FXY-43、FXY-44、FXY-46、FXY-47、FXY-49、FXY-50、FXY-53、FXY-55、FXY-
56。
Containing Na2SeO3The screening that yeast rich in selenium is carried out in the YEPD culture mediums of (final concentration of 0.5g/L), then will be first
It sieves obtained saccharomycete to be cultivated with YEPD culture mediums, every plant of bacterium is divided into blank group and experimental group is cultivated, in experimental group
Middle addition Na2SeO3(final concentration of 0.5g/L) is added without in blank group, after culture for 24 hours, using national standard method (GB
5009.93-2010) --- atomic fluorescence spectrophotometer method measures selenium element content, and it is original to obtain 1 plant of strongest yeast of selenium rich ability
Bacterial strain.Then by this inoculation to YEPD culture mediums, 180 turns/min of shaking flask, 30 DEG C of cultures arrive exponential phase, take bacterium solution
5mL is centrifuged and is washed twice for 7.0 phosphate buffer (PBS) with pH value, then it is about 10 to be diluted to concentration with PBS10A/mL turns
Enter in 10mL centrifuge tubes, 0.8% dithyl sulfate (DES) solution, 30 DEG C of mutagenic treatment 20min, according still further to 2.5 times are added
DES liquor capacities are added 25% hypo solution and terminate reaction, take 200 μ L to be coated on containing Na the bacterium solution after mutagenesis2SeO3
On the YEPD tablets of (final concentration of 0.5g/L), picking single bacterium colony after 30 DEG C of culture 48h obtains a collection of mutagenic strain, finally adopts
The Se content that every plant of mutagenesis saccharomycete is measured with atomic fluorescence spectrophotometer method obtains the strongest mutagenesis saccharomycete of 1 plant of selenium rich ability
Strain measures by 18S ITS RNA sequences and is named as selenium-rich Candida glabrata (Candida with after Physiology and biochemistry identification
Glabrata) FXY-4, the bacterium have been sent to China typical culture collection center on November 6th, 2015 and have carried out preservation, classification
Name：Candida glabrata (Candida glabrata) FXY-4；Deposit number：CCTCC NO：M 2015664；Address：In
Wuhan Wuhan University of state.
Embodiment 2
Yeast strain FXY-4 (deposit numbers：CCTCC NO：M 2015664) 18S ITS rRNA gene sequencings：
(1) extraction of chromosomal DNA (a small amount of)：
1. taking 1.5mL bacterium solutions in 1.5mL Eppendorf pipes, 12 000r/min centrifuge 5min；
2. abandoning supernatant, precipitation is resuspended in 900 μ L phosphate buffers (PBS), and 4 DEG C of 12 000r/min centrifuges 5min；
3. abandoning supernatant, 300TE and 200 μ L 10mg/mL lysozymes, pressure-vaccum mixing, 37 DEG C of incubation 1h, every 15min are added
Reverse mixing is primary；
4. to precipitation be added 600TENS lysates (200mmol/LNaCl, 100mmol/L Tril-HCl pH8.0,
2.0%SDS, 50mmol/L EDTA, 0.5%Triton X-100) and 10 μ L of 20mg/mL Proteinase Ks, reverse mixing, 55 DEG C
1h is incubated, it is primary to overturn mixing every 15min；
5.4 DEG C of 12 000r/min centrifuges 5min, takes supernatant；
6. isometric P ︰ C ︰ I (25 ︰, 24 ︰ 1) are added into supernatant to mix well, 4 DEG C of 12 000r/min centrifuges 10min；
7. repeating step 6；
8. supernatant is transferred in new EP pipes, the 3mol/L sodium acetates of 1/10 volume of addition, the absolute ethyl alcohol of 2 times of volumes,
Mixing, 60min is placed at -20 DEG C, and 4 DEG C of 12 000r/min centrifuges 10min；
9. abandoning supernatant, centrifuge tube is tipped upside down on blotting paper, is air-dried after blotting liquid, 30 μ L sterile waters and 10mg/ is added
ML RNase A0.5 μ L, -20 DEG C of preservations.
The PCR amplification of 1.2 18S ITS rRNA gene orders
(2) PCR amplification
Just to extract and detect qualified chromosomal DNA as template, the primer synthesized by Shanghai Ying Jun biotech firms is utilized
(sense primer ITS1,5'-TCCGTAGGTGAACCTGCGG-3'；Downstream primer ITS4,5'-TCCTCCGCTTATTGATATGC-
The PCR amplification of ITS rRNA gene orders 3') is carried out, PCR amplification system is shown in Table 1.
1 PCR amplification primer of table
| Reagent | Dosage (μ L) |
| Premix Taq enzymes | 25 |
| Sense primer (20 μm of ol/L) | 1 |
| Downstream primer (20 μm of ol/L) | 1 |
| Template DNA (200ng/ μ L) | 1 |
| Deionized water | 22 |
| Total volume | 50 |
PCR amplification condition：
(3) purifying of 18S ITS rRNA gene PCR products
(with reference to Omega Bio-tek companies PCR product purification kit specification)
It is recycled in Eppendorf pipes 1. the product of PCR is cut from corresponding position on agarose gel, adds people
Binding buffer (1g/mL), in 65 DEG C of water-bath 7min until being completely dissolved；
2. solution is transferred in adsorption column, 10000r/min centrifuges 1min, discards waste liquid in collecting pipe；
3. Binding buffer, 10 000r/min the centrifugation 1min of 300 μ L are added, waste liquid in collecting pipe is abandoned；
4. the SPW buffer of 700 μ L are added, 2~3min is placed at room temperature, and 10 000r/min centrifuge 1min, discard receipts
Waste liquid in collector.It is repeated once；
5.10 000r/min void columns centrifuge 2min, discard waste liquid in collecting pipe；
6. adsorption column is put into a clean Eppendorf pipe, the Elution buffer of 30-50 μ L are added,
12000r/min centrifuges 2min and collects product.
(4) sequencing of 18S ITS rRNA genes
18S ITS rRNA gene sequencings are completed by Shanghai Ying Jun companies, and sequencing result is shown in the sequence 1 in sequence table.
(5) 18S ITS rRNA gene order similarity analysis
18S ITS rRNA gene orders after sequencing are passed through into http://www.ncbi.nlm.nih.gov/BLAST/
Alignment programs in webpage carry out homology analysis.
Pass through http:Alignment programs in //www.ncbi.nlm.nih.gov/BLAST/ webpages carry out homology analysis,
Obtain before homology ranking ten strain such as the following table 2, it follows that the strain belongs to candida, we are named as
FXY-4。
The length of yeast FXY-4 18S ITS rRNA gene orders is 1140bp, and specific sequencing result is shown in sequence table
Sequence 1.
2 sequence analysis of table is analyzed
Embodiment 3
Yeast FXY4 (deposit numbers：CCTCC NO：M 2015664), 2 automatic identification and analysis of Bio M é rieux VITEK
Picking strain to be tested lawn is inoculated on YEPD tablets, and 30 DEG C of cultures are dipped to exponential phase with sterilized cotton swabs
Lawn on tablet is scraped after Vitek liquid in kit, then is dissolved in 1.8ml Vitek liquid, mixing is vibrated；Use turbidity
Instrument tune turbidity is to specified range (2MacF)；Bacteria suspension is added in Vitek identification plates with liquid-transfering gun again, bacterium solution does well to fill mirror
Determine hole；Then identification plate is taken out when 30 DEG C of 24~48h of culture, with 2 automatic identification and analysis instrument of Bio M é rieux VITEK to mirror
Fixed board carries out digital independent and analysis, the results are shown in Table 3, this Physiology and biochemistry result and Candida glabrata (Candida
Glabrata) characteristic physiological biochemical reaction is identical.
Table 3：Its physio-biochemical characteristics of yeast FXY-4 are as follows：
Note：Indicate aobvious feminine gender ,+indicate the aobvious positive.
Embodiment 4
Candida glabrata (Candida glabrata) FXY-4 (deposit numbers of the present invention：CCTCC NO：
M2015664 cultural method), described method includes following steps：
(1) by 2ml a concentration of 108~1010The viable bacteria of CFU/ml is inoculated in progress shake flask fermentation training in 100ml culture mediums
It supports, fermentation temperature is 30 DEG C, pH value 6.8, rotating speed 200r/min, and fermentation time is Medium of shaking flask fermentation YEPD for 24 hours
Fluid nutrient medium：Glucose 2%, peptone 2%, yeast powder 1%；
(2) after shake flask fermentation carry out fermentation tank pilot plant test, take 100ml shake flask fermentations seed liquor (seed liquor it is dense
Degree is 108CFU/ml it) is inoculated into 10L fermentation tanks, liquid amount 5L, fermentation temperature is 30 DEG C, pH value 6.8, and mixing speed is
300r/min ferments for 24 hours, ventilation ratio 1:1, wherein fermentation medium is：Lactose 15g, peptone 20g, yeast powder 10g, nitre
Sour potassium 2g, Na2SeO30.5g, deionized water 1000ml, pH 6.5~6.8, after fermentation by fermentation culture be placed in 4 DEG C it is standby
With.
Embodiment 5
One plant of Candida glabrata (Candida glabrata) FXY-4 selenium rich abilities of the present invention are analyzed, and include specifically
Following steps：
(1) in every bottle of YEPD fluid nutrient medium plus 10mL selenium standard liquid (5 ‰), blank group is not added with selenium, in each shaking flask
The strain FXY-4 after ring activation is added, whole shaking flasks cultivates 36h under 30 DEG C, 180r/min environment, the thalline that will have been grown
It is added in centrifuge tube, 5min is centrifuged at 5000r/min, supernatant is outwelled after centrifugation, adds deionized water washing thalline, shake
It swings and is centrifuged again after shaking up, repeatedly aforesaid operations 5 times, thalline is cleaned up altogether, and thalline of the washing after good is put into baking oven
Drying；
(2) the thalline 0.5g (being accurate to 0.1mg) after drying is weighed, is put into 100ml beakers, adds 10mL HNO3It impregnates
Overnight, 2mL HCIO are added4, while making blank control, it shakes up, digests to white cigarette and emitted to the greatest extent, such as in low-temperature heat on electric hot plate
Fruit digestion solution is black or sauce brown, then adds HNO3Continue that digestion disappears to black or sauce brown and solution is in faint yellow, remains
When remaining about 1mL solution, 5mL 6moI/L HCI are added, heat 5~10min of slightly boiling, it is cooling, it washes in 25mL volumetric flasks, uses
6mol/L HCI are diluted to scale.Selenium is measured using national standard method (GB5009.93-2010)-atomic fluorescence spectrophotometer method
Constituent content, it is 5.04mg/g to measure thalline FXY-4 Se contents.
Embodiment 6
A kind of applications of selenium-rich Candida glabrata (Candida glabrata) FXY-4 as fish feed additive,
Application process is as follows：
Experiment is close using 400 weight, and healthy Tilapia mossambica (about 2.00g/ items), fancy carp (about 1.80g/ items) are as reality
Animal is tested, 4 processing groups are randomly divided into, each handles 4 repetitions, it is each to repeat 25.Processing 1 be control group, commercial feed,
Without any medicated premix；Processing 2 is antibiotic group, and a certain amount of antibiotic is added in commercial feed；Processing 3 adds for low amounts
Add selenium-rich Candida glabrata FXY-4 groups, the selenium-rich Candida glabrata FXY-4 fed in commercial feed in the present invention (is raised
Final concentration 5 × 10 in material8CFU/Kg)；Processing 4 adds selenium-rich Candida glabrata FXY-4 groups for a large amount, is mended in commercial feed
To (final concentration 5 × 10 in feed selenium-rich Candida glabrata FXY-4 in the present invention10CFU/Kg), day feeding amount is according to weight
3%, daily feeding 2 times, feeding is after 30 days, measures Tilapia mossambica, fancy carp growth and immune indexes and adds as seen from the experiment
Add selenium-rich Candida glabrata that can significantly improve Tilapia mossambica, fancy carp growth and amynologic index.
Influences of table 4 selenium-rich Candida glabrata (Candida glabrata) FXY-4 to Growth Op Tilapia
| Project | Control group | Antibiotic group | Low amounts adds yeast | A large amount adds yeast |
| Initial average weight //g | 2.00±0.02 | 2.01±0.01 | 2.03±0.03 | 2.02±0.01 |
| End average weight //g eventually | 28.70±1.62 | 29.90±1.88 | 33.77±2.13 | 35.15±2.36 |
Influences of table 5 selenium-rich Candida glabrata (Candida glabrata) FXY-4 to Tilapia mossambica immune system
Influences of table 6 selenium-rich Candida glabrata (Candida glabrata) FXY-4 to fancy carp growth
| Project | Control group | Antibiotic group | Low amounts adds yeast | A large amount adds yeast |
| Initial average weight //g | 1.81±0.02 | 1.80±0.01 | 1.83±0.02 | 1.78±0.01 |
| End average weight //g eventually | 27.70±1.62 | 28.10±1.77 | 32.63±2.27 | 33.20±2.45 |
Influences of table 7 selenium-rich Candida glabrata (Candida glabrata) FXY-4 to fancy carp immune system
Embodiment 7
After feeding Se-enriched yeast FXY-4, the influence to Se content in the flesh of fish
Two kinds of fish (Tilapia mossambica, fancy carp) 2.0g (being accurate to 0.1mg) in embodiment 6 are weighed, is put into 100ml beakers, adds
10mL HNO3Soaked overnight adds 2mL HCIO4, while making blank control, shake up, in low-temperature heat on electric hot plate digest to
White cigarette emits to the greatest extent, if digestion solution is black or sauce brown, adds HNO3Continue digestion to black or sauce brown disappears and solution
In faint yellow, when residue about 1mL solution, 5mL 6moI/L HCI be added, heat 5~10min of slightly boiling, it is cooling, wash 25mL appearances
In measuring bottle, scale is diluted to 6mol/L HCI.National standard method (GB5009.93-2010)-atomic fluorescence spectrophotometer method
Selenium element content is measured, Se content in Tilapia mossambica, the fancy carp flesh of fish is measured and has increased separately 1.99mg/kg, 2.23mg/ compared with control group
Kg reaches the standard that selenium-enriched food Se content is more than 0.2mg/kg.
Claims (3)
1. a kind of selenium-rich Candida glabrata (Candida glabrata) FXY-4, which is characterized in that its deposit number is
CCTCC NO：M 2015664 .
2. a kind of selenium-rich Candida glabrata described in claim 1 (Candida glabrata) FXY-4 cultural method,
It is characterized in that：Described method includes following steps：
（1）By Candida glabrata (Candida glabrata) FXY-4 carries out fermented and cultured in shaking flask, fermentation temperature is
28~30 DEG C, pH value is 6.0~6.8, and rotating speed is 180~200 r/min, and fermentation time is 24~32 h, shake flask fermentation culture
Base is YEPD fluid nutrient mediums：Glucose 2%, peptone 2%, yeast powder 1%；
（2）Pilot scale fermentation is carried out after shake flask fermentation in fermentation tank, shake flask fermentation seed liquor is taken to be inoculated into 10L fermentation tanks
In, inoculum concentration is 6~8%, and the culture medium of 4~7L is filled in fermentation tank, and fermentation temperature is 28~30 DEG C, and pH values are 6.3~6.8,
Mixing speed is 300 r/min, and ferment 28~32h, ventilation ratio 1:1, after fermentation by fermentation culture be placed in 4 DEG C it is standby
It is with, wherein fermentation medium：15 g of lactose, 20 g of peptone, 10 g of yeast powder, potassium nitrate 2 g, Na2SeO3 0.5 g, it goes
Ionized water 1000 ml, pH 6.5~6.8.
3. a kind of selenium-rich Candida glabrata described in claim 1 (Candida glabrata) FXY-4 adds as fish meal
Add the application of agent, which is characterized in that by the selenium-rich Candida glabrata (Candida glabrata) FXY-4 zymotic fluid
In business fish meal, additive amount is 5 × 10 for directly addition6~1010 CFU/Kg fish meals.
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