CN105062905B - One plant of saccharomyces cerevisiae and its application for solid fermentation - Google Patents
One plant of saccharomyces cerevisiae and its application for solid fermentation Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
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- Fodder In General (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the saccharomyces cerevisiaes that one plant is used for solid fermentation, the Strain Designation is saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03, it was preserved in China typical culture collection center on 04 08th, 2015, deposit number is：CCTCC NO:M 2015209.Invention additionally discloses the solid fermentation cultural method of the saccharomyces cerevisiae and its applications of solid fermentation culture.The Wine brewing yeast strain has many advantages, such as that high-biomass, passage are stablized；In solid fermentation, the metabolites such as amino acid, organic acid yield is high compared with other yeast bacteria solid fermentation objects.Saccharomyces cerevisiae BLNJ03 solid fermentation cultures are applied to grass carp, are remarkably improved growth indexes, digestion index and immune indexes；It can increase the output of milk applied to milk cow；It is remarkably improved its production performance applied to beef cattle, mutton sheep.
Description
Technical field
The saccharomyces cerevisiae and its application that the present invention relates to one plant for solid fermentation.
Background technology
Yeast culture refers to the Tiny ecosystem product that saccharomycete is formed on specific culture medium after adequately fermenting,
It is mainly made of yeast cells extra-metabolite, the culture medium to make a variation after everfermentation and a small amount of yeast cells.Metabolism
Product is the general name to extracellular each metabolite, including peptide, organic acid, oligosaccharides, amino acid, flavour enhancing substance and aromatic substance
Deng further including other to substances such as " unknown growth factor " that promote growth of animal beneficial.The function of yeast culture can be summarized
For：Their metabolic activities and maintenance are stimulated by providing additional nutritional substrate to the microorganism lived in animal gastrointestinal tract
Interior ecological environment is stablized relatively.Previous scientific research and production application is it was verified that thin contained in yeast culture
Extracellular metabolism can significantly improve animal production performance, optimize feed nutritive value, improve animal health status.Ferment
Female culture can improve the intestinal flora of animal as feed addictive, inhibit the breeding of pathogenic bacteria, improve the immune of animal
Power enhances the resistivity to disease, while can improve the blood parameters of animal, promotes growth of animal.Therefore, yeast
Culture has very positive meaning for improving breeding performonce fo animals and reducing aquaculture cost.
But the problems such as that there are quality is irregular for current yeast culture product, and using effect is also unstable, there is an urgent need for open
Send out the brand product of super quality and competitive price.By reinforcing the further investigation to the yeast culture mechanism of action, make full use of abundant
Saccharomycete resource, exploitation obtains the new strains that biomass is high, metabolite is abundant, and further passes through optimization for fermentation technology, packet
Liquid and solid fermentation process are included, the ingredient of fermentation medium, the expression of high purpose product, for research and development saccharomyces neoformans training are studied
Produce product are supported to have a very important significance.
Invention content
In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide the wine brewing that one plant is used for solid fermentation
Yeast and its application.
To achieve the above object, the present invention uses following technical proposals：
One plant of saccharomyces cerevisiae for being used for solid fermentation, the Strain Designation are saccharomyces cerevisiae (Saccharomyces
Cerevisiae) BLNJ03, on 04 08th, the 2015 Chinese Typical Representative culture for being preserved in Wuhan University of Wuhan, China city
Collection (CCTCC), deposit number are：CCTCC NO:M 2015209.
The mycology feature of saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03 of the present invention is as follows：The wine
Brewer yeast cell is rounded or oval, and size is (3.5 μm~4.5 μm) × (7.0 μm~13.0 μm), in a manner of polygon budding
Vegetative propagation is carried out, pseudohypha is formed；Bacterium colony is creamy white, the smooth moistening in surface, glossy or matt, neat in edge or bacterium
It is Filamentous；Chmosynthetic heterotrophs, energy glucose fermentation, fructose and mannose, azymic galactolipin, maltose, lactose, melibiose；Energy
Assimilate urea, ammonium salt and nitrate, not decomposing protein and fat；Amphimicrobian under aerobic conditions, carries out aerobic respiration；Nothing
Under the conditions of oxygen, alcoholic fermentation is carried out；30 DEG C of optimum growth temperature, the most suitable growth pH are 7.0.
The present invention also provides the trainings of the solid fermentation of saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03
The method of supporting, step are：The liquid seeds of saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03 are pressed into 4%-6%
The inoculum concentration of (volume fraction) is inoculated in solid fermentation culture medium, and fermentation temperature is 28-30 DEG C, and the aerobic fermentation time is 36-
60h, anaerobic fermentation time are 72-96h.Improvement is optimized to zymotechnique in the present invention, using two step binary states method --- liquid
Body submerged fermentation is combined with solid fermentation；Aerobic fermentation carries out in succession with anaerobic fermentation, makes metabolite in its solid culture
Content is more abundant.
The solid fermentation culture medium is by matrix and water by 1g:(0.5-0.7) mL is formed；Wherein, the group of matrix becomes：
By mass percentage, wheat bran 90%, maize flour 3%, dregs of beans 7%.The present invention is to solid fermentation culture medium mesostroma and water
Ratio (i.e. material-water ratio) is optimized, the results showed that, excessively high and too low material-water ratio is unfavorable for the growth of saccharomycete, this hair
It is bright to control material-water ratio 1:0.5—1:In 0.7 range, viable count is higher in the culture cultivated in the range.
The preparation method of the liquid seeds of saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03, specifically
Steps are as follows：
(1) the saccharomycete fluid nutrient medium of 1/4-1/5 volumes is packed into triangular flask, sterilizing is connect after cooling with oese
Kind two ring solid slope saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03,30 DEG C of shaking table 200rpm cultivate 20h,
Obtain first order seed saccharomyces cerevisiae bacterium solution；
(2) the saccharomycete fluid nutrient medium of 2/5-3/5 volumes is added into seeding tank, the temperature that disappears in fact is 121 DEG C, and the time is
30min；When culture medium temperature is reduced to 30 DEG C, first order seed saccharomyces cerevisiae bacterium solution, culture temperature are inoculated with by 1.5% (volume fraction)
28-30 DEG C of degree, tank press 0.05MPa, speed of agitator 70-90rpm, cultivate 14-20h；
In step (1) and step (2), the group of the saccharomycete fluid nutrient medium becomes：Glucose 2%, peptone 1%,
Yeast extract 0.5%, dipotassium hydrogen phosphate 0.2%, bubble enemy 0.05%, the percentage composition are mass percentage, and pure water is matched
System, pH value 7.0.
In step (1), the condition of sterilizing is：121 DEG C of sterilizing 30min.
The present invention still further provides the fermentation training of saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03
Support application of the object in aquatic livestock and/or ruminant cultivation.
The present invention also provides a kind of feed addictives, with saccharomyces cerevisiae (Saccharomyces cerevisiae)
The fermentation culture medium of BLNJ03 is active ingredient.
Specifically, the feed addictive is the fermentation in saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03
The high activity Candida utilis dry ferment that 4 ‰ (mass fractions) are added in culture mixes, saccharomycete in mixture after compounding
Viable count is not less than 5 × 108cfu/g。
Beneficial effects of the present invention：
The present invention has filtered out one plant of saccharomyces cerevisiae BLNJ03 for solid fermentation, which has high-biomass, passes
The advantages that in generation, stablizes；In solid fermentation, the metabolites such as amino acid, organic acid yield is compared with other yeast bacteria solid fermentation objects
It is high.Saccharomyces cerevisiae BLNJ03 solid fermentation cultures are applied to grass carp, be remarkably improved growth indexes, digestion index and are immunized
Index；It can increase the output of milk applied to milk cow；It is remarkably improved its production performance applied to beef cattle, mutton sheep.
Description of the drawings
Fig. 1：BLNJ03 saccharomyces cerevisiae chromosome XII sequence electrophoretograms；
Fig. 2：The growth curve that saccharomyces cerevisiae BLNJ03 is cultivated in fermentation tank；
Fig. 3：Influences of the initial pH of culture medium to solid fermentation viable count.
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 separation screening of saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03
1 materials and methods
1.1 material
1.1.1 sampling position and sample type
Respectively from acquiring pedotheque under grape trellis from the two of Pingdu Daze Mount endroit.
1.1.2 key instrument equipment
1.1.3 culture medium
1.1.3.1 isolation medium
Potato dextrose agar (PDA culture medium)：Sucrose 20g, potato 200g, streptomysin 20mg, agar
Powder 15g, 1000mL, pH7.0,121 DEG C of sterilizing 20min of water.
1.1.3.2 pure medium
Yeast extract powder peptone dextrose solid medium (YPD culture mediums)：Glucose 4g, peptone 4g, yeast extract 2g, fine jade
Cosmetics 15g, 100mL, pH4.5,115 DEG C of sterilizing 20min of distilled water.
1.1.3.3 liquid fermentation medium
Glucose 20g, peptone 10g, yeast extract 2g, potassium dihydrogen phosphate 1g, water 1000mL, 7.0,121 DEG C of sterilizings of pH value
20min。
1.2 test method
1.2.1 the separation and purifying of saccharomycete
The separation of saccharomycete is carried out using enrichment culture method.Sample is opened in superclean bench, with the spoon of sterilizing
Sample l0g or so is obtained, is added in the triangular flask with bead that one fills 40mL sterile waters, 30 DEG C of shaking 30min will
Sample fully breaks up, mixing.Then the liquid for drawing 1mL is added in the PDA liquid medium containing streptomysin, 30 DEG C of shaking table trainings
It supports 1 day, the culture solution for drawing 1mL is inoculated into a new PDA liquid medium containing streptomysin, 30 DEG C of shaking table cultures 1-2 days.
Culture solution is dipped with transfer needle, and 4 rides are carried out on the solid PDA medium containing streptomysin.Culture is inverted in 30 DEG C of incubators
2-3 days, the different saccharomycete bacterium colony of picking form carried out scribing line purifying on YPD culture mediums, and purified bacterial strain is accessed
Inclined-plane is spare.
1.2.2 the measurement of yeast bacteria biomass
The saccharomycete being separated to is respectively connected in liquid fermentation medium, 30 DEG C of shaking table culture 20h take zymotic fluid
3500g centrifuges 10min, collects thalline, and distillation water washing 3 times collects thalline, weighed after freeze-drying up to (g/100mL).
1.2.3 bacterial strain screening
15 plants of single strains that bacterium colony growth is rapid, gram stain microscopy meets saccharomycete feature are picked out, are given birth to after measured
Object amount experiment after, filter out 3 plants of biomass the higher person, PDA slant mediums pass 5 generations after (observe its stability), and with it is first
Beginning bacterial strain ferments in triangular flask respectively, and secondary screening is carried out by index of biomass, filters out the bacterium that 1 plant of biomass is high and stablizes
Strain.
1.2.4 bacterial strain is identified
Identification method：Strain idenfication uses the PCR identification methods of chromosome XII sequence, and the specific method is as follows：
The primer sequence of saccharomycete chromosome XII sequence gene fragment amplifications is：CXS1：5'-
CACAGAAATCTCTCACCG-3 ' (as shown in SEQ ID NO.1 in sequence table), CXS2：5'-TTATGATATGCTTAAGTTC-
3 ' (as shown in SEQ ID NO.2 in sequence table), sequence is synthesized by Shanghai biotechnology Services Co., Ltd.PCR reacts
System is：4 μ L, 5 × Prime STAR Buffer of dNTP Mixture 10 μ L, each 0.6 μ L of upstream and downstream primer, genome mould
0.8 μ L, Prime STAR HS DNA Polymerase of plate, 0.6 μ L, 50 μ L are supplemented to aseptic deionized water.PCR cycle is joined
Number is：95℃5min；94 DEG C of 1min, 52 DEG C of 15s, 72 DEG C of 2min, 30 cycles；72℃10min.By BLNJ03 bacterial strains
Chromosome XII sequence gene target fragments gel extractions (TAKARA plastic recovery kits), send TAKARA to be sequenced,
Sequencing result logs in Genbank blast and compares online.
2 test results
2.1 bacterial strain screening
The selection result of bacterial strain is shown in Table 1.
1 high-biomass yeast strain the selection result of table
By table 1 as it can be seen that it is 2,3, No. 10 bacterial strains that biomass is higher in this 15 plants of bacterial strains, be respectively designated as BLNJ02,
BLNJ03、BLNJ10。
2.2 stability test
By above-mentioned experiment sieving to three plants of bacterial strains carry out liquid fermentation culture, and it is foster to be commissioned to train through continuous 5, measures its biology
Amount, as a result proves that this three plants of bacterium stabilities are preferable, this shows that 3 saccharomycetes screened all have good inheritance stability
Property, and the biomass highest of BLNJ03 bacterial strains, still select BLNJ03 bacterial strains to carry out molecular biology identification, and as follow-up
Test strain.The result is shown in tables 2.
2 yeast strain genetic stability test result of table
2.3 molecular biology identification
The molecular Biological Detection of S. cervisiae：BLNJ03 zymotic fluid 1mL are taken, total DNA, PCR amplification are extracted
Chromosome XII sequence gene orders, PCR product electrophoresis result is as shown in Figure 1, the results show that big in molecular weight
It is small to obtain a good band of specificity for 1000bp or so, it is consistent with expected results.
By the chromosome XII sequence gene target fragments gel extraction of BLNJ03 bacterial strains, (TAKARA glue returns
Receive kit), send TAKARA to be sequenced, sequencing results are logged in Genbank blast to be compared online, the results showed that BLNJ03
With number be CP006468.1, CP006465.1, CP006464.1 bacterial strain homology it is 99%.
Chromosome XII sequence gene sequencing results are as shown in SEQ ID NO.3 in sequence table.
Be saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03 through screening obtained Strain Designation, in
It is preserved in China typical culture collection center on 04 08th, 2015, deposit number is：CCTCC NO:M 2015209.
The mycology feature of saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03 is as follows：
The brewing yeast cell is rounded or oval, and size is (3.5 μm~4.5 μm) × (7.0 μm~13.0 μm),
Vegetative propagation is carried out in a manner of polygon budding, forms pseudohypha；Bacterium colony is creamy white, the smooth moistening in surface, glossy or unglazed
Pool, neat in edge or mycelioid；Chmosynthetic heterotrophs, can glucose fermentation, fructose and mannose, azymic galactolipin, maltose,
Lactose, melibiose；Urea, ammonium salt and nitrate can be assimilated, not decomposing protein and fat；Amphimicrobian, under aerobic conditions, into
Row aerobic respiration；Under oxygen free condition, alcoholic fermentation is carried out；30 DEG C of optimum growth temperature, the most suitable growth pH are 7.0.
Embodiment 2：The optimization of saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03 fermentation conditions
The liquid deep layer fermenting of 1 saccharomycete
1.1 instrument and equipment：
Electric heating constant temperature shaking table, 50 liters of seeding tanks, 500 liters of fermentation tanks, constant temperature electric heating incubator etc..
1.2 test material：
Culture medium：Glucose 2%, peptone 1%, yeast extract 0.5%, dipotassium hydrogen phosphate 0.2%, bubble enemy 0.05%, pH
Value 7.0.(percentage composition is mass fraction)
1.3 test method
1.3.1 prepared by first order seed bacterium solution
Using 5000mL triangular flasks, loading amount 1000mL/5000mL triangular flasks, 121 DEG C of sterilizing 30min are inoculated with after cooling and make
Brewer yeast BLNJ03 is first order seed bacterium solution in 30 DEG C of shaking table 200rpm cultures 20h.
1.3.2 seeding tank ferments
50L seed tank culture base loading amounts are 30L；Disappear 121 DEG C of temperature in fact, time 30min；Wait for that culture medium temperature is reduced to 30 DEG C
When be inoculated with saccharomyces cerevisiae BLNJ03 first order seed bacterium solutions, 28-30 DEG C of cultivation temperature, tank press 0.05MPa, speed of agitator 80rpm, training
16h is supported, obtains liquid seeds, you can be transferred to fermentation tank culture.
1.3.3 500 liters of ferment tanks
500 liters of fermentation tank culture medium loading amounts are 350L；Disappear 121 DEG C of temperature in fact, time 30min；Wait for that culture medium temperature is reduced to
It is inoculated at 30 DEG C, 28-30 DEG C of cultivation temperature, tank presses 0.05MPa.
Sampling in two hours is primary during fermentation, and microscopy simultaneously counts yeast viable count, and 20h puts tank, is used for down as liquid seeds
The solid fermentation of one step.
The growth curve that saccharomyces cerevisiae BLNJ03 is cultivated in fermentation tank is as shown in Figure 2.
2 saccharomyces cerevisiae solid fermentation production process are studied
Influence of the 2.1 liquid seeds inoculum concentrations to solid fermentation viable count
Culture medium：Wheat bran 90%, maize flour 3%, dregs of beans 7% (being mass fraction), material-water ratio 1g:0.7mL, pH value
7.0, inoculum concentration is respectively 2%, 4%, 6%, 8%, 10% (volume fraction), and 30 DEG C of culture 48h dry, measure and live at room temperature
Bacterium number and water content.It the results are shown in Table 3.
Influence of 3 inoculum concentration of table to solid fermentation viable count
Test result shows that within the scope of experimental design, influence of the inoculum concentration to viable count is little, considering cost etc.
Factor, it is proposed that inoculum concentration is advisable with 4%~6%.
Influences of the 2.2 initial pH of fermentation to solid fermentation viable count
The initial pH of yeast bacteria solid fermentation culture medium is adjusted to 4.5,5.0,5.5,6.0,6.5,7.0,121 by experiment respectively
DEG C sterilizing 30min, 30 DEG C of culture 48h, count viable count, influence results of the initial pH of culture medium to solid fermentation be shown in Fig. 3 after inoculation.
Test result confirms that initial pH value of medium is best with 7.0.
Influence of 2.3 culture medium prescriptions to solid fermentation viable count
2.3.1 influence of the soybean meal content to yeast culture crude protein content
Dregs of beans exists in the solid fermentation of yeast as important nitrogen source, in addition, the height of its content is trained with yeast
The crude protein content for supporting object is closely related.The content of dregs of beans in culture medium is set as 5% by experiment respectively, 7%, 15%, 20%,
25%, 30%, 35% (being mass fraction), other raw material types of culture medium and ratio are identical, 30 DEG C of aerobic fermentation 48h, survey
Determine yeast viable count and the crude protein content in culture.Test result is shown in Table 4.
Influence of 4 soybean meal content of table to viable count and crude protein content
As shown in Table 4, the crude protein content of yeast culture rises with the raising of soybean meal content, and still, viable count exists
Soybean meal content is little up to variation before 20%, and when culture medium soybean meal content meets and exceeds 25%, viable count declines rapidly.Analysis
The reason for this is that being improved with soybean meal content, broth viscosity increases, and ventilatory capacity declines, and causes Yeast Growth slow.Consider
The factors such as real cost of production select within 7% as best dregs of beans additive amount.
2.3.2 orthogonal test screens complex solid fermentation medium
It is carried out using Three factors-levels orthogonal test.500mL triangular flask loading amount 20g, material-water ratio 1:0.5, pH value 7.0,
Inoculum concentration 6%, it is parallel to do two.Orthogonal test designs table is shown in Table 5, and orthogonal experiments are shown in Table 6.
5 Orthogonal Experiment and Design of table
6 orthogonal experiments of table
Experiment results proved, medium component influence factor maize flour > wheat bran > dregs of beans, optimum formula A2B2C3, i.e.,
Wheat bran 90%, maize flour 3%, dregs of beans 7%.
2.3.3 influence of the material-water ratio to solid fermentation viable count
Fermentation medium material-water ratio is adjusted to 1 respectively:0.3,1:0.4,1:0.5,1:0.6,1:0.7,1:0.8,1:0.9,
1:1(g:ML), 500mL triangular flasks loading amount 20g, inoculum concentration are 6% (v/m), and 30 DEG C of culture 48h are dried, counted, measured moisture content.
It the results are shown in Table 7.
Influence of 7 material-water ratio of table to the viable count that ferments
Test result shows that excessively high and too low material-water ratio is unfavorable for the growth of saccharomycete, and material-water ratio is 1:0.5—1:
Viable count is higher in 0.7 range, it is proposed that material-water ratio is set to 1 in production:0.7.
Influence of the 2.4 aerobic fermentation periods to viable count
Saccharomyces cerevisiae seed liquor is inoculated in solid fermentation culture medium, aerobe fermentation of acting charitably is piled into, 12h starts after inoculation
Sampling samples 1 time per 12h, counts yeast viable count.It the results are shown in Table 8.
Influence of the 8 aerobic fermentation period of table to viable count
As shown in Table 8, extending at any time, yeast viable count gradually increases in culture medium, reaches highest to 48h viable counts,
It is 3.9 × 108cfu/g.Later, extend at any time, yeast viable count is in the trend that maintains an equal level in culture medium, it is thus determined that best aerobic
Fermentation time is 48h.
Influence of the 2.5 anaerobic fermentation periods to nutritional ingredient and digestibility
Saccharomyces cerevisiae BLNJ03 seed liquors are inoculated in yeast solids fermentation medium, are compacted after aerobic fermentation 48h close
Envelope carries out anaerobic fermentation, and anaerobic fermentation starts to sample for 24 hours, per sampling 1 time for 24 hours, measures amino acid, nucleotide in yeast culture
Content and digestibility index.It the results are shown in Table 9.
Influence of the 9 anaerobic fermentation period of table to amino acid, nucleotide content and digestibility
As shown in Table 9, with anaerobic fermentation time lengthening, amino acid and nucleotide content increase in yeast culture, digestion
Rate improves, and high value 11.33%, 28.31mg/g and 89.05% are reached to 96h, considers production cost and fermentation time
Continue to extend the factors such as mould easy to pollute, determines that the best anaerobic fermentation time is 96h.
To sum up, the optimal conditions of saccharomyces cerevisiae BLNJ03 solid fermentation process are：Solid fermentation culture medium be wheat bran 90%,
Maize flour 3%, dregs of beans 7%, initial pH value 7.0, material-water ratio 1:0.7, inoculum concentration 4%~6%, anaerobism is sent out after aerobic fermentation 48h
Ferment 96h.
The nutritional ingredient of 3 yeast cultures detects
By saccharomyces cerevisiae BLNJ03, candida utili (Candida utilis) CUM (candida utili CUM in
It is preserved on 04 19th, 2010 China typical culture collection center, deposit number is CCTCC M 2010090；It is documented in
Another patent " one plant of saccharomycete and its application with the tolerance of stronger Organic Selenium, enrichment and conversion capability " (CN of applicant
101864369 B) in), press same medium formula and zymotechnique respectively and carry out solid fermentation, after yeast is trained respectively
Object low temperature drying is supported, and is crushed.Buy the similar saccharomyces cerevisiae solid culture products measure viable count in market, nutrient composition content
And digestibility.It the results are shown in Table 10.
10 yeast solids culture viable count of table, nutrient composition content and digestibility compare
As shown in Table 10, saccharomyces cerevisiae BLNJ03 solid cultures and commercially available Cultures of S. cerevisiae, candida utili
Culture is compared, and the basic application component such as crude fibre, crude fat is related to culture medium prescription, is not much different；Protein content omits
There is raising, this phenomenon is related with mycoprotein；The content of amino acid is than commercially available Cultures of S. cerevisiae, candida utili culture
Object is high by 30% or so, organic acid content be commercially available Cultures of S. cerevisiae, 1.5-2 times of candida utili culture, explanation
Saccharomyces cerevisiae BLNJ03 strains are more excellent, have advantage in terms of metabolite in solid fermentation；Meanwhile fermentation of the invention
Technique is more advanced, and two step binary states method --- liquid deep layer fermenting is combined with solid fermentation；Aerobic fermentation is successive with anaerobic fermentation
It carries out, keeps metabolite content in its solid culture more abundant.
Embodiment 3：Influence of the yeast culture to Growth of Grass Carps Ctenopharyngodon Idellus, digestion and immune performance
1 materials and methods
Fish is used in 1.1 experiments：43 ± 5g grass carps, 200 tail；
1.2 experiment groupings：
Four groups are divided into, every group of experiment 50 tail of fish.Control group feeds basal diet；Test I groups, BLNJ03 Yeast Cultivations
Object compounds the candida utili dry powder of 4 ‰ (mass fractions), compounds mixture saccharomycete viable count up to 5 × 108Cfu/g is pressed
3 ‰ ratio of mass fraction is added to basal diet；II groups are tested, BLNJ03 Yeast Cultivations are added in the basal diet of feeding
Object, adding proportion are 3 ‰ (mass fractions)；Competing product group adds the competing product yeast bought in the market in the basal diet of feeding
Culture, adding proportion are 3 ‰ (mass fractions).
1.3 experimental animals are raised
1.3.1 pre-feeding period
It is fed by the 3% of fish body weight, early, middle and late right place is fed, observation feeding situation and residual bait rate, according to reality
Situation carries out appropriate adjustment and determines feeding volume.
1.3.2 just raising the phase
Feeding management is carried out according to the feeding volume that pre-feeding period obtains, 10 weeks by a definite date.Sampling is primary every 2 weeks.
1.4 sample collections measure
After feeding experiment, stop eating for 24 hours.Measure weight, the net weight of every group of grass carp.Every group is randomly selected 9 tail grass carp sterns
Fin venous blood collection, blood stand 1h and wait for its solidification at room temperature first, set after 4 DEG C of refrigerators keep 4h, under the conditions of 4 DEG C with
4000r/min centrifuges 10min, collects upper serum.Grass carp after blood sampling is weighed in the enteron aisle of operation acquisition on ice, liver, middle kidney, and
Buffer solution is added and takes supernatant after being ground on ice.Calculate feed coefficient, rate of body weight gain, specific growth rate.Measure enteron aisle egg
White enzyme and amylase index.
Calculation formula：
Rate of body weight gain (%)=100 × (the last first counterpoise of counterpoise -)/first counterpoise
Specific growth rate (%)=100 × (the first counterpoises of the ends Ln counterpoise-Ln)/raising number of days
Feed coefficient=every group feeds feed total amount/every group of fish body total augment weight amount
Digest index：
Protease activity determination method：Forint phenol method.
Amylase activity assay method：Iodine-starch colorimetric method.
Histone builds up total protein detection reagent box using Nanjing and measures.
Immune indexes：
Lysozyme activity assay method：Micrococcus lysodeikticus method.
2 results
2.1 growth indexes
2.1.1 feed coefficient
Influence of the yeast culture to feed of grass carp coefficient is as described in Table 11.
Influence of 11 yeast culture of table to feed of grass carp coefficient
As seen from the above table, positive to raise 10 weeks, control group weight gain 708.2g, compound the experiment of candida utili culture
I group weight gain 806g, compared with control group weight 97.8g；BLNJ03 yeast cultures group (experiment II groups) weight gain 799g is more right
According to a group weight 90.8g；Competing product group weight gain 753g, compared with control group weightening 44.8g.Control group, experiment I groups, experiment II groups, competing product
Four groups of feed coefficients of group are respectively 1.93,1.69,1.70,1.81.It is found that the yeast culture for adding the present invention in feed is apparent
Feed coefficient is reduced, food utilization efficiency is improved, improves Growth of Grass Carps Ctenopharyngodon Idellus speed.
2.1.2 rate of body weight gain
Influence of the yeast culture to grass carp rate of body weight gain is as shown in table 12.
Influence of 12 yeast culture of table to grass carp rate of body weight gain
As shown in Table 12, positive to raise 10 weeks, the rate of body weight gain for compounding candida utili group and BLNJ03 yeast culture groups is in
Grow steadily trend, is significantly improved compared with control group and competing product group.
2.1.3 specific growth rate
Influence of the yeast culture to grass carp specific growth rate is as shown in table 13.
Influence of 13 yeast culture of table to grass carp specific growth rate
As shown in Table 13, positive to raise 10 weeks, compound the particular growth of candida utili group and BLNJ03 yeast culture groups
Rate is in rising trend.Due to the randomness of sampling, control group compounds Candida utilis ferment with competing product group in trend, general trend is replaced
Female group and BLNJ03 yeast culture group is better than other two groups.The yeast culture of the present invention is improving grass carp growth rate and specific
Equal significant effect in growth rate, it is with obvious effects to the growth promoting function of grass carp.
2.2 digestion index determinings
Protease digestion vitality test (folin's methods), as a result as described in Table 14.
Influence of 14 yeast culture of table to grass carp intestinal protease activity
As shown in Table 14,10 weeks sampling determinations just are being raised the results show that preceding, Midgut protein enzymatic activity, compounds Candida utilis ferment
Female culture group, BLNJ03 yeast cultures group and competing product group are all remarkably higher than control group (P < 0.05), and compound Candida utilis
Yeast culture group, BLNJ03 yeast culture groups are significantly higher than competing product group (P < 0.05).Yeast culture can promote gastrointestinal tract
Function improves intestinal protein enzymatic activity, promotes to digest and assimilate, and improves nutritive digestibility in feed, and the yeast of the present invention
Culture effect is better than competing product.
2.3 immune indexes measure
2.3.1 influence of the yeast culture to grass carp lysozyme activity
Micrococcus lysodeikticus method measures experiment Blood of Ctenopharyngodon lysozyme activity, the results are shown in Table 15.
Influence of 15 yeast culture of table to grass carp lysozyme activity
As shown in Table 15, the 8th week lysozyme activity, compounding candida utili culture group, BLNJ03 yeast cultures
Group is significantly higher than control group (P < 0.05), and competing product group is higher than control group, but difference is not notable；10th week lysozyme activity, compounding
Candida utili culture group, BLNJ03 yeast cultures group and competing product group are all remarkably higher than control group (P < 0.05).Thus
Infer, blood lysozyme activity can be improved in yeast and its culture, inhibits the flourish of harmful bacteria, and compound Candida utilis ferment
Female culture group, the effect of BLNJ03 yeast culture groups become apparent.
2.3.2 influence of the yeast culture to grass carp immune organ
Index and spleen index, renal index measurement result are shown in Table 16.
Influence of 16 yeast culture of table to grass carp immune organ
As shown in Table 16,10 weeks compounding candida utili culture groups are fed and BLNJ03 yeast culture group spleens refer to
Number, renal index are significantly higher than control group and competing product group (P < 0.05).It can be seen that the yeast culture of the present invention can promote
Development of Immune Organs improves the effect of index of immunity is to reach strengthen immunity.
2.4 saccharomyces cerevisiae BLNJ03 cultures verify the protectiveness of grass carp --- and-infected by Aeromonas hydrophila is tested
It is positive to raise 10 weeks after the test, take the control group of feeding basal diet to be added on the basis of feeding basal diet
The 15 tail grass carp of test group of 3 ‰ BLNJ03 yeast cultures carries out attacking poison with 3 kinds of concentration Aeromonas hydrophila intraperitoneal injections respectively
Experiment.It the results are shown in Table 17.
17 acute challenge test result of table
As shown in Table 17, Aeromonas hydrophila is 1 × 109Under cfu/mL concentration, control group is all dead in the 1st day,
The 1st day dead 3 tail of BLNJ03 yeast cultures group, the 2nd day dead 1 tail；Aeromonas hydrophila is 1 × 108Under cfu/mL concentration,
The 2nd day dead 2 tail of control group, the 3rd day dead 1 tail, the 3rd day dead 1 tail of BLNJ03 yeast cultures group, the 4th day dead 1 tail；
Aeromonas hydrophila is 1 × 107Under cfu/mL concentration, the 4th day dead 1 tail of control group, BLNJ03 yeast culture groups are without death.
This explanation, under the conditions of acute infection, saccharomyces cerevisiae BLNJ03 cultures can delay to fall ill, hence it is evident that improve the anti-disease energy of grass carp
Power.
3 conclusions
The yeast culture of the present invention can significantly improve grass carp Protease Activities In Digestive Tube；Improve grass carp specific growth rate and
Rate of body weight gain improves efficiency of feed utilization, reduces feed coefficient；Improve blood lysozyme activity, Immune Organs Index and premunition.
Embodiment 4：Application of the yeast culture on ruminant
Influence of 1 yeast culture to milk cow production performance
1.1 materials and methods
1.1.1 experimental animal
The holstein cow of 40 lactation mid-terms, it is desirable that age, daily yielding, lactation number of days, parity are close.
1.1.2 experiment grouping and feeding management
40 lactation mid-term milk cows are randomly divided into 4 groups, every group 10.Be set to control group, experiment I group, experiment II group,
Competing product group.Control group feeds basal diet；I groups are tested, BLNJ03 Yeast Cultivations are added on the basis of feeding basal diet
Object, adding proportion are 5 ‰ (mass fractions)；II groups are tested, BLNJ03 yeast cultures compound 4 ‰ (mass fraction) Candida utilis
Yeast dry powder compounds in mixture saccharomycete viable count up to 5 × 108Cfu/g, the ratio for being 5 ‰ in mass fraction are added to base
Plinth daily ration；Competing product group, adds the competing product yeast culture bought in the market, and adding proportion is 5 ‰.
Experiment daily ration is voluntarily prepared by pasture, and using TMR feeding technics, concentrated feed is had by Shandong Bora Li Laibafu feeds
Limit company provides, and coarse fodder is hay and ensiling, and essence is slightly than about 40：60, experiment schedules to last 30d.
1.1.3 testing index and method
The output of milk：Daily morning and every group of output of milk per cow head is recorded at dusk, calculate test group and right during experiment
According to the average output of milk per cow head daily of group.
Milk-quality measures：Sample detecting each group milk quality when off-test, including butterfat percnetage, protein ratio, milk sugar rate,
The detection of the indexs such as non-fat solid content.Instrument is Unihub analysis of milk composition instrument.
1.2 results and analysis
As shown in Table 18, I group (BLNJ03 yeast cultures group) and II group of experiment (compounding candida utili group) are tested
The output of milk be significantly higher than competing product group and control group (P < 0.05)；From the point of view of milk composition index, BLNJ03 yeast cultures group and
Also significantly (P < 0.05) is higher than control group and competing product group to butterfat, lactoprotein, the lactose of compounding candida utili group, illustrates this
The yeast culture of invention has effects that significantly improve milk production of cow, and the effect of compounding candida utili group is slightly better than
BLNJ03 yeast culture groups；Meanwhile BLNJ03 yeast cultures and compounding candida utili all have improvement butterfat, newborn egg
In vain, the effect of lactose, the effect with competing product group is in significant difference (P < 0.05).
Influence of 18 yeast culture of table to milk cow production performance and milk composition
Note：Identical lowercase person, which is indicated, with column data shoulder indicates that difference between the two is not notable (P > 0.05), it is different small
Female person that writes indicates significant difference (P < 0.05) between the two, similarly hereinafter.
1.3 conclusion
The yeast culture of the present invention has preferable using effect in terms of improving milk production of cow and improving milk-quality.
Influence of 2 yeast cultures to beef cattle production performance
2.1. materials and methods
2.1.1 experimental animal
40 5 monthly age Simmental beef cattles, it is desirable that weight is close.
2.1.2 experiment grouping and feeding management
Beef cattle is randomly divided into 4 groups, every group 10, respectively control group, I group of experiment, experiment II group, competing product group.Control
Group：Feed basal diet；Test I groups：BLNJ03 yeast cultures are added on the basis of feeding basal diet, adding proportion is
5‰；Test II groups：BLNJ03 yeast cultures compound 4 ‰ (mass fraction) candida utili dry powder, compound ferment in mixture
Female bacterium viable count is up to 5 × 108Cfu/g is added to basal diet in 5 ‰ ratios；Competing product group：Add the competing product ferment bought in the market
Female culture, adding proportion are 5 ‰.
Experiment daily ration is voluntarily prepared by pasture, and using TMR feeding technics, concentrated feed is had by Shandong Bora Li Laibafu feeds
Limit company provides, and coarse fodder is hay and ensiling, and essence is slightly than about 40：60.Preliminary trial period 5d records the health status of ox, preliminary trial period
After start formal test, experiment schedules to last 55d
2.1.3 testing index and method
Production performance index：On-test and end early morning on the same day each test group and control group weigh a body on an empty stomach respectively
Weight, calculates total augment weight and the average daily gain of beef cattle.
Serum antioxidant indices：The 30th day of experimental period, 2h jugular vein blood collections 10mL, 3500r/min centrifugations before being raised in morning
15min takes serum keeping in -20 DEG C of refrigerators, measures Antioxidant Indexes malonaldehyde (MDA), reductive glutathione (GSH) contains
Amount, total antioxidant capacity (T-AOC), superoxide dismutase (SOD) vigor.
2.2 test results and analysis
2.2.1 to the influence of beef cattle production performance
Known by table 19, experiment I group (BLNJ03 yeast cultures group) and experiment II group (compound candida utili group) and
The total augment weight of competing product group and average daily gain are all remarkably higher than control group (P < 0.05), test I group of (BLNJ03 yeast culture
Group) and II group of experiment (compounding candida utili group) total augment weight and average daily gain be above competing product group, wherein testing I group
Compared with competing product group, total augment weight improves 10.31%, average daily gain and improves 11.11%, and II group of experiment is compared with competing product group, always
Weightening improves 9.03%, average daily gain and improves 10.10%, illustrates that the yeast culture of the present invention has and improves beef cattle growth
Effect.
Influence of 19 yeast culture of table to beef cattle production performance
2.2.2 to the influence of beef cattle Serum antioxidant indices
As shown in Table 20, BLNJ03 yeast cultures group, the Content of MDA of compounding candida utili group and competing product group
It is substantially less than control group (P < 0.05), the MDA of BLNJ03 yeast culture groups is also below competing product group, but difference not significantly (P
> 0.05)；Testing I groups, the SOD in serum for testing II groups, GSH, T-AOC, significantly (P < 0.05) is higher than competing product group and control group,
The SOD in serum of competing product group, GSH, T-AOC are also significantly greater than control group (P < 0.05).Illustrate that the yeast culture of the present invention has
The apparent effect for improving body activities of antioxidant enzymes, can effectively remove free radical, reduce active oxygen and damaged caused by body
Wound, to alleviate it is hot and cold etc. it is various stress, improve Abwehrkraft des Koepers.
Influence of 20 yeast culture of table to beef cattle anti-oxidation function
2.3 conclusion：
2.3.1 yeast culture of the invention has preferable using effect in terms of improving beef cattle growth.
2.3.2 yeast culture of the invention has preferably in terms of alleviating beef cattle and stress, enhance immunity of organisms and makes
Use effect.
Influence of 3 yeast cultures to mutton sheep production performance
3.1 materials and methods
3.1.1 experimental animal
The Small-fat-tail sheep of close (15 ± 3) kg of 200 weight.
3.1.2 the selection, grouping and feeding management of experimental animal
Small-fat-tail sheep is randomly divided into 4 groups, every group 50, respectively control group, I group of experiment, experiment II group, competing product group.
Control group：Feed basal diet；Test I groups：BLNJ03 yeast cultures are added on the basis of feeding basal diet, add ratio
Example is 5 ‰；Test II groups：BLNJ03 yeast cultures compound 4 ‰ (mass fraction) candida utili dry powder, compound mixture
Middle saccharomycete viable count is up to 5 × 108Cfu/g is added to basal diet in 5 ‰ ratios；Competing product group：Addition is bought competing in the market
Product yeast culture, adding proportion are 5 ‰.
Experiment daily ration is voluntarily prepared by pasture, and using TMR feeding technics, concentrated feed is had by Shandong Bora Li Laibafu feeds
Limit company provides, and coarse fodder is hay and ensiling, and essence is slightly than about 40：60, experiment sets preliminary trial period 7d, positive to try phase 30d.
3.1.3 testing index and method
The detection of production performance index：On-test and each test group early morning on the same day of end weigh a weight on an empty stomach,
Calculate total augment weight, the daily gain of mutton sheep.
The detection of Biochemical Indices In Serum：Blood sample is acquired on an empty stomach in off-test early morning the previous day, with vacuum anticoagulant blood-collecting pipe
15min is centrifuged through jugular vein blood collection about 10mL, 3500r/min, isolates blood plasma, -20 DEG C preserve, for plasma glucose, in vain
The measurement of albumen, urea nitrogen, total protein, instrument are Shanghai China of section automatic clinical chemistry analyzer.
3.2. test results and analysis
3.2.1 influence of the yeast culture to mutton sheep production performance
As shown in Table 21 BLNJ03 yeast cultures group and compounding candida utili group sheep total augment weight only, averagely increase day by day
It is above competing product group again, experiment I groups, the total augment weight of experiment II groups sheep, average daily gain are all remarkably higher than control group (P <
0.05).Illustrate that yeast culture has effects that improve mutton sheep weightening, and the effect of the yeast culture of the present invention is better than competing
Product.
Influence of 21 yeast culture of table to mutton sheep production performance
3.2.2 influence of the yeast culture to mutton sheep Biochemical Indices In Serum
As shown in Table 22, BLNJ03 yeast cultures group, the serum of compounding candida utili group and competing product group sheep only
Urea nitrogen is substantially less than control group (P < 0.05), and BLNJ03 yeast cultures group, compounding candida utili group are substantially less than competing
Product group (P < 0.05)；BLNJ03 yeast cultures group, seralbumin, IgG, the IgA for compounding candida utili group are notable
Higher than competing product group and control group (P < 0.05), competing product group is also significantly greater than control group (P < 0.05).Illustrate yeast culture energy
By increasing the quantity of rumen microorganism, and then increase the utilization to the ammonia that nitrogen substance degradation generates in daily ration, causes to reduce
Enter the ammonia content in blood through urea cycle, and synthesis and the antibody level of mutton sheep protein can be improved.
Influence of 22 yeast culture of table to mutton sheep physiochemical indice
3.3 conclusion：
3.3.1 yeast culture of the present invention has preferable using effect in terms of improving mutton sheep weightening.
3.3.2 yeast culture of the present invention can reduce the content of serum urea nitrogen, while improving seralbumin and resisting
Body content, and function and effect in this regard are better than competing product.
Claims (6)
1. one plant of saccharomyces cerevisiae for being used for solid fermentation, which is characterized in that the Strain Designation is saccharomyces cerevisiae
(Saccharomyces cerevisiae) BLNJ03 has been deposited on 04 08th, 2015 big positioned at Wuhan, China city Wuhan
China typical culture collection center, deposit number are：CCTCC NO：M 2015209.
2. the solid fermentation cultural method of saccharomyces cerevisiae described in claim 1, which is characterized in that step is：By saccharomyces cerevisiae
The liquid seeds of (Saccharomyces cerevisiae) BLNJ03 are inoculated in solid fermentation culture medium by the inoculum concentration of 4-6%
In, fermentation temperature is 28-30 DEG C, and the aerobic fermentation time is 36-60h, and the anaerobic fermentation time is 72-96h；
The solid fermentation culture medium is by matrix and water by 1g:(0.5-0.7) mL is formed；Wherein, the group of matrix becomes：By matter
Measure percentages, wheat bran 90%, maize flour 3%, dregs of beans 7%；The saccharomyces cerevisiae (Saccharomyces cerevisiae)
The preparation method of the liquid seeds of BLNJ03, steps are as follows：
(1) to sterilizing, saccharomycete liquid inoculation of medium saccharomyces cerevisiae (Saccharomyces after cooling
Cerevisiae) BLNJ03, shaking table culture obtain first order seed saccharomyces cerevisiae bacterium solution；
(2) first order seed saccharomyces cerevisiae bacterium solution is inoculated in the saccharomycete fluid nutrient medium in seeding tank, cultivation temperature 28-30
DEG C, mixing speed 70-90rpm, 14-20h is to get saccharomyces cerevisiae (Saccharomyces cerevisiae) BLNJ03 for culture
Liquid seeds；
In step (1) and step (2), the group of the saccharomycete fluid nutrient medium becomes：Glucose 2%, peptone 1%, yeast
Cream 0.5%, dipotassium hydrogen phosphate 0.2%, bubble enemy 0.05%, the percentage composition are mass percentage, pH value 7.0.
3. the fermentation by saccharomyces cerevisiae culture that the cultural method described in claim 2 is prepared.
4. application of the fermentation by saccharomyces cerevisiae culture in aquatic livestock and/or ruminant cultivation described in claim 3.
5. a kind of feed addictive, which is characterized in that with the fermentation by saccharomyces cerevisiae culture described in claim 3 be effectively at
Point, which formed by the fermentation by saccharomyces cerevisiae culture compounding candida utili dry powder described in claim 3,
Saccharomycete viable count is greater than or equal to 5 × 10 in mixture after compounding8cfu/g；
Candida utili was preserved in China typical culture collection center, deposit number CCTCC on 04 19th, 2010
M 2010090。
6. feed addictive as claimed in claim 5, which is characterized in that the feed addictive is described in claim 3
The candida utili dry powder of fermentation by saccharomyces cerevisiae culture compounding 4 ‰ forms.
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| CN111676145B (en) * | 2020-06-09 | 2021-05-28 | 青岛玛斯特生物技术有限公司 | Saccharomyces cerevisiae and application thereof in aquaculture |
| CN114350534B (en) * | 2022-01-17 | 2023-12-29 | 沈阳丰美生物技术有限公司 | Saccharomyces cerevisiae, fattening sheep biological feed and preparation method and application thereof |
| CN114680229A (en) * | 2022-03-09 | 2022-07-01 | 湖北丰甜生物科技有限公司 | Fermentation process of fishing yeast culture |
| CN115927021B (en) * | 2022-07-06 | 2024-06-21 | 中粮集团有限公司 | Saccharomyces cerevisiae, fermentation inoculant and application thereof |
| CN116850267B (en) * | 2023-07-13 | 2024-03-15 | 山东宝来利来生物工程股份有限公司 | Yeast cell wall-antibacterial peptide A3 composite preparation and preparation method and application thereof |
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