CN104342372A - Method for producing yeast autolysate by probiotic fermentation - Google Patents
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Abstract
The invention provides a novel method for producing yeast autolysate by probiotic fermentation, which is particularly implemented by carrying out mixed fermentation on Bacillus subtilis K1 and yeast cells. The collection number of the Bacillus subtilis K1 is CCTCC NO:M2014396. After 48 hours of fermentation, the wall breaking ratio of the yeast cells in the fermentation tank filled with the Bacillus subtilis K1 is up to 95% above, thereby obtaining unexpected technical effects. Compared with the traditional technique, the method provided by the invention greatly shortens the technical process, reduces the production cost, and has obvious wall breaking effect and wide application prospects.
Description
Technical field
The present invention relates to technical field of microbe application, be specifically related to a kind of method utilizing probiotics fermention to produce yeast hydrolyate.
Technical background
Yeast, especially cereuisiae fermentum and bread yeast are the single celled eukaryotic microorganisms with Important Economic value.Yeast cell matter contains abundant nutritive substance, as protein, Yeast Nucleic Acid, vitamin B group and abundant amino acid etc., flavour nucleotide wherein by a large amount of for food seasoning and health product raw material, also by a large amount of phagostimulant for animal cultivation.β-1,3 dextran in yeast cells wall can strengthen mammiferous immune vigor, reduce cholesterol and blood fat, promotion wound healing etc., is a kind of good biological response modifiers.Because yeast cells wall is very thick, not through the Whole yeast cells of broken wall, above-mentioned substance cannot discharge and play a role how efficiently, at low cost by breaking yeast cellule membrane, therefore, be the matter of utmost importance that each primary yeast technique of deep processing byproduct will solve.
Yeast cell wall thickness is about 0.1-0.3 μm, and structure is tough and tensile, divides three layers from outside to inside, and main component has dextran, mannosans, protein, chitin, lipid etc.Wherein the content of dextran and mannosans accounts for about 30% of cell walls dry weight respectively.Conventional breaking yeast cellule membrane method has: acid and alkali hydrolysis broken wall method, chemical broken wall method, mechanical process, self-dissolving broken wall method, enzymically hydrolyse method etc.
1. acid and alkali hydrolysis method broken wall be in yeast thalline add acid or alkali after hydrolyzable method, conventional acid, alkali are hydrochloric acid, sulfuric acid, NaOH etc., this method is cheap, amino acid yield is high, but the product micro-nutrient composition loss after hydrolysis is large, taste bad will, color and luster is bad, environmental pollution is large, and in, environmental protection pressure more and more higher to the safety requirements of food today more and more, this method is eliminated gradually.
2. chemical broken wall method: best with toluene shell-broken effect, but toxicity is large, and the shell-broken effects such as chloroform, ethyl acetate, p-Hydroxybenzoate are general, and there is the residual problem of chemical toxic substances.
3. mechanical process: comprise high-pressure homogenization, pearl mill method, supersonic method etc., its broken wall efficiency can reach more than 90%, but requires high to appointed condition, and will operate at low temperatures, in case shear heating and make protein denaturation, affect enzymolysis efficiency, be only suitable for laboratory operation.
4. self-dissolving broken wall method: the method for cell walls is decomposed in the comprehensive action of the various enzymes (various protein, dextranase, amylase, cellulase etc.) utilizing yeast thalline to contain itself.Because playing a role of enzyme needs certain condition, and endobacillary enzyme majority is in proenzyme state, if not by activation of zymogen, then enzyme is also difficult to play a role.Need lower temperature and longer time by self-dissolving completely, about need 3-7 days, the easy microbiological contamination of production process, has a strong impact on generation quality.
5. enzymatic broken wall method: carry out cell wall breakdown by additional enzyme.Have data to show, when β-1,3 dextranase and papoid are combined, the cell walls of 93% is by enzymolysis.Wherein, β-1,3 dextranase mainly acts on the dextran of cell walls, causes cell rupture, and papoid is mainly used in the hydrolysis accelerating albumen.Enzymatic shell-broken must be noted that to add the kind of enzyme, ratio and order, the hydrolysis result that could realize.
In current actual production, most widely used in conjunction with enzymatic broken wall method with self-dissolving broken wall, but these class methods still exist many defects, as large in enzyme dosage, enzyme cost is high, complex technical process etc., greatly limit scale operation and the application of yeast byproduct.
Summary of the invention
The present invention, for solving prior art problem, provides a kind of method utilizing probiotics fermention to produce yeast hydrolyate.Described method be by subtilis (
bacillus subtilis) carry out mixed fermentation with yeast, realize the broken wall treatment to yeast cell during the fermentation, technological process is simple, and successful, effectively can reduce production cost.
Applicant screens the novel subtilis of a strain from the mud of brew-house's sewage draining exit, and this bacterial strain effectively can abolish yeast cells wall, thus facilitates the present invention.
One aspect of the present invention provides a kind of method of breaking yeast cellule membrane, by subtilis and yeast cell are carried out mixed fermentation to realize.
Described subtilis is subtilis K1(
bacillus subtilisk1), be preserved in the China typical culture collection center of Wuhan, China Wuhan University on September 3rd, 2014, deposit number is CCTCC NO:M2014396.
Described mixed fermentation process, be specially: first sterilised yeast suspension is moved in fermentor tank, then add sterilized sulfate solution, DAP solution, glucose solution respectively, make the total reducing sugars concentration in sterilised yeast suspension be 2.5%-5%, the final concentration of ammonia nitrogen is 0.5-1.5%; Add the subtilis K1 after activation by the volume ratio of 5-10% again, 50 DEG C of fermentation 36h-48h, namely obtain breaking yeast cellule membrane liquid.
Present invention also offers a kind of preparation method of yeast hydrolyate, is the yeast lysate that aforesaid method obtains is carried out concentrated or spraying dry to realize.
beneficial effect
The subtilis K1 that the present invention screens effectively can abolish yeast cells wall, by by subtilis K1 and yeast cell mixed fermentation, after 48h, the sporoderm-broken rate adding yeast cell in the fermentor tank of subtilis K1 reaches more than 95%, achieves unexpected technique effect.The present invention directly by subtilis K1 and yeast cell mixed fermentation, carries out broken wall treatment to yeast cells wall, compared with traditional technology, substantially reduces technological process, reduces production cost, and shell-broken effect is remarkable, has a extensive future.
Embodiment
embodiment 1 take yeast cells wall as the screening of the bacterial strain of nutritive substance
1, substratum
1) solid selective medium: yeast cells wall powder 1%(w/v), yeast extract paste 0.05%(w/v), ammonium di-hydrogen phosphate 0.1%(w/v) and, sulfate of ammoniac 0.1%(w/v), iron(ic) chloride 0.01%(w/v) and, magnesium sulfate 0.01%(w/v), agar 1%(w/v).
Take said components in proportion, dissolve with tap water, adjust ph 6-7,121 DEG C of sterilizing 20min, make solid culture plate for subsequent use.
2) fitting of fluids substratum: yeast cells wall powder 1%(w/v), yeast extract paste 0.05%(w/v), ammonium di-hydrogen phosphate 0.1%(w/v) and, sulfate of ammoniac 0.1%(w/v), iron(ic) chloride 0.01%(w/v) and, magnesium sulfate 0.01%(w/v).
Take said components in proportion, dissolve with tap water, adjust ph 6-7,121 DEG C of sterilizing 20min.
, screening
Sample: the mud of Qingdao of Shandong province Qingdao Beer Brewery sewage draining exit.
1) enrichment culture: sample thief 10g, adds 250mL fitting of fluids substratum, 250rpm, 55 DEG C, constant-temperature shaking culture 3-5 days;
2) select to cultivate: the nutrient solution 0.1mL obtained after getting above-mentioned enrichment culture, applying solid selective medium, cultivate 48 hours for 55 DEG C.According to bacterium colony size, filter out 8 strain dominant strains altogether, respectively called after K1, K2, K3 ..., K8.
the screening of embodiment 2 broken wall bacterial strain
1) will the beautiful filtering and impurity removing of yeast slurry 100 order yarn of Qingdao Beer Brewery be taken from, measure moisture content, and regulate moisture to obtain the sterilised yeast suspension of moisture 70%-95%.Detect yeast cell situation under microscope, in result display sterilised yeast suspension, the yeast cell of more than 99% is very complete, non-broken wall.
2) by K1, K2, K3 ..., K8 bacterial strain is inoculated in 100mL liquid selective medium respectively, 250rpm, 55 DEG C of constant-temperature shaking culture 48 hours; Then add above-mentioned sterilised yeast suspension 50mL, 250rpm, 55 DEG C of constant-temperature shaking culture 48 hours, the every 8 hours nutrient solutions that take a morsel, examine under a microscope the broken wall situation of yeast cell, calculate the sporoderm-broken rate of yeast cell.Blank group is set, i.e. 100mL liquid selective medium and sterilised yeast suspension 50mL simultaneously, does not add any bacterial strain.Concrete outcome is in table 1.
In table 1 48h, different strains is on the impact of breaking yeast cellule membrane rate
| Bacterial strain | 8h | 16h | 24h | 32h | 40h | 48h |
| Blank | 0 | 0 | 3.1% | 5.3% | 6.2% | 6.8% |
| K1 | 6.5% | 18.2% | 30.5% | 45% | 70.6% | 85.2% |
| K2 | 1.5% | 6% | 19.4% | 36.1% | 44% | 45.8% |
| K3 | 0 | 1.2% | 7.5% | 15.1% | 34.2% | 40.7% |
| K4 | 7.2% | 16.5% | 28.4% | 40% | 52.5 | 69.6% |
| K5 | 3.4% | 10.2% | 21% | 32.5% | 38.2% | 44.1% |
| K6 | 3.1% | 18.3% | 35.5% | 50.2% | 61.9% | 70.3% |
| K7 | 0 | 1.5% | 10.4% | 17.2% | 25.5% | 29.3% |
| K8 | 0 | 2.2% | 12.8% | 19.5% | 24.3% | 26.8% |
As can be seen from the data of table 1, when not adding any bacterial strain, the efficiency of the yeast cell self-dissolving broken wall of blank group is very low, and in 48 hours, sporoderm-broken rate just reaches 6.8%; And by adding K1 respectively, K2, K3 ..., K8 bacterial strain and yeast cell mixed culture, can make the sporoderm-broken rate of yeast cell generally improve 20%-80%, wherein the highest to add the sporoderm-broken rate of yeast cell after K1 bacterial strain mixed culture again, reaches 85.2%.
the qualification of embodiment 3 broken wall bacterial strain K1
1) the physiology and chemistry characteristic of K1 bacterial strain:
Bacterium colony is flat, and surface irregularity is opaque, dirty white, gemma size 0.6-0.9 × 1.0-1.5 μm, oval or column, middle life or near middle raw; Gram-positive, V-P tests the positive, and Starch Hydrolysis test is positive, and indole test is positive, can utilize glucose, pectinose, wood sugar and N.F,USP MANNITOL, growth temperature range 20-45 DEG C, pH scope 5-9.
2) molecular biology identification of K1 bacterial strain:
Adopt molecular biological method to identify the K1 bacterial strain that above-mentioned screening obtains, record its 16s rDNA sequence, and carry out blast comparison in GenBank nucleic acid database.In conjunction with biological characteristics and the 16srDNA comparison result of K1, applicant confirm K1 bacterial strain be subtilis (
bacillus subtilis), called after subtilis K1(
bacillus subtilisk1).
Applicant on September 3rd, 2014 by above-mentioned subtilis K1(
bacillus subtilisk1) be preserved in the China typical culture collection center of Wuhan, China Wuhan University, deposit number is CCTCC NO:M2014396.
the preparation of embodiment 4 yeast hydrolyate
4.1 bacterial strain activation
Get the subtilis K1 of freezen protective; Draw 100 μ L subtilis K1 under aseptic condition, be inoculated in 100mL nutrient broth medium (pH7.2), 30 DEG C-60 DEG C, 15-24h cultivated by 150-300rpm shaking table, works as OD
600namely=0.4 stop cultivating, and obtains activated spawn.
breaking yeast cellule membrane
Yeast slurry: the beer yeast slurry taking from Qingdao Beer Brewery.
Carry out filtering and impurity removing with 100 order yarns are beautiful to yeast slurry, add water adjustment, makes its moisture content within the scope of 70%-95%, obtain sterilised yeast suspension.Detect yeast cell situation under microscope, in result display sterilised yeast suspension, the yeast cell of more than 99% is very complete, non-broken wall.
Above-mentioned sterilised yeast suspension moved in fermentor tank, then add sterilized sulfate solution, DAP solution, glucose solution respectively, make the total reducing sugars concentration in sterilised yeast suspension be 2.5%-5%, the final concentration of ammonia nitrogen is 0.5-1.5%; Add the subtilis K1 after activation by the volume ratio of 5-10% again, 50 DEG C of fermentation 36h-48h, control stirring velocity and ventilation in fermenting process.Every 8h sampling, detect breaking yeast cellule membrane situation under the microscope respectively.
Detected result shows, after fermentation 48h, the sporoderm-broken rate adding yeast cell in the fermentor tank of subtilis K1 reaches more than 95%, thus the subtilis K1 and yeast cell mixed fermentation that the present invention are screened are described, effectively can improve the sporoderm-broken rate of yeast cell, achieve unexpected technique effect.
the preparation of yeast hydrolyate
Above-mentioned yeast lysate is warming up to boiling, maintains 10-20h, moisture is evaporated; Be about about 55% when yeast lysate is concentrated into moisture content, namely obtain yeast hydrolysis cream.The yeast that takes a morsel hydrolysis cream, detect its total nitrogen, Nucleotide, amino acid whose content respectively, concrete outcome is in table 2;
Or further yeast lysate is concentrated into after moisture content is about 40%-50%, carries out spraying dry (inlet temperature 120-180 DEG C, air outlet temperature 70-100 DEG C), namely obtain yeast hydrolysis powder.The yeast that takes a morsel hydrolysis powder, measure total nitrogen wherein, Nucleotide, aminoacids content respectively, concrete outcome sees the following form 3.
Table 2 yeast hydrolysis cream detected result
Table 3 yeast hydrolysis powder detected result
As can be seen from the data of table 2 and table 3, in the yeast hydrolysis cream that the present invention prepares or yeast hydrolysis powder, in the born of the same parents such as amino acid, Nucleotide, nutrition content is high, further demonstrate that the method for the invention is good to the shell-broken effect of yeast cell.Described method, by by subtilis K1 and yeast cell mixed fermentation, realizes effective broken wall of yeast cell, nutritive substance in born of the same parents is all discharged, obtains unexpected technique effect.
The present invention directly by subtilis K1 and yeast cell mixed fermentation, carries out broken wall treatment to yeast cells wall, substantially reduces technological process, reduces production cost, and shell-broken effect is remarkable, has a extensive future.
Claims (4)
1. a method for breaking yeast cellule membrane, is characterized in that, by subtilis and yeast cell are carried out mixed fermentation to realize.
2. method as claimed in claim 1, it is characterized in that, described subtilis is subtilis K1(
bacillus subtilisk1), deposit number is CCTCC NO M2014396.
3. method as claimed in claim 1, it is characterized in that, described mixed fermentation is specially: first move in fermentor tank by sterilised yeast suspension; Then add sterilized sulfate solution, DAP solution, glucose solution respectively, make the total reducing sugars concentration in sterilised yeast suspension be 2.5%-5%, the final concentration of ammonia nitrogen is 0.5-1.5%; Add the subtilis K1 after activation by the volume ratio of 5-10% again, 50 DEG C of fermentation 36h-48h, namely obtain breaking yeast cellule membrane liquid.
4. a preparation method for yeast hydrolyate the yeast lysate that method described in claim 3 obtains is carried out concentrated or spraying dry to realize.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108782948A (en) * | 2018-06-01 | 2018-11-13 | 东莞市银华生物科技有限公司 | One primary yeast source functional albumen and preparation method thereof |
| CN109303195A (en) * | 2017-07-28 | 2019-02-05 | 安琪酵母股份有限公司 | A kind of yeast cell wall and its preparation method and application of alternative antibiotic |
| CN111154752A (en) * | 2020-02-12 | 2020-05-15 | 黑龙江八一农垦大学 | Preparation method of PCR template DNA of yeast monoclonal colony |
| CN112155120A (en) * | 2020-09-30 | 2021-01-01 | 河北科技大学 | Micro-ecological synbiotic preparation for feed, preparation method and application |
-
2014
- 2014-10-15 CN CN201410541496.3A patent/CN104342372B/en active Active
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109303195A (en) * | 2017-07-28 | 2019-02-05 | 安琪酵母股份有限公司 | A kind of yeast cell wall and its preparation method and application of alternative antibiotic |
| CN109303195B (en) * | 2017-07-28 | 2021-09-07 | 安琪酵母股份有限公司 | Yeast cell wall capable of replacing antibiotics and preparation method and application thereof |
| CN108782948A (en) * | 2018-06-01 | 2018-11-13 | 东莞市银华生物科技有限公司 | One primary yeast source functional albumen and preparation method thereof |
| CN108782948B (en) * | 2018-06-01 | 2021-09-03 | 湛江银恒生物科技有限公司 | Yeast-derived functional protein and preparation method thereof |
| CN111154752A (en) * | 2020-02-12 | 2020-05-15 | 黑龙江八一农垦大学 | Preparation method of PCR template DNA of yeast monoclonal colony |
| CN112155120A (en) * | 2020-09-30 | 2021-01-01 | 河北科技大学 | Micro-ecological synbiotic preparation for feed, preparation method and application |
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