CN104450522A - Beer yeast cell disruption method adopting synergetic enzyme and mechanical disruption - Google Patents
Beer yeast cell disruption method adopting synergetic enzyme and mechanical disruption Download PDFInfo
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Abstract
The invention discloses a beer yeast cell disruption method adopting synergetic enzyme and mechanical disruption. The beer yeast cell disruption method comprises the following steps: (1) mixing beer yeast paste with water, thereby preparing 30-50% beer yeast suspension; (2) adding 0.3-0.5% of composite enzyme into the beer yeast suspension, and performing homogenization crushing for 2-3 times in a high-pressure homogenization crusher at the pressure of 1400-1600bar and the outlet temperature of 15-25 DEG C, wherein the composite enzyme comprises the following components in parts by weight: 1.5-5 parts of protease and 0.5-2 parts of cellulose. The beer yeast cell disruption method is short in disruption time, high in disruption rate, low in equipment utilization rate, free of organic solvent, acid or alkali, free of environmental pollution, low in production cost and capable of achieving continuous production.
Description
Technical field
The invention belongs to biological technical field.Relate to a kind of breaking yeast cellule membrane method particularly.
Background technology
Beer waste yeast is in beer production, and brewage through Primary Fermentation and secondary fermentation the muddy yeast that operation produces, be otherwise known as beer yeast slurry.From the eighties in 20th century, along with China's brewing industry develops rapidly, the beer waste yeast of generation is also increasing.By on average often producing the beer yeast slurry 1.5 tons calculating that 100 kilolitre beer produce moisture content 75% ~ 80%, within 2011, beer production is 489.88 hundred million liters, just has the beer yeast slurry of 73.48 ten thousand tons.This part cereuisiae fermentum Fender part is outside roughing uses as feed at present, and other have all directly emitted as refuse, become a large waste of foodstuffs industry.Therefore, the comprehensive utilization of beer waste yeast and high-valued exploitation are come into one's own day by day.
Cereuisiae fermentum is a kind of single celled eukaryotic microorganism, cell wall thickness is about 0.1 ~ 0.3 μm, structure is tough and tensile, and main component has dextran, mannosans, protein, chitin and lipid etc., and wherein the content of dextran and mannosans accounts for about 30% of dry cell weight respectively; Containing abundant nutritive substance in cerevisiae matter, as multiple nutritional components such as protein, nucleic acid, VITAMIN, carbohydrate, lipid material, mineral substance.But because cerevisiae wall construction complexity is tough and tensile, broken wall is comparatively difficult, has had a strong impact on the utilization of cerevisiae wall and kytoplasm effective constituent.At present, conventional wall-breaking method has polishing, supersonic method, acid-base method, enzyme process or the simple series connection use of several method.Wherein polishing is the common method of cerevisiae crushing experiment room, but needs ball mill, and equipment price format high throughput is little, needs to repeat fragmentation, is not suitable for suitability for industrialized production.Also there is heat radiation difficulty in supersonic method, easily produces the problem of localized hyperthermia, be only suitable for laboratory scale.Acid-base method broken wall is the common method of current suitability for industrialized production, needs to use a large amount of strong acid and strong bases, produces a large amount of waste water.Enzymatic cleavage broken wall has very outstanding advantage compared with other physico-chemical processes, and as energy consumption is low, reaction conditions is gentle, low to nutritive ingredient destructiveness, environmental protection.But existing enzymatic shell-broken due to the specific aim of enzyme poor, be difficult to reach desirable shell-broken effect.The enzyme process of existing report is also the autolytic process in conjunction with cereuisiae fermentum, and rely on the enzyme system in cerevisiae to carry out broken wall, enzymolysis time is long, easily causes the amount reproduction of miscellaneous bacteria, and hold facility rate is high.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, the cerevisiae wall-breaking method that a kind of enzyme and mechanical breaking-wall method are worked in coordination with is provided.
Technical scheme of the present invention is summarized as follows:
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 30% ~ 50%;
(2) be the ratio of 0.3%-0.5% in prozyme addition, prozyme joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher in the pressure of 1400-1600bar, temperature out under the condition of 15-25 DEG C, carry out homogeneous broken wall 2-3 time; Described prozyme comprises by weight: 1.5 ~ 5 parts, proteolytic enzyme, cellulase 0.5 ~ 2 part.
Preferred: the enzyme work of described proteolytic enzyme is 86000 ~ 180000U/ml; The enzyme work of described cellulase is 80 ~ 550U/ml.
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 30% ~ 50%;
(2) be the ratio of 0.3%-0.5% in prozyme addition, prozyme joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher in the pressure of 1400-1600bar, temperature out under the condition of 15-25 DEG C, carry out homogeneous broken wall 2-3 time; Described prozyme comprises by weight: 1.5 ~ 5 parts, proteolytic enzyme, cellulase 0.5 ~ 2 part, chitinase 0.5 ~ 1 part.
Preferred: the enzyme work of described proteolytic enzyme is 50000 ~ 155000U/ml; The enzyme work of described cellulase is 80 ~ 400U/ml, and the enzyme work of chitinase is 70 ~ 170U/ml.
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 30% ~ 50%;
(2) be the ratio of 0.3%-0.5% in prozyme addition, prozyme joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher in the pressure of 1400-1600bar, temperature out under the condition of 15-25 DEG C, carry out homogeneous broken wall 2-3 time; Described prozyme comprises by weight: 1.5 ~ 5 parts, proteolytic enzyme, cellulase 0.5 ~ 2 part, chitinase 0.5 ~ 1 part, mannase 0.3 ~ 2 part.
Preferably: described proteolytic enzyme is 5 parts, described cellulase is 2 parts, and described chitinase is 0.5 part, and described mannase is 0.3 part.
Preferred: the enzyme work of described proteolytic enzyme is 50000 ~ 100000U/ml; The enzyme work of described cellulase is 50 ~ 400U/ml, and the enzyme work of chitinase is 30 ~ 100U/ml, and the enzyme work of mannase is 35000 ~ 250000U/ml.
Preferably: the enzyme of described proteolytic enzyme is lived as 100000U/ml; The enzyme of described cellulase is lived as 200U/ml, and the enzyme of chitinase is lived as 30U/ml, and the enzyme of mannase is lived as 35000U/ml.
In aforesaid method, pressure the best is 1500bar.
Advantage of the present invention:
Broken time of the present invention is short, and sporoderm-broken rate is high, and hold facility rate is low, not with an organic solvent acid or alkali, not to environment; Production cost is low, can realize continuous prodution.
Accompanying drawing explanation
Fig. 1 is the impact of self-dissolving time on sporoderm-broken rate;
Fig. 2 is that broken wall number of times and broken wall pressure are on the impact of sporoderm-broken rate;
Fig. 3 is that enzyme concentration and broken wall number of times are on the impact of sporoderm-broken rate;
Fig. 4 is that cereuisiae fermentum mass concentration and broken wall number of times are on the impact of sporoderm-broken rate;
Fig. 5 is the impact that homogenization pressure is lived on enzyme.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, simultaneously in order to simple and clearly object, hereafter suitably eliminates the description of known technology, in order to avoid the description of those unnecessary details impact to the technical program.Need be illustrated, the proteolytic enzyme selected by the present invention, cellulase, mannase and chitinase are commercially available.
Beer waste yeast pre-treatment: by beer waste yeast removal of impurities after filtration, centrifugal except after drinking, be the NaHCO of 0.5% by mass concentration
3aqueous solution soaking 0.5h debitterize, with water cleaning to supernatant liquor clarification, at 6000 revs/min, centrifugal 20 minutes, collecting precipitation was beer yeast slurry.
Embodiment 1(contrasts)
Suspended in water by beer yeast slurry and obtain the cereuisiae fermentum suspension that mass concentration is 15%, at pH=5, carry out self-dissolving hydrolysis at 50 DEG C, Fig. 1 is shown in the impact of self-dissolving time and sporoderm-broken rate.As shown in Figure 1, during self-dissolving 24h, sporoderm-broken rate reaches the highest under this condition, is 55%.
Embodiment 2(contrasts)
Beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%;
Adopt high-pressure homogeneous crusher to carry out broken wall, temperature out is under the condition of 20 DEG C, and broken wall pressure and broken wall number of times (broken wall, twice broken wall, three broken walls) are shown in Fig. 2 to the impact of sporoderm-broken rate.
Along with the increase of broken wall pressure, sporoderm-broken rate rises, and under the pressure of 1800-1900bar, one time sporoderm-broken rate is about 75%, and secondary sporoderm-broken rate reaches 85%; As required, sporoderm-broken rate reaches more than 90%, then need to carry out three broken walls.
Embodiment 3
Prozyme I is made up of following raw material by weight: 5 parts, proteolytic enzyme, cellulase 2 parts, chitinase 0.5 part, mannase 0.3 part.
The enzyme of proteolytic enzyme is lived as 100000U/ml, and the enzyme of cellulase is lived as 200U/ml, and the enzyme of chitinase is lived as 30U/ml, and the enzyme of mannase is lived as 35000U/ml.
Embodiment 4
Complex Ⅱ is made up of following raw material by weight: protease 3 .5 part, cellulase 0.5 part, chitinase 1 serving part, mannase 2 parts.
The enzyme of proteolytic enzyme is lived as 82000U/ml, and the enzyme of cellulase is lived as 50U/ml, and the enzyme of chitinase is lived as 100U/ml, and the enzyme of mannase is lived as 250000U/ml.
Embodiment 5
Prozyme III is made up of following raw material by weight: 1.5 parts, proteolytic enzyme, cellulase 1.8 parts, chitinase 0.6 part, mannase 1 part.
The enzyme of proteolytic enzyme is lived as 50000U/ml, and the enzyme of cellulase is lived as 400U/ml, and the enzyme of chitinase is lived as 75U/ml, and the enzyme of mannase is lived as 175000U/ml.
Embodiment 6
Prozyme IV is made up of following raw material by weight: 1.5 parts, proteolytic enzyme, cellulase 2 parts.
The enzyme of proteolytic enzyme is lived as 86000U/ml, and the enzyme of cellulase is lived as 550U/ml.
Embodiment 7
Prozyme V is made up of following raw material by weight: 5 parts, proteolytic enzyme, cellulase 0.5 part.
The enzyme of proteolytic enzyme is lived as 180000U/ml, and the enzyme of cellulase is lived as 80U/ml.
Embodiment 8
Prozyme VI is made up of following raw material by weight: protease 3 part, cellulase 1 part.
The enzyme of proteolytic enzyme is lived as 150000U/ml, and the enzyme of cellulase is lived as 220U/ml.
Embodiment 9
Prozyme VII is made up of following raw material by weight: 1.5 parts, proteolytic enzyme, cellulase 1.8 parts, chitinase 1 serving part.
The enzyme of proteolytic enzyme is lived as 50000U/ml, and the enzyme of cellulase is lived as 400U/ml, and the enzyme of chitinase is lived as 170U/ml.
Embodiment 10
Prozyme VIII is made up of following raw material by weight: 5 parts, proteolytic enzyme, cellulase 2 parts, chitinase 0.5 part.
The enzyme of proteolytic enzyme is lived as 133000U/ml, and the enzyme of cellulase is lived as 267U/ml, and the enzyme of chitinase is lived as 70U/ml.
Embodiment 11
Prozyme Ⅸ is made up of following raw material by weight: 4.5 parts, proteolytic enzyme, cellulase 0.5 part, chitinase 0.8 part.
The enzyme of proteolytic enzyme is lived as 155000U/ml, and the enzyme of cellulase is lived as 80U/ml, and the enzyme of chitinase is lived as 100U/ml.
Embodiment 12
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%, divide four parts;
(2) be the ratio of 0.3% in prozyme I, IV, VII addition, respectively prozyme I, IV, VII joined in above-mentioned cereuisiae fermentum suspension, stir, not add prozyme sample for contrast.In high-pressure homogeneous crusher, in the pressure of 1500bar, temperature out under the condition of 20 DEG C, carry out broken wall, broken wall number of times on the impact of sporoderm-broken rate in table 1.
Table 1 different composite enzyme and broken wall number of times are on the impact of sporoderm-broken rate
Result shows, and the control group that a sporoderm-broken rate of prozyme I, IV, VII does not add prozyme improves 32.5%, 19.5% and 23.8% respectively; The control group that secondary sporoderm-broken rate does not add prozyme improves 21.2%, 7.3% and 13.4% respectively.
Embodiment 13
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%, divide four parts;
(2) in Complex Ⅱ, V, VIII addition is the ratio of 0.5%, respectively Complex Ⅱ, V, VIII is joined in above-mentioned cereuisiae fermentum suspension, stirs, not add prozyme sample for contrast.In high-pressure homogeneous crusher, in the pressure of 1400bar, temperature out under the condition of 15 DEG C, carry out broken wall, broken wall number of times on the impact of sporoderm-broken rate in table 2.
Table 2 different composite enzyme and broken wall number of times are on the impact of sporoderm-broken rate
Result shows, Complex Ⅱ, V, the sporoderm-broken rate of VIII control group that do not add prozyme improves 33.5%, 24.5% and 29% respectively; The control group that secondary sporoderm-broken rate does not add prozyme improves 19.2%, 11.1% and 16.8% respectively.
Embodiment 14
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension four parts that mass concentration is 30%;
(2) be the ratio of 0.5% in prozyme III, VI, Ⅸ addition, respectively prozyme III, VI, Ⅸ joined in above-mentioned cereuisiae fermentum suspension, stir, not add prozyme sample for contrast.In high-pressure homogeneous crusher, in the pressure of 1600bar, temperature out under the condition of 25 DEG C, carry out broken wall, broken wall number of times on the impact of sporoderm-broken rate in table 3.
Table 3 different composite enzyme and broken wall number of times are on the impact of sporoderm-broken rate
Result shows, and the control group that a sporoderm-broken rate of prozyme III, VI, Ⅸ does not add prozyme improves 34.5%, 18.6% and 23.9% respectively; The control group that secondary sporoderm-broken rate does not add prozyme improves 21.2%, 7.3% and 15.3% respectively.
Embodiment 15
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%;
(2) be the ratio of 0.3% in prozyme I addition, prozyme I is joined in described cereuisiae fermentum suspension, stirs, carry out broken wall at high-pressure homogeneous crusher, temperature out is under the condition of 20 DEG C, and Fig. 2 is shown in broken wall pressure and the impact of broken wall number of times on sporoderm-broken rate.
Result shows, and 1500bar, one time sporoderm-broken rate reaches more than 80%, and secondary sporoderm-broken rate reaches more than 95%.
Embodiment 16
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%;
(2) respectively in the ratio that addition is 0.00%, 0.10%, 0.20%, 0.30%, 0.40%, 0.50%, prozyme I is joined in cereuisiae fermentum suspension, after stirring, in high-pressure homogeneous crusher, in the pressure of 1500bar, temperature out under the condition of 20 DEG C, carry out broken wall, Fig. 3 is shown in enzyme concentration and the impact of broken wall number of times on sporoderm-broken rate.
Along with the increase of enzyme concentration, sporoderm-broken rate has significant change, and prozyme I consumption is 0.3% time, and one time sporoderm-broken rate is 80%, and secondary sporoderm-broken rate reaches 95%; Prozyme I consumption is 0.4% time, and one time sporoderm-broken rate is 85%, and secondary sporoderm-broken rate reaches 97%; Prozyme I consumption is 0.5% time, and one time sporoderm-broken rate is 85%, and secondary sporoderm-broken rate reaches 95%.
Embodiment 17
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry is mixed with water to make mass concentration be respectively 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, the cereuisiae fermentum suspension of 60%;
(2) be the ratio of 0.3% in prozyme I addition, prozyme I is joined in described cereuisiae fermentum suspension respectively, stirs, in high-pressure homogeneous crusher, under 1500bar, carry out broken wall, Fig. 4 is shown in beer yeast slurry concentration and the impact of broken wall number of times on sporoderm-broken rate.
Result shows, and beer yeast slurry mass concentration is between 10% ~ 55%, and one time sporoderm-broken rate reaches more than 70%; Beer yeast slurry concentration is between 30% ~ 50%, and secondary sporoderm-broken rate reaches more than 95%.
Embodiment 18
Prozyme I adopts high-pressure homogeneous crusher to process, and temperature out controls at 20 DEG C, and the enzyme activity determination of proteolytic enzyme adopts folin's methods, and cellulase adopts filter paper enzyme activity measuring method.Processing pressure is shown in Fig. 5 to the impact that enzyme is lived.
Primary treatment process, from 1100bar, enzyme is lived, and loss beginning is obvious to be increased, and after 1600bar, rate of loss has increase, and loss speed is very fast.But affect little in 1300 ~ 1600bar processing pressure on the rate of loss that enzyme is lived after being through secondary treatment, adopt the pressure treatment enzyme being greater than 1600bar loss alive higher.Therefore 1400 ~ 1600bar processing pressure is adopted.
Embodiment 19
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%; Treatment capacity is 2L;
(2) be the ratio of 0.3% in prozyme I addition, prozyme I is joined in described beer yeast slurry suspension, in high-pressure homogeneous crusher (treatment capacity 10L/h) in the pressure of 1500bar, temperature out under the condition of 15 DEG C, carry out homogeneous broken wall 2 times; 95.9% is reached through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Embodiment 20
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%; Treatment capacity is 2L;
(2) be the ratio of 0.3% in prozyme IV addition, prozyme IV is joined in described beer yeast slurry suspension, in high-pressure homogeneous crusher (treatment capacity 10L/h) in the pressure of 1500bar, temperature out under the condition of 15 DEG C, carry out homogeneous broken wall 2 times; 84.5% is reached through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Substitute the present embodiment prozyme IV with prozyme V, other same the present embodiment, reach 85.5% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Substitute the present embodiment prozyme IV with prozyme VI, other same the present embodiment, reach 84.9% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Embodiment 21
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 30%; Treatment capacity is 2L;
(2) be the ratio of 0.3% in prozyme VII addition, prozyme VII is joined in described beer yeast slurry suspension, in high-pressure homogeneous crusher (treatment capacity 10L/h) in the pressure of 1400bar, temperature out under the condition of 25 DEG C, carry out homogeneous broken wall 2 times; 89.3% is reached through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 30min.
Substitute the present embodiment prozyme VII with prozyme VIII, other same the present embodiment, reach 88.1% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 30min.
Substitute the present embodiment prozyme VII with prozyme Ⅸ, other same the present embodiment, reach 88.6% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 30min.
Embodiment 22
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%; Treatment capacity is 2L;
(2) be the ratio of 0.5% in prozyme I addition, prozyme I is joined in described beer yeast slurry suspension, in high-pressure homogeneous crusher (treatment capacity 10L/h) in the pressure of 1500bar, temperature out under the condition of 25 DEG C, carry out homogeneous broken wall 2 times; 97.7% is reached through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Substitute the present embodiment prozyme I with prozyme IV, other same the present embodiment, reach 86.4% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Substitute the present embodiment prozyme I with prozyme VII, other same the present embodiment, reach 91.2% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Embodiment 23
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 30%; Treatment capacity is 2L;
(2) be the ratio of 0.3% in prozyme I addition, prozyme I is joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher (treatment capacity 10L/h) in the pressure of 1500bar, temperature out under the condition of 15 DEG C, carry out homogeneous broken wall 2 times; 95.12% is reached through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 30min.
Substitute the present embodiment prozyme I with prozyme IV, other same the present embodiment, reach 85.8% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 30min.
Substitute the present embodiment prozyme I with prozyme VII, other same the present embodiment, reach 90.3% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 30min.
Embodiment 24
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 30%; Treatment capacity is 2L;
(2) be the ratio of 0.5% in prozyme I addition, prozyme I is joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher (treatment capacity 10L/h) in the pressure of 1500bar, temperature out under the condition of 20 DEG C, carry out homogeneous broken wall 2 times; 97.23% is reached through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 30min.
Substitute the present embodiment prozyme I with prozyme IV, other same the present embodiment, reach 86.1% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 30min.
Substitute the present embodiment prozyme I with prozyme VII, other same the present embodiment, reach 90.6% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 30min.
Embodiment 25
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%; Treatment capacity is 2L;
(2) be the ratio of 0.3% in Complex Ⅱ addition, Complex Ⅱ is joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher (treatment capacity 10L/h) in the pressure of 1500bar, temperature out under the condition of 20 DEG C, carry out homogeneous broken wall 2 times; 95.8% is reached through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Cellulase selected by various embodiments of the present invention is acid fiber enzyme, substitutes the cellulase in the present embodiment in Complex Ⅱ with neutral cellulase, other same the present embodiment, reaches 95.12% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Embodiment 26
The cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are worked in coordination with, comprises the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 50%; Treatment capacity is 2L;
(2) be the ratio of 0.3% in prozyme III addition, prozyme III is joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher (treatment capacity 10L/h) in the pressure of 1500bar, temperature out under the condition of 15 DEG C, carry out homogeneous broken wall 2 times; 96.2% is reached through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Proteolytic enzyme selected by various embodiments of the present invention is aspartic protease, with the proteolytic enzyme in prozyme III in neutral protein enzymes extraction the present embodiment, other same the present embodiment, reaches 95.26% through blood counting chamber microscopic inspection sporoderm-broken rate; Completing broken time is 35min.
Proteinase activity is defined as: under certain temperature and pH value condition, and 1min caseinhydrolysate produces 1 μ g tyrosine, is 1 enzyme activity unit.Measuring method is according to folin's methods in GB/T 23527 protease preparation.
Cellulase activity is defined as: at 50 DEG C, and under appointment pH condition (acidic cellulase pH 4.8, neutral cellulase pH 6.0), lh is hydrolyzed filter paper substrate, and producing the reducing sugar amount being equivalent to lmg glucose, is 1 enzyme activity unit.Measuring method is according to filter paper enzyme activity measuring method in QB 2583 cellulase preparation.
Mannase is lived and is defined as: refer at the standard conditions (pH 5.3,50 DEG C), and the amount that per minute hydrolysis substrate produces enzyme needed for 1 μ g seminose is defined as mannosans activity unit (U) measuring method employing DNS method.
Chitinase enzyme is lived and is defined as: referring to that at the standard conditions (pH 6.0,45 DEG C) decomposition tobacco brown spot pathogen per hour produces the enzyme amount of 1 μ g 2-Acetamido-2-deoxy-D-glucose, is 1 enzyme activity unit (U).And measuring method is with reference to Haitao Zhang, Wang Ting, etc. chitinase producing strains Screening and Identification and the research of product enzyme performance. Chinese biological engineering magazine, 2010,30(8): 82-87
Claims (9)
1. the cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are collaborative, is characterized in that comprising the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 30% ~ 50%;
(2) be the ratio of 0.3%-0.5% in prozyme addition, prozyme is joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher in the pressure of 1400-1600bar, temperature out under the condition of 15-25 DEG C, carry out homogeneous broken wall 2-3 time; Described prozyme comprises by weight: 1.5 ~ 5 parts, proteolytic enzyme, cellulase 0.5 ~ 2 part.
2. the cerevisiae wall-breaking method that a kind of enzyme according to claim 1 and mechanical breaking-wall method are collaborative, is characterized in that the enzyme work of described proteolytic enzyme is 86000 ~ 180000U/ml; The enzyme work of described cellulase is 80 ~ 550U/ml.
3. the cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are collaborative, is characterized in that comprising the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 30% ~ 50%;
(2) be the ratio of 0.3%-0.5% in prozyme addition, prozyme is joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher in the pressure of 1400-1600bar, temperature out under the condition of 15-25 DEG C, carry out homogeneous broken wall 2-3 time; Described prozyme comprises by weight: 1.5 ~ 5 parts, proteolytic enzyme, cellulase 0.5 ~ 2 part, chitinase 0.5 ~ 1 part.
4. the cerevisiae wall-breaking method that a kind of enzyme according to claim 3 and mechanical breaking-wall method are collaborative, is characterized in that the enzyme work of described proteolytic enzyme is 50000 ~ 155000U/ml; The enzyme work of described cellulase is 80 ~ 400U/ml, and the enzyme work of chitinase is 70 ~ 170U/ml.
5. the cerevisiae wall-breaking method that enzyme and mechanical breaking-wall method are collaborative, is characterized in that comprising the steps:
(1) beer yeast slurry and water are mixed and made into the cereuisiae fermentum suspension that mass concentration is 30% ~ 50%;
(2) be the ratio of 0.3%-0.5% in prozyme addition, prozyme is joined in described cereuisiae fermentum suspension, in high-pressure homogeneous crusher in the pressure of 1400-1600bar, temperature out under the condition of 15-25 DEG C, carry out homogeneous broken wall 2-3 time; Described prozyme comprises by weight: 1.5 ~ 5 parts, proteolytic enzyme, cellulase 0.5 ~ 2 part, chitinase 0.5 ~ 1 part, mannase 0.3 ~ 2 part.
6. the cerevisiae wall-breaking method that a kind of enzyme according to claim 5 and mechanical breaking-wall method are collaborative, it is characterized in that described proteolytic enzyme is 5 parts, described cellulase is 2 parts, and described chitinase is 0.5 part, and described mannase is 0.3 part.
7. the cerevisiae wall-breaking method that a kind of enzyme according to claim 5 or 6 and mechanical breaking-wall method are worked in coordination with, is characterized in that the enzyme work of described proteolytic enzyme is 50000 ~ 100000U/ml; The enzyme work of described cellulase is 50 ~ 400U/ml, and the enzyme work of chitinase is 30 ~ 100U/ml, and the enzyme work of mannase is 35000 ~ 250000U/ml.
8. the cerevisiae wall-breaking method that a kind of enzyme according to claim 7 and mechanical breaking-wall method are collaborative, is characterized in that the enzyme of described proteolytic enzyme is lived as 100000U/ml; The enzyme of described cellulase is lived as 200U/ml, and the enzyme of chitinase is lived as 30U/ml, and the enzyme of mannase is lived as 35000U/ml.
9. the cerevisiae wall-breaking method that a kind of enzyme according to claim 1,3 or 5 and mechanical breaking-wall method are worked in coordination with, is characterized in that described pressure is 1500bar.
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