CN104450522B - 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.More particularly to a kind of breaking yeast cellule membrane method.
Background technology
Beer waste yeast is that in beer production, the pureed ferment produced by operation is brewageed in Jing main fermentations and after fermentation
Mother, be otherwise known as beer yeast slurry.From the eighties in 20th century, as China's brewing industry is developed rapidly, the beer of generation gives up
Yeast is also increasing.Based on averagely often producing 100 kilolitre beer and producing 1.5 tons of the beer yeast slurry of moisture content 75%～80%
Calculate, beer production is 489.88 hundred million liters within 2011, just there is 73.48 ten thousand tons of beer yeast slurry.Current this part beer yeast slurry
Except a part is in addition to roughing is used as feed, other are directly emitted as waste, become the one of food industry
Big discarded object.Therefore, comprehensive utilization and high-valued exploitation to beer waste yeast is increasingly subject to pay attention to.
Brewer's yeast is a kind of single celled eukaryotic microorganism, and cell wall thickness is about 0.1～0.3 μm, and structure is tough and tensile, mainly
The content that composition has glucan, mannosan, protein, chitin and lipid etc., wherein glucan and mannosan is accounted for respectively
30% or so of dry cell weight；Containing abundant nutriment, such as protein, nucleic acid, vitamin, carbon in beer yeast cells matter
The multiple nutritional components such as hydrate, lipid material, mineral matter.But, because beer yeast cells wall construction complexity is tough and tensile, break
Wall is more difficult, has had a strong impact on the utilization of beer yeast cells wall and kytoplasm active ingredient.At present, conventional wall-breaking method has
Polishing, supercritical ultrasonics technology, acid-base method, enzyme process or several method are simply used in series.Wherein polishing is that brewer's yeast is thin
The common method of born of the same parents' crushing experiment room, however it is necessary that ball mill, equipment price format high throughput is little, needs to repeat broken, is not suitable for
Industrialized production.Supercritical ultrasonics technology there is also radiating difficulty, be also easy to produce the problem of localized hyperthermia, be only suitable for laboratory scale.Soda acid
Method broken wall is the common method of current industrialized production, needs to use substantial amounts of strong acid and strong base, produces substantial amounts of waste water.Enzyme process splits
Solution broken wall has the advantages that very prominent compared with other physico-chemical processes, and such as energy consumption is low, and reaction condition is gentle, broken to nutrient content
Bad low degree, environmental protection.But existing enzymatic shell-broken is poor due to the specific aim of enzyme, it is difficult to reach preferable shell-broken effect.
The enzyme process of existing report is also the autolytic process for combining brewer's yeast, and by the enzyme system in beer yeast cells broken wall, enzyme are carried out
The solution time is long, easily causes the amount reproduction of miscellaneous bacteria, and hold facility rate is high.
The content of the invention
The purpose of the present invention is to overcome the deficiencies in the prior art, there is provided the brewer's yeast that a kind of enzyme is cooperateed with mechanical breaking-wall method is thin
Born of the same parents' wall-breaking method.
Technical scheme is summarized as follows：
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 30%～50%；
（2）It is the ratio of 0.3%-0.5% in complex enzyme addition, complex enzyme is added to into the brewer's yeast suspension
In, the pressure, outlet temperature in high-pressure homogeneous disintegrating machine in 1400-1600bar carries out homogeneous under conditions of 15-25 DEG C
Broken wall 2-3 time；The complex enzyme includes by weight：1.5～5 parts of protease, 0.5～2 part of cellulase.
Preferably：The enzyme activity of the protease is 86000～180000U/ml；The enzyme activity of the cellulase be 80～
550U/ml。
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 30%～50%；
（2）It is the ratio of 0.3%-0.5% in complex enzyme addition, complex enzyme is added to into the brewer's yeast suspension
In, the pressure, outlet temperature in high-pressure homogeneous disintegrating machine in 1400-1600bar carries out homogeneous under conditions of 15-25 DEG C
Broken wall 2-3 time；The complex enzyme includes by weight：1.5～5 parts of protease, 0.5～2 part of cellulase, chitinase
0.5～1 part.
Preferably：The enzyme activity of the protease is 50000～155000U/ml；The enzyme activity of the cellulase be 80～
400U/ml, the enzyme activity of chitinase is 70～170U/ml.
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 30%～50%；
（2）It is the ratio of 0.3%-0.5% in complex enzyme addition, complex enzyme is added to into the brewer's yeast suspension
In, the pressure, outlet temperature in high-pressure homogeneous disintegrating machine in 1400-1600bar carries out homogeneous under conditions of 15-25 DEG C
Broken wall 2-3 time；The complex enzyme includes by weight：1.5～5 parts of protease, 0.5～2 part of cellulase, chitinase
0.5～1 part, 0.3～2 part of mannase.
Preferably：The protease is 5 parts, and the cellulase is 2 parts, and the chitinase is 0.5 part, the sweet dew
Dextranase is 0.3 part.
Preferably：The enzyme activity of the protease is 50000～100000U/ml；The enzyme activity of the cellulase be 50～
400U/ml, the enzyme activity of chitinase is 30～100U/ml, and the enzyme activity of mannase is 35000～250000U/ml.
Preferably：The enzyme activity of the protease is 100000U/ml；The enzyme activity of the cellulase be 200U/ml, chitin
The enzyme activity of matter enzyme is 30U/ml, and the enzyme activity of mannase is 35000U/ml.
In said method, pressure is most preferably 1500bar.
Advantages of the present invention：
Broken time of the present invention is short, and sporoderm-broken rate is high, and hold facility rate is low, organic solvent or acid or alkali is not used, not to ring
Border pollutes；Low production cost, it is possible to achieve continuous prodution.
Description of the drawings
Fig. 1 is impact of the self-dissolving time to sporoderm-broken rate；
Fig. 2 is the impact of broken wall number of times and broken wall pressure to sporoderm-broken rate；
Fig. 3 is the impact of enzyme concentration and broken wall number of times to sporoderm-broken rate；
Fig. 4 is the impact of brewer's yeast mass concentration and broken wall number of times to sporoderm-broken rate；
Fig. 5 is impact of the homogenization pressure to enzyme activity.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described, while for purposes of simplicity and clarity, under
Text suitably eliminates the description of known technology, in case those unnecessary details affect the description to the technical program.Need to add
To illustrate, the protease, cellulase, mannase and chitinase selected by the present invention is commercially available.
Beer waste yeast pre-treatment：It is 0.5% with mass concentration by beer waste yeast through filtering miscellaneous, centrifugation except after drinking
NaHCO3The de- hardship of aqueous solution soaking 0.5h, is cleaned to supernatant clarification with water, at 6000 revs/min, is centrifuged 20 minutes, collects heavy
Form sediment, be beer yeast slurry.
Embodiment 1（Contrast）
Beer yeast slurry is suspended in water the brewer's yeast suspension for obtaining that mass concentration is 15%, in pH=5, at 50 DEG C
Self-dissolving hydrolysis is carried out, the self-dissolving time sees Fig. 1 with the impact of sporoderm-broken rate.As shown in figure 1, under this condition self-dissolving 24h when sporoderm-broken rate
Highest is reached, is 55%.
Embodiment 2（Contrast）
Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 50%；
Broken wall is carried out using high-pressure homogeneous disintegrating machine, outlet temperature under conditions of 20 DEG C, broken wall pressure and broken wall number of times
（Broken wall, twice broken wall, three broken walls）Fig. 2 is shown in impact to sporoderm-broken rate.
With the increase of broken wall pressure, sporoderm-broken rate rises, and under the pressure of 1800-1900bar, one time sporoderm-broken rate is 75%
Left and right, secondary sporoderm-broken rate reaches 85%；Such as require that sporoderm-broken rate reaches more than 90%, then need to carry out three broken walls.
Embodiment 3
Complex enzyme I is made up of by weight following raw materials：5 parts of protease, 2 parts of cellulase, 0.5 part of chitinase,
0.3 part of mannase.
The enzyme activity of protease is 100000U/ml, and the enzyme activity of cellulase is 200U/ml, and the enzyme activity of chitinase is 30U/
Ml, the enzyme activity of mannase is 35000U/ml.
Embodiment 4
Complex Ⅱ is made up of by weight following raw materials：Protease 3 .5 parts, 0.5 part of cellulase, chitinase 1 serving
Part, 2 parts of mannase.
The enzyme activity of protease is 82000U/ml, and the enzyme activity of cellulase is 50U/ml, and the enzyme activity of chitinase is 100U/
Ml, the enzyme activity of mannase is 250000U/ml.
Embodiment 5
Complex enzyme III is made up of by weight following raw materials：1.5 parts of protease, 1.8 parts of cellulase, chitinase
0.6 part, 1 part of mannase.
The enzyme activity of protease is 50000U/ml, and the enzyme activity of cellulase is 400U/ml, and the enzyme activity of chitinase is 75U/
Ml, the enzyme activity of mannase is 175000U/ml.
Embodiment 6
Complex enzyme IV is made up of by weight following raw materials：1.5 parts of protease, 2 parts of cellulase.
The enzyme activity of protease is 86000U/ml, and the enzyme activity of cellulase is 550U/ml.
Embodiment 7
Complex enzyme V is made up of by weight following raw materials：5 parts of protease, 0.5 part of cellulase.
The enzyme activity of protease is 180000U/ml, and the enzyme activity of cellulase is 80U/ml.
Embodiment 8
Complex enzyme VI is made up of by weight following raw materials：Protease 3 part, 1 part of cellulase.
The enzyme activity of protease is 150000U/ml, and the enzyme activity of cellulase is 220U/ml.
Embodiment 9
Complex enzyme VII is made up of by weight following raw materials：1.5 parts of protease, 1.8 parts of cellulase, chitinase 1 serving
Part.
The enzyme activity of protease is 50000U/ml, and the enzyme activity of cellulase is 400U/ml, and the enzyme activity of chitinase is 170U/
ml。
Embodiment 10
Complex enzyme VIII is made up of by weight following raw materials：5 parts of protease, 2 parts of cellulase, 0.5 part of chitinase.
The enzyme activity of protease is 133000U/ml, and the enzyme activity of cellulase is 267U/ml, and the enzyme activity of chitinase is 70U/
ml。
Embodiment 11
Complex enzyme Ⅸ is made up of by weight following raw materials：4.5 parts of protease, 0.5 part of cellulase, chitinase
0.8 part.
The enzyme activity of protease is 155000U/ml, and the enzyme activity of cellulase is 80U/ml, and the enzyme activity of chitinase is 100U/
ml。
Embodiment 12
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 50%, divide four parts；
（2）In the ratio that the addition of complex enzyme I, IV, VII is 0.3%, respectively complex enzyme I, IV, VII is added to into above-mentioned beer
In brewer yeast suspension, stir, not add compound enzyme sample as control.In high-pressure homogeneous disintegrating machine, 1500bar's
Pressure, outlet temperature carry out broken wall under conditions of 20 DEG C, and impact of the broken wall number of times to sporoderm-broken rate is shown in Table 1.
The impact of the different composite enzyme of table 1 and broken wall number of times to sporoderm-broken rate
As a result show, a sporoderm-broken rate of complex enzyme I, IV, VII does not add the control group of complex enzyme to be respectively increased
32.5%th, 19.5% and 23.8%；Secondary sporoderm-broken rate does not add the control group of complex enzyme to be respectively increased 21.2%, 7.3% and 13.4%.
Embodiment 13
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 50%, divide four parts；
（2）In Complex Ⅱ, the ratio that V, VIII addition is 0.5%, Complex Ⅱ, V, VIII are added to respectively above-mentioned
In brewer's yeast suspension, stir, not add compound enzyme sample as control.In high-pressure homogeneous disintegrating machine, in 1400bar
Pressure, outlet temperature under conditions of 15 DEG C, carry out broken wall, impact of the broken wall number of times to sporoderm-broken rate is shown in Table 2.
The impact of the different composite enzyme of table 2 and broken wall number of times to sporoderm-broken rate
As a result show, Complex Ⅱ, V, VIII sporoderm-broken rate do not add the control group of complex enzyme to be respectively increased
33.5%th, 24.5% and 29%；Secondary sporoderm-broken rate does not add the control group of complex enzyme to be respectively increased 19.2%, 11.1% and 16.8%.
Embodiment 14
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into four parts of the brewer's yeast suspension that mass concentration is 30%；
（2）In the ratio that the addition of complex enzyme III, VI, Ⅸ is 0.5%, complex enzyme III, VI, Ⅸ is added to respectively above-mentioned
In brewer's yeast suspension, stir, not add compound enzyme sample as control.In high-pressure homogeneous disintegrating machine, in 1600bar
Pressure, outlet temperature under conditions of 25 DEG C, carry out broken wall, impact of the broken wall number of times to sporoderm-broken rate is shown in Table 3.
The impact of the different composite enzyme of table 3 and broken wall number of times to sporoderm-broken rate
As a result show, a sporoderm-broken rate of complex enzyme III, VI, Ⅸ does not add the control group of complex enzyme to be respectively increased
34.5%th, 18.6% and 23.9%；Secondary sporoderm-broken rate does not add the control group of complex enzyme to be respectively increased 21.2%, 7.3% and 15.3%.
Embodiment 15
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 50%；
（2）In the ratio that the addition of complex enzyme I is 0.3%, complex enzyme I is added in the brewer's yeast suspension, is stirred
Mix uniform, in high-pressure homogeneous disintegrating machine broken wall is carried out, under conditions of 20 DEG C, broken wall pressure and broken wall number of times are to broken for outlet temperature
Fig. 2 is shown in the impact of wall rate.
As a result show, 1500bar, one time sporoderm-broken rate reaches more than 80%, and secondary sporoderm-broken rate reaches more than 95%.
Embodiment 16
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast 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%, by complex enzyme I
Pressure, outlet temperature in being added to brewer's yeast suspension, after stirring, in high-pressure homogeneous disintegrating machine, in 1500bar
Under conditions of 20 DEG C, broken wall is carried out, Fig. 3 is shown in the impact of enzyme concentration and broken wall number of times to sporoderm-broken rate.
With the increase of enzyme concentration, sporoderm-broken rate has significant change, and at 0.3%, one time sporoderm-broken rate is the consumption of complex enzyme I
80%, secondary sporoderm-broken rate reaches 95%；At 0.4%, one time sporoderm-broken rate is 85% to the consumption of complex enzyme I, and secondary sporoderm-broken rate reaches 97%；
At 0.5%, one time sporoderm-broken rate is 85% to the consumption of complex enzyme I, and secondary sporoderm-broken rate reaches 95%.
Embodiment 17
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry is mixed with water and is respectively prepared mass concentration for 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60% brewer's yeast suspension；
（2）In the ratio that the addition of complex enzyme I is 0.3%, complex enzyme I is added separately to into the brewer's yeast suspension
In, stir, in high-pressure homogeneous disintegrating machine, broken wall, beer yeast slurry concentration and broken wall number of times pair are carried out under 1500bar
Fig. 4 is shown in the impact of sporoderm-broken rate.
As a result show, between 10%～55%, one time sporoderm-broken rate reaches more than 70% to beer yeast slurry mass concentration；Beer
Between 30%～50%, secondary sporoderm-broken rate reaches more than 95% to brewer yeast mud concentration.
Embodiment 18
Complex enzyme I is processed using high-pressure homogeneous disintegrating machine, outlet temperature control at 20 DEG C, the enzyme activity determination of protease
Using folin's methods, cellulase adopts filter paper enzyme activity assay method.Fig. 5 is shown in impact of the processing pressure to enzyme activity.
Single treatment process, under 1100bar, enzyme activity loss starts substantially to increase, the great increasing of loss late to after 1600bar
Plus, loss speed is very fast.But after after-treatment the loss late of enzyme activity is affected not in 1300～1600bar processing pressures
Greatly, it is higher using the pressure treatment enzyme activity loss more than 1600bar.Therefore 1400～1600bar processing pressures are adopted.
Embodiment 19
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 50%；Treating capacity is 2L；
（2）In the ratio that the addition of complex enzyme I is 0.3%, complex enzyme I is added in the beer yeast slurry suspension,
Pressure, outlet temperature in high-pressure homogeneous disintegrating machine (treating capacity 10L/h) in 1500bar is carried out under conditions of 15 DEG C
Matter broken wall 2 times；The micro- detection sporoderm-broken rate of menses ball count plate reaches 95.9%；Broken time is completed for 35min.
Embodiment 20
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 50%；Treating capacity is 2L；
（2）In the ratio that the addition of complex enzyme IV is 0.3%, complex enzyme IV is added to into the beer yeast slurry suspension
In, the pressure, outlet temperature in high-pressure homogeneous disintegrating machine (treating capacity 10L/h) in 1500bar is carried out under conditions of 15 DEG C
Homogeneous broken wall 2 times；The micro- detection sporoderm-broken rate of menses ball count plate reaches 84.5%；Broken time is completed for 35min.
The present embodiment complex enzyme IV is substituted with complex enzyme V, other same the present embodiment, the micro- detection of menses ball count plate is broken
Wall rate reaches 85.5%；Broken time is completed for 35min.
The present embodiment complex enzyme IV is substituted with complex enzyme VI, other same the present embodiment, the micro- detection of menses ball count plate is broken
Wall rate reaches 84.9%；Broken time is completed for 35min.
Embodiment 21
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 30%；Treating capacity is 2L；
（2）In the ratio that the addition of complex enzyme VII is 0.3%, complex enzyme VII is added to into the beer yeast slurry suspension
In, the pressure, outlet temperature in high-pressure homogeneous disintegrating machine (treating capacity 10L/h) in 1400bar is carried out under conditions of 25 DEG C
Homogeneous broken wall 2 times；The micro- detection sporoderm-broken rate of menses ball count plate reaches 89.3%；Broken time is completed for 30min.
The present embodiment complex enzyme VII is substituted with complex enzyme VIII, other same the present embodiment, the micro- detection of menses ball count plate is broken
Wall rate reaches 88.1%；Broken time is completed for 30min.
The present embodiment complex enzyme VII is substituted with complex enzyme Ⅸ, other same the present embodiment, the micro- detection of menses ball count plate is broken
Wall rate reaches 88.6%；Broken time is completed for 30min.
Embodiment 22
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 50%；Treating capacity is 2L；
（2）In the ratio that the addition of complex enzyme I is 0.5%, complex enzyme I is added in the beer yeast slurry suspension,
Pressure, outlet temperature in high-pressure homogeneous disintegrating machine (treating capacity 10L/h) in 1500bar is carried out under conditions of 25 DEG C
Matter broken wall 2 times；The micro- detection sporoderm-broken rate of menses ball count plate reaches 97.7%；Broken time is completed for 35min.
The present embodiment complex enzyme I, other same the present embodiment, the micro- detection broken wall of menses ball count plate are substituted with complex enzyme IV
Rate reaches 86.4%；Broken time is completed for 35min.
The present embodiment complex enzyme I, other same the present embodiment, the micro- detection broken wall of menses ball count plate are substituted with complex enzyme VII
Rate reaches 91.2%；Broken time is completed for 35min.
Embodiment 23
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 30%；Treating capacity is 2L；
（2）In the ratio that the addition of complex enzyme I is 0.3%, complex enzyme I is added in the brewer's yeast suspension,
In high-pressure homogeneous disintegrating machine (treating capacity 10L/h) 1500bar pressure, outlet temperature under conditions of 15 DEG C, carry out homogeneous
Broken wall 2 times；The micro- detection sporoderm-broken rate of menses ball count plate reaches 95.12%；Broken time is completed for 30min.
The present embodiment complex enzyme I, other same the present embodiment, the micro- detection broken wall of menses ball count plate are substituted with complex enzyme IV
Rate reaches 85.8%；Broken time is completed for 30min.
The present embodiment complex enzyme I, other same the present embodiment, the micro- detection broken wall of menses ball count plate are substituted with complex enzyme VII
Rate reaches 90.3%；Broken time is completed for 30min.
Embodiment 24
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 30%；Treating capacity is 2L；
（2）In the ratio that the addition of complex enzyme I is 0.5%, complex enzyme I is added in the brewer's yeast suspension,
In high-pressure homogeneous disintegrating machine (treating capacity 10L/h) 1500bar pressure, outlet temperature under conditions of 20 DEG C, carry out homogeneous
Broken wall 2 times；The micro- detection sporoderm-broken rate of menses ball count plate reaches 97.23%；Broken time is completed for 30min.
The present embodiment complex enzyme I, other same the present embodiment, the micro- detection broken wall of menses ball count plate are substituted with complex enzyme IV
Rate reaches 86.1%；Broken time is completed for 30min.
The present embodiment complex enzyme I, other same the present embodiment, the micro- detection broken wall of menses ball count plate are substituted with complex enzyme VII
Rate reaches 90.6%；Broken time is completed for 30min.
Embodiment 25
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 50%；Treating capacity is 2L；
（2）In the ratio that Complex Ⅱ addition is 0.3%, Complex Ⅱ is added in the brewer's yeast suspension,
Pressure, outlet temperature in high-pressure homogeneous disintegrating machine (treating capacity 10L/h) in 1500bar is carried out under conditions of 20 DEG C
Matter broken wall 2 times；The micro- detection sporoderm-broken rate of menses ball count plate reaches 95.8%；Broken time is completed for 35min.
Cellulase selected by various embodiments of the present invention is acid fiber enzyme, and with neutral cellulase the present embodiment is substituted
Cellulase in middle Complex Ⅱ, other same the present embodiment, the micro- detection sporoderm-broken rate of menses ball count plate reaches 95.12%；It is complete
It is 35min into broken time.
Embodiment 26
The beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, comprises the steps：
（1）Beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 50%；Treating capacity is 2L；
（2）In the ratio that the addition of complex enzyme III is 0.3%, complex enzyme III is added in the brewer's yeast suspension,
Pressure, outlet temperature in high-pressure homogeneous disintegrating machine (treating capacity 10L/h) in 1500bar is carried out under conditions of 15 DEG C
Matter broken wall 2 times；The micro- detection sporoderm-broken rate of menses ball count plate reaches 96.2%；Broken time is completed for 35min.
Protease selected by various embodiments of the present invention is acid protease, is answered with neutral protein enzymes extraction the present embodiment
Protease in synthase III, other same the present embodiment, the micro- detection sporoderm-broken rate of menses ball count plate reaches 95.26%；Complete broken wall
Time is 35min.
Proteinase activity is defined as：Under uniform temperature and pH value condition, 1min caseinhydrolysates produce 1 μ g tyrosine,
As 1 enzyme activity unit.Assay method is according to folin's methods in the protease preparations of GB/T 23527.
Cellulase activity is defined as：At 50 DEG C, it is intended that (acidic cellulase pH 4.8, neutral cellulase under the conditions of pH
PH 6.0), lh hydrolysis filter paper substrates produce the reduction sugar amount equivalent to lmg glucose, are 1 enzyme activity unit.Measure side
Method is according to filter paper enzyme activity assay method in the cellulase preparations of QB 2583.
Mannosan enzyme activity is defined as：Refer at the standard conditions（5.3,50 DEG C of pH）, hydrolysis substrate 1 μ of generation per minute
The amount of enzyme needed for g mannoses is defined as a mannosan active unit（U）Assay method adopts DNS methods.
Chitinase enzyme activity is defined as：Refer at the standard conditions（6.0,45 DEG C of pH）Decompose tobacco brown spot pathogen per hour
The enzyme amount of 1 μ g 2-Acetamido-2-deoxy-D-glucoses is produced, is 1 enzyme activity unit (U).And assay method reference Haitao Zhang, Wang Ting,
Deng. chitinase producing strains Screening and Identification and producing enzyme performance study. Chinese biological engineering magazine, 2010,30（8）:82-87.
Claims (9)
1. the beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, is characterized in that comprising the steps：
(1) beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 30%～50%；
(2) it is the ratio of 0.3%-0.5% in complex enzyme addition, complex enzyme is added in the brewer's yeast suspension,
Pressure, outlet temperature in high-pressure homogeneous disintegrating machine in 1400-1600bar carries out homogeneous broken wall under conditions of 15-25 DEG C
2-3 time；The complex enzyme includes by weight：1.5～5 parts of protease, 0.5～2 part of cellulase.
2. the beer yeast cells wall-breaking method that a kind of enzyme according to claim 1 is cooperateed with mechanical breaking-wall method, is characterized in that
The enzyme activity of the protease is 86000～180000U/mL；The enzyme activity of the cellulase is 80～550U/mL.
3. the beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, is characterized in that comprising the steps：
(1) beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 30%～50%；
(2) it is the ratio of 0.3%-0.5% in complex enzyme addition, complex enzyme is added in the brewer's yeast suspension,
Pressure, outlet temperature in high-pressure homogeneous disintegrating machine in 1400-1600bar carries out homogeneous broken wall under conditions of 15-25 DEG C
2-3 time；The complex enzyme includes by weight：1.5～5 parts of protease, 0.5～2 part of cellulase, chitinase 0.5～1
Part.
4. the beer yeast cells wall-breaking method that a kind of enzyme according to claim 3 is cooperateed with mechanical breaking-wall method, is characterized in that
The enzyme activity of the protease is 50000～155000U/mL；The enzyme activity of the cellulase be 80～400U/mL, chitinase
Enzyme activity be 70～170U/mL.
5. the beer yeast cells wall-breaking method that a kind of enzyme is cooperateed with mechanical breaking-wall method, is characterized in that comprising the steps：
(1) beer yeast slurry and water are mixed and made into into the brewer's yeast suspension that mass concentration is 30%～50%；
(2) it is the ratio of 0.3%-0.5% in complex enzyme addition, complex enzyme is added in the brewer's yeast suspension,
Pressure, outlet temperature in high-pressure homogeneous disintegrating machine in 1400-1600bar carries out homogeneous broken wall under conditions of 15-25 DEG C
2-3 time；The complex enzyme includes by weight：1.5～5 parts of protease, 0.5～2 part of cellulase, chitinase 0.5～1
Part, 0.3～2 part of mannase.
6. the beer yeast cells wall-breaking method that a kind of enzyme according to claim 5 is cooperateed with mechanical breaking-wall method, is characterized in that
The protease is 5 parts, and the cellulase is 2 parts, and the chitinase is 0.5 part, and the mannase is 0.3 part.
7. the beer yeast cells wall-breaking method that a kind of enzyme according to claim 5 or 6 is cooperateed with mechanical breaking-wall method, its feature
The enzyme activity for being the protease is 50000～100000U/mL；The enzyme activity of the cellulase be 50～400U/mL, chitin
The enzyme activity of enzyme is 30～100U/mL, and the enzyme activity of mannase is 35000～250000U/mL.
8. the beer yeast cells wall-breaking method that a kind of enzyme according to claim 7 is cooperateed with mechanical breaking-wall method, is characterized in that
The enzyme activity of the protease is 100000U/mL；The enzyme activity of the cellulase is 200U/mL, and the enzyme activity of chitinase is 30U/
ML, the enzyme activity of mannase is 35000U/mL.
9. the beer yeast cells wall-breaking method that a kind of enzyme according to claim 1,3 or 5 is cooperateed with mechanical breaking-wall method, it is special
Levy is that the pressure is 1500bar.
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| 酵母细胞破壁技术研究与应用进展;杨翠竹等;《食品科技》;20060731(第07期);全文 * |
| 酶法破碎微生物细胞的研究进展;崔丁维等;《微生物学通报》;20101120;第37卷(第11期);全文 * |
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