CN109182131A - Plant cell wall wall-breaking method based on enzyme bacterium combination technology - Google Patents
Plant cell wall wall-breaking method based on enzyme bacterium combination technology Download PDFInfo
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Abstract
The present invention provides a kind of plant cell wall wall-breaking methods based on enzyme bacterium combination technology, a variety of enzyme viabilities can be constantly generated using fungi, in conjunction with existing enzyme, wall processing is carried out brokenly to the cell wall of plant cell at normal temperatures and pressures, so that intracellular substance can sufficiently discharge, and the activity for capableing of effective guarantee intraor extracellular substance is not damaged with structure, the method that it is used is as follows: Step1, take pectase X-Y%, amylase X-Y%, mannase X-Y%, cellulase X-Y%, laccase X-Y%, protease X-Y%, saccharomycete X-Y%, aspergillus X-Y% and the water of surplus are added to the container mixing；Step2, plant haulm and/or blade are added into container；Step3, mixture in container is stirred or is vibrated, speed is 100-120 revs/min, and control container internal temperature is 40-45 DEG C.
Description
Technical field
The present invention relates to plant cell breakage field, specially a kind of plant cell wall based on enzyme bacterium combination technology
Wall-breaking method.
Background technique
It is so-called to cell wall broken wall, i.e., the process broken the cell wall of cell using physics or chemical method is passed through
It is extracellular that the process can be such that intracellular effective nutritional ingredient is released, can be more efficient to this convenient for other bodies
The absorption of part nutriment.
The cell wall of plant cell is divided into 3 layers, i.e. intercellular layer (middle layer), primary wall and secondary wall.Intercellular layer is adjacent thin
Born of the same parents stick together to form tissue.In intercellular layer two sides, the main composition of cell wall is primary wall: cellulose, hemicellulose,
Lignin and pectic substance composition, cell wall breaking technology is to utilize enzymatic isolation method, chemical method, physical method or several sides at present
Method is used cooperatively.These methods have different disadvantages, such as the shortcomings that enzymatic isolation method: 1, the reaction of various enzymes more using the type of enzyme
Condition is inconsistent；2, in the use process of enzyme, enzyme is constantly consumed, supplement that enzyme and supplement enzyme amount it is uncertain, may not have
When reaching experiment purpose, enzyme has been run out of；3, it is excessively high using a large amount of enzyme cost, it is unfavorable for industrialized production.Chemical method
Disadvantage: 1, chemical method uses acid or alkali to have huge shadow to intracellular other compositions although cell wall can be destroyed more
It rings；2, using acid or alkali, it is unfavorable for the extraction of needed nutrient matter；The shortcomings that physical method, has: 1, either with high temperature or
Person's low temperature wall-breaking method all can greatly reduce the activity of the active substance of intraor extracellular, the especially protein substances such as enzyme, no
It can avoid that denaturation occurs and structure change；2, although mechanical wall-breaking method can be not enough with broken wall, while mechanical broken
High temperature may be generated when wall, will affect the activity of intraor extracellular active material；
So, it is believed that, breaking-wall cell must be carried out under normal temperature and pressure normality, as far as possible holding intraor extracellular substance
Activity, maximum possible improve cell effective component bioavilability.
Summary of the invention
The present invention provides the methods that the cell wall of a kind of pair of plant cell carries out broken wall, can be constant using fungi
A variety of enzyme viabilities are generated, in conjunction with existing enzyme, wall processing is carried out brokenly to the cell wall of plant cell at normal temperatures and pressures, so that
Intracellular substance can sufficiently discharge, and the activity for capableing of effective guarantee intraor extracellular substance is not damaged with structure.
The method that it is used is as follows:
Step1, pectase 1%-5%, amylase 1%-5%, mannase 1%-5%, cellulose are taken in mass ratio
Enzyme 1%-5%, laccase 1%-5%, protease 1%-5%, saccharomycete 5%-10%, aspergillus 5%-10% and surplus water be added
It is mixed in container, while controlling vessel temp is 40-50 DEG C；
Step2, plant haulm, root tuber, fruit and/or blade are added into container；
Step3, mixture in container is stirred or is vibrated, speed is 100-120 revs/min, is controlled inside container
Temperature is 40-45 DEG C；
The technical effect that the above method can be generated is as follows:
1, by the way of enzyme bacterium structure, the enzyme amount that phase fungal metabolite generates before the reaction is less, reaction can not be opened largely
In the case where dynamic, degradation reaction is carried out using cell wall of the enzyme being added early period to plant cell, so as to make rapid reaction
Starting improves reaction efficiency.
2, the growth course of fungi is the release process of purpose enzyme, therefore the case where the enzyme that early period is added constantly consumes
Under, the purpose enzyme of fungi release can be such that the enzyme in container is endlessly supplemented, to make entire reaction process that can hold
The continuous time is longer, therefore does not need to be supplemented enzyme amount in reaction process, and enough enzymes have been effectively ensured to the drop of plant cell wall
Solution reaction.
3, enzyme used in the above method is consistent with the growth conditions of fungi, can reach to thin at normal temperatures and pressures
The purpose of cell wall breakage, the effective guarantee activity and structural intergrity of intraor extracellular ingredient.
4, the procurement cost of fungi is far below the production cost of enzyme, therefore the technology combined using enzyme bacterium, can effectively drop
Low production cost.
Detailed description of the invention
Fig. 1 is dark green tea histiocyte of tea leaf using the schematic diagram before the processing of the cell wall degradation method of the present embodiment.
Fig. 2 is schematic diagram of the dark green tea histiocyte of tea leaf after cell wall degradation method processing through this embodiment.
The profile of cell is relatively clear in Fig. 1, illustrates that cell wall breakage rate is not high, cell is more complete；And it can be seen in Fig. 2
Out, the soft edge of cell, cell show fluffy, then illustrate that the breakage rate of cell wall is higher, lead to a large amount of cell contents
Object is released in cell liquid.
Specific embodiment
Following embodiment is described in further details the present invention by taking the pharmacological for improving dark green tea as an example.
Dark green tea is unique microbial fermentation tea among big tea system of China six.In recent years, dark green tea is in reducing blood lipid, blood glucose, blood
Pressure and anti-oxidant etc. effect increasingly obtain the approval of people, about the exploration of wherein active material and grinding for the mechanism of action
Studying carefully just becomes new hot spot.
Dark green tea exist into the cell largely can reduce human blood lipid, blood glucose, blood pressure substance, while further including a large amount of human bodies
Required nutriment, so needing the substance as much as possible for keeping dark green tea intracellular to obtain to improve the pharmacological of dark green tea
The step of release, most critical is to break the cell wall of dark green tea.
In the prior art, the typical process flow of dark green tea is to finish, just rub, pile fermentation, rub, bake again, passes through these sides
Method can play certain effect to the breakage of dark green tea cell wall, be released intracellular matter, but as above-mentioned background
It is described in technology, using high temperature or low temperature wall-breaking method, the activity of the active substance of intraor extracellular can be greatly reduced, especially
The protein substances such as enzyme, it is inevitable that denaturation and structure change occurs；Although mechanical wall-breaking method can be with broken wall, not
It is enough abundant, while high temperature may also can be generated when mechanical breaking-wall method, it will affect the activity of intraor extracellular active material.
It is to be degraded using enzymatic isolation method to cell wall merely there are also a kind of mode, to realize the mesh of cell wall breakage
, but since the ingredient of cell wall is more, need the type of enzyme more, and the reaction condition of various enzymes is inconsistent, it is unfavorable
In large-scale production；And enzyme is constantly consuming, and the amount of consumption can not detect again, so can not carry out to the enzyme of consumption accurate
Supplement, possible reaction, which is not over enzyme and has just consumed, to be over, effect needed for production is not achieved.
For these reasons, the technology that following embodiment uses enzyme bacterium to combine, can be produced in the metabolic process using strain
Raw corresponding enzyme viability carries out the purpose enzyme in reaction vessel to continue supplement, to make to the cell wall degradation of dark green tea
Process can obtain effectively lasting.
In the examples below, made using pectase, amylase, mannonase cellulase, laccase, protease etc.
It degrades for purpose enzyme to dark green tea cell wall collective effect, can continue to generate lignin in saccharomycete and aspergillus metabolic process
The purpose of degrading enzyme, hemicellulase, cellulase enzyme, for participating in degradation to cell wall and being carried out to consumed enzyme
Supplement.
For the shell-broken effect of cell wall, the extracellular starch of detection, reduced sugar, crude protein, small peptide, thick fibre are generallyd use
The content and pH value of dimension are judged.
Embodiment one
Pectase 1.1%, amylase 1.3%, mannase 1.0%, cellulase 3.2%, laccase are chosen first
3.2%, the water of protease 2.0%, saccharomycete 6.8%, aspergillus 6.8% and surplus is added to the container mixing, while to infuse
The temperature of meaning control container enzymatic mixture is at 40 DEG C；Then dark green tea blade is added into container, then mixture in container is carried out
Stirring or oscillation, speed are 100 revs/min, and control container internal temperature is 40 DEG C.Make the cell wall of dark green tea blade in the work of enzyme
It gradually degrades under, to keep cell wall damaged, cellular content is released.
After a period of time, starch, reduced sugar, crude protein in detection container, small peptide, coarse-fibred content, it is especially desirable to
Starch, reduced sugar and coarse-fibred content are detected, concrete content such as following table, following data is with the calculating of 10% moisture.
| Starch/% | Reduced sugar/% | Crude protein/% | Small peptide/% | Crude fibre/% | pH | |
| Before enzymatic hydrolysis | 11.80 | 7.63 | 24.91 | 24.09 | 27.05 | 5.76 |
| After enzymatic hydrolysis | 2.71 | 16.08 | 25.69 | 28.84 | 16.75 | 5.58 |
It can be seen that starch by upper table information and the case where content is greatly reduced all occur after enzymatic hydrolysis in crude fibre,
And the amount of reduced sugar is then significantly increased, and illustrates that starch is gradually being converted into reduced sugar, and crude fibre is gradually degraded as fine fibre.It forms sediment
Powder and crude fibre are all the important components of plant cell wall, and content reduction then illustrates that plant cell wall has been degraded, i.e.,
It realizes and the broken wall of dark green tea blade cell wall is acted on.
Embodiment two
Choose pectase 1.8%, amylase 2 .0%, mannase 1.3%, cellulase 2.0%, laccase 2.0%,
Protease 1.5%, saccharomycete 7.0%, aspergillus 8.0% and surplus water be added to the container mixing；Simultaneously it is noted that control
The temperature of container enzymatic mixture is at 45 DEG C；Then into container be added dark green tea blade, then mixture in container is stirred or
Oscillation, speed are 100 revs/min, and control container internal temperature is 45 DEG C.Make the cell wall of dark green tea blade under the action of enzyme by
It gradually degrades, to keep cell wall damaged, cellular content is released.
After a period of time, starch, reduced sugar, crude protein in detection container, small peptide, coarse-fibred content, it is especially desirable to
Starch, reduced sugar and coarse-fibred content are detected, concrete content such as following table, following data is with the calculating of 10% moisture.
| Starch/% | Reduced sugar/% | Crude protein/% | Small peptide/% | Crude fibre/% | pH | |
| Before enzymatic hydrolysis | 15.61 | 6.81 | 26.33 | 26.12 | 31.54 | 5.68 |
| After enzymatic hydrolysis | 3.92 | 17.09 | 27.18 | 29.52 | 20.82 | 5.54 |
It can be seen that starch by upper table information and the case where content is greatly reduced all occur after enzymatic hydrolysis in crude fibre,
And the amount of reduced sugar is then significantly increased, and illustrates that starch is gradually being converted into reduced sugar, and crude fibre is gradually degraded as fine fibre.It forms sediment
Powder and crude fibre are all the important components of plant cell wall, and content reduction then illustrates that plant cell wall has been degraded, i.e.,
It realizes and the broken wall of dark green tea blade cell wall is acted on.
Embodiment three
Choose pectase 3.0%, amylase 3.2%, mannase 2.5%, cellulase 1.4%, laccase 1.2%,
Protease 1.1%, saccharomycete 9.0%, aspergillus 7.5% and surplus water be added to the container mixing；Simultaneously it is noted that control
The temperature of container enzymatic mixture is at 50 DEG C；Then into container be added dark green tea blade, then mixture in container is stirred or
Oscillation, speed are 100 revs/min, and control container internal temperature is 50 DEG C.Make the cell wall of dark green tea blade under the action of enzyme by
It gradually degrades, to keep cell wall damaged, cellular content is released.
After a period of time, starch, reduced sugar, crude protein in detection container, small peptide, coarse-fibred content, it is especially desirable to
Starch, reduced sugar and coarse-fibred content are detected, concrete content such as following table, following data is with the calculating of 10% moisture.
| Starch/% | Reduced sugar/% | Crude protein/% | Small peptide/% | Crude fibre/% | pH | |
| Before enzymatic hydrolysis | 13.20 | 7.96 | 25.01 | 26.12 | 30.36 | 5.71 |
| After enzymatic hydrolysis | 2.99 | 16.93 | 26.28 | 28.84 | 18.12 | 5.60 |
It can be seen that starch by upper table information and the case where content is greatly reduced all occur after enzymatic hydrolysis in crude fibre,
And the amount of reduced sugar is then significantly increased, and illustrates that starch is gradually being converted into reduced sugar, and crude fibre is gradually degraded as fine fibre.It forms sediment
Powder and crude fibre are all the important components of plant cell wall, and content reduction then illustrates that plant cell wall has been degraded, i.e.,
It realizes and the broken wall of dark green tea blade cell wall is acted on.
A dark green tea prepared to the method for embodiment three through the foregoing embodiment, then it is dry by being formed after the processes such as drying
Dry tealeaves, then brewed and drunk with water, since cell wall has obtained breakage, cellular content is largely discharged into cell
Outside, so drinking again after brewing at this time, human body can be made to obtain more from intracellular nutriment, to make dark green tea
Pharmacological be significantly improved.
Example IV
Comparative example of the present embodiment as embodiment three, it is in the present embodiment, thin to dark green tea only with enzymatic isolation method
Cell wall carries out degradation treatment, chooses pectase 3.0%, amylase 3.2%, mannase 2.5%, cellulase 1.4%, paint
The water of enzyme 1.2%, protease 1.1% and surplus is added to the container mixing；Simultaneously it is noted that controlling container enzymatic mixture
Temperature is at 50 DEG C；Then dark green tea blade is added into container, then mixture in container is stirred or is vibrated, speed 100
Rev/min, control container internal temperature is 50 DEG C.The cell wall of dark green tea blade is set gradually to degrade under the action of enzyme, to make
Cell wall is damaged, and cellular content is released.
After a period of time, starch, reduced sugar, crude protein in detection container, small peptide, coarse-fibred content, it is especially desirable to
Starch, reduced sugar and coarse-fibred content are detected, concrete content such as following table, following data is with the calculating of 10% moisture.
| Starch/% | Reduced sugar/% | Crude protein/% | Small peptide/% | Crude fibre/% | pH | |
| Before enzymatic hydrolysis | 12.86 | 8.35 | 22.18 | 25.33 | 31.11 | 5.69 |
| After enzymatic hydrolysis | 10.78 | 10.14 | 24.35 | 27.20 | 27.56 | 5.57 |
By embodiment three and example IV comparison as can be seen that in example IV, starch, reduced sugar and crude fibre are in enzyme
Content before Xie Houyu enzymatic hydrolysis is all not much different, and illustrates that the amount that Starch Conversion is reduced sugar is little, crude fibre does not also drop largely
Solution is fine fibre.Starch and crude fibre are all the important components of plant cell wall, digest the changes of contents of front and back less then
The degree for illustrating that plant cell wall is degraded is not high, i.e., little to the broken wall function and effect of dark green tea blade cell wall.
And found during the present embodiment is implemented, after reaction carries out a period of time, detect above-mentioned in container
Constituent content gradually tends to balance, and is no longer changed, and illustrates that the enzyme digestion reaction in container at this time is over, enzyme amount is complete
It totally disappeared consumption.And be then not in the phenomenon that such reaction stops in embodiment one to three, because fungi in the metabolic process can
Enough continual generation purpose enzymes extend the reaction time, so can be to thin so that purpose enzyme be enable effectively to be supplemented
The degradation of cell wall is more sufficiently thoroughly.
Embodiment five
The present embodiment selects radix tetrastigme to be tested, and radix tetrastigme is the distinctive valuable ingredient of traditional Chinese medicine of China, root tuber, fruit and complete
Grass can be used as medicine, and be distributed mainly on the province such as Zhejiang Province, China, Anhui, Fujian, have clearing lung-heat clearing heat and detoxicating, cool in nature, anti-cancer protect liver etc.
Multiple efficacies are preferred especially with the produced radix tetrastigme drug effect in Zhejiang, civil always as paediatrics key medicine simply, are had very high medicinal
Value and economic value, radix tetrastigme are also known as " civil god's medicine ".
The general concocting method of Chinese medicine is broadly divided into: being impregnated, boiling, sand is fried, fried, quenching.There are some Chinese medicines also to add
Enter some auxiliary materials to be processed, such as use vinegar, with honey, impregnates frying with boys' urine etc. with alum with ginger juice.These above-mentioned processings
The main purpose of method is released the content of crude drug cell is as much as possible, is improved medicinal material and is imitated to the treatment of disease
Fruit.
But as described in background technique, there are many defects for these above-mentioned concocting methods, lead to the pharmacological of medicinal material
It is not high.The present embodiment carries out pre-processing to radix tetrastigme using enzyme bacterium combination technology, improves radix tetrastigme root tuber, fruit and herb
The breakage rate of cell wall improves the pharmacological of radix tetrastigme with this so that intracellular active principle be made to be released.
Choose pectase 2.1%, amylase 2 .8%, mannase 2.1%, cellulase 1.8%, laccase 1.5%,
Protease 2.0%, saccharomycete 7.6%, aspergillus 7.5% and surplus water be added to the container mixing；Simultaneously it is noted that control
The temperature of container enzymatic mixture is at 45 DEG C；Then root tuber, fruit and the herb of radix tetrastigme are added into container, then is mixed in container
It closes object to be stirred or vibrate, speed is 100 revs/min, and control container internal temperature is 45 DEG C.Make the cell wall of dark green tea blade
It gradually degrades under the action of enzyme, to keep cell wall damaged, cellular content is released.
After a period of time, starch, reduced sugar, crude protein in detection container, small peptide, coarse-fibred content, it is especially desirable to
Starch, reduced sugar and coarse-fibred content are detected, concrete content such as following table, following data is with the calculating of 10% moisture.
| Starch/% | Reduced sugar/% | Crude protein/% | Small peptide/% | Crude fibre/% | pH | |
| Before enzymatic hydrolysis | 31.20 | 20.51 | 65.30 | 66.07 | 70.36 | 5.73 |
| After enzymatic hydrolysis | 6.33 | 43.59 | 67.12 | 67.93 | 47.16 | 5.52 |
It can be seen that starch by upper table information and the case where content is greatly reduced all occur after enzymatic hydrolysis in crude fibre,
And the amount of reduced sugar is then significantly increased, and illustrates that starch is gradually being converted into reduced sugar, and crude fibre is gradually degraded as fine fibre.It forms sediment
Powder and crude fibre are all the important components of plant cell wall, and content reduction then illustrates that plant cell wall has been degraded, i.e.,
It realizes and the broken wall of dark green tea blade cell wall is acted on.
The radix tetrastigme medicinal material prepared by the above method, then the finished product dry by formation after the processes such as drying, then lead to
Medicinal material processing process is crossed, since cell wall has obtained breakage, cellular content has largely been discharged into extracellularly, so through big gun
It is drunk again after system, human body can be made to obtain more from intracellular nutriment, so that the pharmacological of clover be made to obtain
To significantly improving.
Embodiment six
The present embodiment is tested by taking corn stover as an example, and corn stover is that the grain, economy and feeding being made for based on feed are also used as
Object, corn stover are also the important resources of production of work, agricultural production.As one kind, corn stover nutrition rich in and can
The chemical component utilized can be used as the raw material of livestock feed.The substance release for keeping corn stover intracellular effectively improves
The nutritional ingredient of livestock feed.
Choose pectase 2.8%, amylase 3.0%, mannase 2.8%, cellulase 1.6%, laccase 1.0%,
Protease 1.6%, saccharomycete 8.7%, aspergillus 6.9% and surplus water be added to the container mixing；Simultaneously it is noted that control
The temperature of container enzymatic mixture is at 50 DEG C；Then corn stover fragment is added into container, then mixture in container is stirred
It mixes or vibrates, speed is 100 revs/min, and control container internal temperature is 50 DEG C.Make the cell wall of corn stover fragment in enzyme
It gradually degrades under effect, to keep cell wall damaged, cellular content is released.
After a period of time, starch, reduced sugar, crude protein in detection container, small peptide, coarse-fibred content, it is especially desirable to
Starch, reduced sugar and coarse-fibred content are detected, concrete content such as following table, following data is with the calculating of 10% moisture.
| Starch/% | Reduced sugar/% | Crude protein/% | Small peptide/% | Crude fibre/% | pH | |
| Before enzymatic hydrolysis | 21.20 | 15.06 | 33.13 | 34.22 | 38.66 | 5.70 |
| After enzymatic hydrolysis | 9.89 | 24.13 | 34.71 | 36.01 | 26.02 | 5.63 |
It can be seen that starch by upper table information and the case where content is greatly reduced all occur after enzymatic hydrolysis in crude fibre,
And the amount of reduced sugar is then significantly increased, and illustrates that starch is gradually being converted into reduced sugar, and crude fibre is gradually degraded as fine fibre.It forms sediment
Powder and crude fibre are all the important components of plant cell wall, and content reduction then illustrates that plant cell wall has been degraded, i.e.,
It realizes and the broken wall of corn stover cell wall is acted on.
The corn stover prepared through the foregoing embodiment, then formed after processes by drying etc. dry and secondary operation at
Feed, since cell wall has obtained breakage, cellular content has largely been discharged into extracellularly, so straw feed can make
Livestock obtains more from intracellular nutriment, so that the nutritive value of corn stover be made to be significantly improved.
Data based on the above embodiments can be seen that the either blade of dark green tea, corn stover or the block of radix tetrastigme
Root, fruit and herb, using enzyme bacterium combination technology to the shell-broken effect of cell wall compared to enzymatic hydrolysis before, starch and coarse-fibred
Content can all substantially reduce, and the content of reduced sugar can then greatly increase, and illustrate that the damaged effect of cell wall is good.
And from embodiment one to four as can be seen that being broken using enzyme bacterium combination technology of the present invention to plant cell wall
Wall effect will be much better than simple enzymatic isolation method to the shell-broken effect of plant cell wall.
Therefore effect of the enzyme bacterium combination technology of the present invention in terms of the pharmacological for improving Chinese medicine is very prominent
, main reason is that enzyme bacterium combination technology can increase substantially the breakage rate of plant cell wall, so that the content of cell
Object as much as possible can be released effectively, and have in Chinese medicine medicative substance exactly released it is intracellular
It is tolerant.Therefore improving Chinese medicine cell wall breakage rate is where improving the key point of Chinese medicine pharmacological.
These are only the preferred embodiment of the present invention, is not intended to restrict the invention, for those skilled in the art
For member, the invention may be variously modified and varied.All within the spirits and principles of the present invention, it is made it is any modification,
Equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (2)
1. the plant cell wall wall-breaking method based on enzyme bacterium combination technology, it is characterised in that: the enzyme includes pectase, starch
Enzyme, mannonase cellulase, laccase and protease, the bacterium bag include saccharomycete and aspergillus, and method and step is as follows:
Step1, pectase 1%-5%, amylase 1%-5%, mannase 1%-5%, cellulase are taken in mass ratio
1%-5%, laccase 1%-5%, protease 1%-5%, saccharomycete 5%-10%, aspergillus 5%-10% and surplus water be added and hold
It is mixed in device, while controlling vessel temp is 40-50 DEG C；
Step2, plant haulm, root tuber, fruit and/or blade are added into container；
Step3, mixture in container is stirred or is vibrated, speed is 100-120 revs/min, controls container internal temperature
It is 40-45 DEG C.
2. plant cell wall breaking method according to claim 1, it is characterised in that: in Step2, be added plant haulm it
Before, first plant haulm or blade are crushed and form chunky shape.
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| CN110195018A (en) * | 2019-06-21 | 2019-09-03 | 寿碧霞 | A method of utilizing more kinds of enzyme enzymatic hydrolysis animals and plants of N+1 and microorganism wall |
| CN110664909A (en) * | 2019-10-29 | 2020-01-10 | 刘本德 | Wall-breaking extraction method of Chinese herbal medicine |
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Application publication date: 20190111 |