CN115633718A - Method for improving plant milk enzymolysis efficiency through low-consumption physical double-field cooperation - Google Patents
Method for improving plant milk enzymolysis efficiency through low-consumption physical double-field cooperation Download PDFInfo
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention discloses a method for improving the enzymolysis efficiency of plant milk by low-consumption physical double-field synergy, which comprises the steps of dissolving enzyme in water, stirring in an ice bath to obtain an enzyme solution; carrying out low-temperature plasma treatment on the enzyme liquid, then carrying out soaking and water filtration on flaxseeds, grinding the flaxseeds by a colloid mill to obtain flaxseed homogenate, carrying out pretreatment on the flaxseed homogenate by using a pulsed electric field, adding the enzyme liquid after CP treatment into flaxseed plant milk after PEF treatment for enzymolysis, carrying out enzyme deactivation and filtration to obtain the flaxseed plant milk with high total solid content, high protein content and total phenol content and moderate viscosity. After the low-consumption physical double-field treatment, the edible quality of the linseed plant milk is greatly improved, the total phenol content, the protein content and the solid content are respectively improved by 66 percent, 17 percent and 12 percent, and the viscosity is reduced by 49 percent. The double-field coupling enzymolysis of the invention embodies synergistic interaction and has great application potential in the fields of green, clean labels and high-quality plant milk processing.
Description
Technical Field
The invention relates to the technical field of functional food processing, in particular to a method for improving the enzymolysis efficiency of plant milk by low-consumption physical double-field synergy.
Background
Enzymes are present in all naturally occurring life forms, including plants, animals and microorganisms. The use of enzymes in the food industry is mainly aimed at modifying the functional properties of products and/or controlling or improving food processing. The enzyme can replace a synthetic catalyst, reduces the use of toxic reagents, generates less energy consumption, is environment-friendly, and belongs to the low-carbon and low-consumption technology. At present, the global agricultural food generates 25 percent of carbon emission (125 hundred million tons), and the first place is that the national requirements of 'carbon emission reduction', 'carbon peak' for environmental protection and the improvement effect of enzyme on the functions of the product, including texture, appearance, nutritional quality, quality guarantee period and safety, are widely accepted by the food processing industry, so that the application of the enzyme in the agricultural product processing industry, particularly in the food industry, is becoming more attractive.
However, the enzyme is a special protein with catalytic activity and high specificity, and has very high substrate specificity, so that the instability of the enzymolysis process is increased due to the complex and variable processing conditions of the industrial process and the mixing of multiple substrates, so that the industrial efficiency of the enzyme is reduced, and the price of the enzyme per se is higher than that of a synthetic catalyst, which puts higher requirements on the improvement of the enzymolysis efficiency and the optimization of the enzyme hydrolysis process. The enzymolysis reaction speed/enzymolysis efficiency mainly depends on 2 factors such as mass transfer efficiency and enzyme molecule conformation. The method has the potential of improving the enzymolysis efficiency by using low-consumption, green and environment-friendly physical field treatment.
The low temperature plasma (CP) is the fourth state of matter, is a system composed of a large number of molecules, atoms, ions, electrons and other particles, can generate active particles with higher energy such as active oxygen and active nitrogen under lower energy consumption, and can keep enough high activity at lower temperature (less than 50 ℃), thereby influencing enzyme molecule conformation to different degrees and further changing enzyme catalysis efficiency. Non-heat treatment equipment and process are efficient and novel methods for improving the performance of plant milk. Pulsed Electric Field (PEF), as a low-consumption, non-thermal, novel processing technology, produces high-energy particles by inducing plant stress stimulation reactions on the one hand, and also causes structural changes in food products, for example, reversible or irreversible pores in cell membranes are formed by electric permeabilization effects, thereby promoting accumulation of nutrients including proteins and phenols in food systems, and thus can be used for plant material processing, such as extraction of plant compounds, preparation of high-quality fruit juice/plant milk and other beverages. In addition, the pretreatment of the reaction substrate by the PEF has an effect of improving the efficiency of contacting the enzyme with the substrate, and thus has the potential of promoting the improvement of the enzymatic hydrolysis efficiency. Ultrasound (US) is a non-thermal technique that has received much attention due to its unique cavitation effect and excellent properties, and studies have shown that US treatment can increase the structural flexibility of colloidal macromolecules such as proteins, polysaccharides, etc., and has a potential effect in improving the interaction between enzymes and substrates.
At present, the plasma technology is applied to a plurality of fields and plays a positive role, such as aerospace, biology, medical treatment and the like, wherein the atmospheric pressure plasma equipment is simple and convenient, has low cost and can be widely applied to the fields of food processing and the like. Meanwhile, the application range of the pulse electric field and the ultrasonic wave is wide, the energy consumption is low, the novel green processing technology is adopted, and the application of the pulse electric field and the ultrasonic wave in the food processing field is more and more extensive at home and abroad.
Therefore, providing a method for improving the plant lactolysis efficiency by low-consumption physical double-field synergy is a technical problem which needs to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of the above, the invention provides a method for improving the plant milk enzymolysis efficiency by low-consumption physical double-field synergy. The method has the characteristics of simple and convenient operation and high efficiency.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for improving the plant milk enzymolysis efficiency by low-consumption physical double-field synergy comprises the following steps:
(1) Preparing an enzyme solution: weighing enzyme, adding water for mixing, stirring in an ice bath, and uniformly mixing to obtain an enzyme solution for later use;
(2) Low-temperature plasma treatment: sucking the enzyme liquid into a clean culture dish, placing the culture dish below a nozzle of a plasma generator, treating the enzyme liquid with stable plasma, collecting the treated enzyme liquid, and sealing and storing at 2-8 ℃ for later use;
(3) Preparing linseed homogenate: mixing and soaking microwave degummed semen Lini with water, removing water, adding water into soaked semen Lini, and colloid milling for 4-12min to collect plant milk;
(4) Pretreating flaxseed homogenate by a pulsed electric field: subjecting the plant milk prepared in the step (3) to a pulse electric field to obtain a treatment solution;
(5) Enzymolysis: and (3) adding 0.1-0.5wt% of the enzyme solution prepared in the step (1) or the step (2) into the treatment solution, inactivating the enzyme after enzymolysis, and filtering to obtain the linseed plant milk with high protein content, high solid content and moderate viscosity.
Further, in the step (1), the enzyme is one or a mixture of more of cellulase, protease, phytase, hemicellulase, saccharifying enzyme and bromelain; the mass ratio of the enzyme to the water is 1-5.
The beneficial effect of adopting the further scheme is that: by adopting the scheme of the invention, the solid enzyme powder can be fully dissolved, and the method is suitable for the processing requirement of low-temperature plasma equipment on the sample.
Further, the pulse discharge voltage of the low-temperature plasma treatment in the step (2) is 16-20kV, the frequency is 200-2000Hz, the pulse width is 100-200ns, and the treatment time is 15-60s.
The beneficial effect of adopting the further scheme is that: in the scheme of the invention, the low-temperature plasma generates active particles with higher energy under low energy consumption, thereby influencing the molecular conformation of the enzyme and changing the activity of the enzyme. The cellulase is treated by low-temperature plasma for 15s, and the enzyme activity of the cellulase can be improved by 89.56%.
Further, the mass ratio of the flax seeds degummed by the microwave to the water in the step (3) is 1:5-10, and the soaking time is 0.5-3h.
The beneficial effect of adopting the above-mentioned further scheme lies in: the flax seeds soaked in the ratio can quickly absorb water to swell and soften, and the pulping efficiency is improved.
Further, the electric field intensity of the pulse electric field treatment in the step (4) is 1.3-3.7kV/cm, the pulse frequency is 3-9 times, the pulse width is 100-500ns, the frequency is 500-1000Hz, and the flow rate is 40-80rpm.
The beneficial effect of adopting the further scheme is that: the pulse electric field has the function of improving the contact efficiency of enzyme and substrate when used for pretreating linseed homogenate, and is favorable for assisting enzymolysis to promote the release of total phenols in linseed plant milk.
Further, the pulsed electric field in step (4) may be replaced with ultrasound
The beneficial effect of adopting the further scheme is that: the ultrasonic pretreatment of the linseed homogenate has the effect of improving the contact efficiency of enzyme and a substrate, and is favorable for assisting enzymolysis to promote the release of protein in linseed plant milk.
Further, the amount of the enzyme solution added in the step (5) is 0.1-0.5wt%.
Further, in the step (5), the enzymolysis temperature is 45-55 ℃, and the enzymolysis time is 30-75min; inactivating enzyme at 90-100 deg.C for 10-15min; filtering and sieving to obtain 120-200 mesh powder.
The beneficial effect of adopting the further scheme is that: the enzyme hydrolyzes glycosidic bonds in the plant cell wall layer to promote the dissolution of cell contents, and the edible quality of the flaxseed plant milk is greatly improved by the low-consumption physical double-field coupling enzyme method, wherein the total phenol content, the protein content and the solid content are respectively improved by 66 percent, 17 percent and 12 percent, so that the flaxseed plant milk is endowed with higher nutritional value.
Compared with the prior art, the invention has the beneficial effects that:
1. after the low-consumption physical double-field treatment, the edible quality of the linseed plant milk is greatly improved, wherein the total phenol content, the protein content and the solid content are respectively improved by 66%, 17% and 12%, and the linseed plant milk is endowed with higher nutritional value.
2. Compared with the single conventional technology, the synergistic effect is reflected by low-consumption physical double-field coupling enzymolysis, the preparation process is simple and convenient, the reaction process is easy to control, the production period is short, the production cost is low, and the method belongs to a green, clean label and high-quality plant milk processing mode.
3. The invention firstly provides a method for improving the enzymolysis efficiency of the plant milk by low-consumption physical double-field coupling, on one hand, the activity of the enzyme is directly improved through a first physical field, the enzymolysis efficiency is directly improved, the reaction time is shortened, on the other hand, the contact between the enzyme and a substrate is promoted through treating the plant milk by a second physical field, and the enzymolysis effect is indirectly further improved. The research of the invention finds that after low-consumption physical double-field treatment, the edible quality of the linseed plant milk is greatly improved, wherein the total phenol content, the protein content and the solid content are respectively improved, and the viscosity is reduced by 49%. Data show that the double-field coupling enzymolysis embodies synergistic interaction and has great application potential in the fields of green, clean labels and high-quality plant milk processing.
Drawings
FIG. 1 shows the enzyme activity and the improvement rate of cellulase after low temperature plasma treatment for various periods of time;
fig. 2A and 2B show the total phenol content and viscosity of flaxseed milk prepared by assisting cellulase at low temperature plasma, respectively;
fig. 3A and 3B show the solid content and protein content of flaxseed milk prepared by low temperature plasma assisted bromelain, respectively;
fig. 4 shows the total phenol content after the pulsed electric field with different electric field strengths is used for independently treating or assisting enzymolysis of the linseed milk;
fig. 5A and 5B show the solid content of the flaxseed milk prepared by assisting bromelain with different ultrasonic powers and different ultrasonic times, respectively;
fig. 6A and fig. 6B show protein content and solid content of linseed plant milk prepared by low-temperature plasma and ultrasonic coupled enzyme method, respectively;
FIGS. 7A and 7B show the total phenolic content and viscosity of linseed plant milk prepared by low temperature plasma and pulsed electric field coupled enzymatic method, respectively;
fig. 8 is a schematic process flow diagram of linseed milk preparation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1 first physical field increases enzyme Activity
1.1. Materials and reagents
Cellulase CTS, weifang kang Den Biotech Co., ltd; cellulase (CL) activity assay kit, AKSU043M, beijing Box Biotechnology Ltd.
1.2. Main apparatus and equipment
Digital display constant temperature water bath, HH-6, hongkong instruments and factories of Jintancity; multifunctional microplate reader, spectraMAXM2, milo molecular instruments (shanghai) ltd, pulsed power-driven plasma discharge device.
1.3. Experimental methods and results
Green physical field low-temperature plasma treatment of cellulase to improve cellulase activity
(1) Preparing an enzyme solution: 15g of cellulase is weighed and added into 50mL of water to be uniformly mixed, and the mixture is stirred for 30min in ice bath.
(2) Treating enzyme liquid by low-temperature plasma: 1mL of enzyme solution is absorbed into a clean culture dish (the diameter is 35 mm), marked and placed below a nozzle of a plasma generator, the pulse discharge voltage is 18kV, the frequency is 1000Hz, and the pulse width is 200ns. When the plasma is stable (i.e. no obvious filiform spark), starting a timer, setting the treatment time to be 60s, and collecting the treated cellulase, and sealing and storing at 4 ℃ for later use.
(3) The enzyme activity was determined using a BOXBIO cellulase activity detection kit.
Enzyme liquid pretreatment: centrifuging the enzyme solution after CP treatment at 4 ℃ for 10min, and taking the supernatant to be tested.
The determination step comprises: and sequentially adding 50 mu L of a first reagent, 200 mu L of a second reagent, 50 mu L of water and 50 mu L of a supernatant of the crude enzyme solution into the measuring tube, replacing the crude enzyme solution in the control tube with the corresponding inactivated crude enzyme solution, and keeping other reagents and the addition amount unchanged. Mixing, saccharifying in 50 deg.C water bath for 30min, immediately boiling water bath for 15min, and sealing to prevent water loss to obtain saccharified solution. Sucking 10 μ L of the saccharified solution, adding 30 μ L of reagent III, developing in boiling water bath for 15min (sealing to prevent water loss), and cooling to room temperature. The standard tube saccharification solution was replaced with 10. Mu.L of standard dilution, and the remaining reagents and amounts added were kept constant. Absorbing 200 mu L of reaction liquid into a 96-well plate, measuring the light absorption value at 540nm, and recording as A measurement, A contrast, A standard and A blank; calculate Δ a assay = a assay-a control, Δ a standard = a standard-a blank.
Calculation of Cellulase (CL) activity: definition of units: the catalytic production of 1. Mu.g glucose per minute per mL of liquid sample is defined as one unit of enzyme activity.
CL(U/mL)＝1000×x×V Anti-total /(V Sample (A) ×T)＝233.33×x
Note that: v Anti-total : the total volume of the reaction system is 0.35mL; v Sample (A) : adding 0.05mL of crude enzyme solution into the reaction system; t: saccharification time, 30min;1000: unit conversion factor, 1mg/mL =1000 μ g/mL.
As shown in figure 1, after the CP is used for treating the cellulase for different time, the enzyme activity is increased firstly and then reduced, and when the CP treatment time is 15s, the enzyme activity can be improved by 89%, the improvement of the enzyme activity can be caused by that active particles influence the enzyme molecular conformation to different degrees, the active sites of the enzyme are exposed, and the enzyme activity is improved.
Example 2 first physical field increases the actual enzymatic efficiency of enzymes in the processing of plant milk
2.1. Materials and reagents
Cellulase CTS, weifang kang Den Biotech Co., ltd; bromelain, beijing solibao science and technology ltd; dry degummed flax seeds, food grade, ministry of agricultural sciences in Gansu province; sea sand, concentrated sulfuric acid, boric acid and the like are purchased from chemical reagents of national drug group, inc.; the Kjeldahl nitrogen-fixing high-efficiency catalytic tablet and the Folin phenol are all from source leaf organisms.
2.2. Main apparatus and equipment
A plasma discharge device driven by a pulse power supply; colloid mill, horizontal 60 type, shenzhen, leitong Engineers, inc.; digital display constant temperature water bath, HH-6, hongkong instruments and factories of Jintancity; electrothermal blowing dry box, 101-1AB, tester instruments, inc. of Tianjin; full-automatic Kjeldahl apparatus, K9860, ocean Energy future science and technology group, inc.; digital viscometer, SNB-1, shanghai precision scientific instruments Inc.
2.3. Experimental methods and results
2.3.1A method for improving the enzymolysis efficiency of cellulase in the processing of plant milk by applying low-temperature plasma to the cellulase, which comprises the following steps:
(1) Preparing an enzyme solution: weighing 15g of cellulose CTS, adding 50mL of water, uniformly mixing, and stirring in ice bath for 30min.
(2) Treating enzyme liquid by low-temperature plasma: 1mL of enzyme solution was aspirated into a clean petri dish (diameter 35 mm) and labeled, and placed under a plasma generator nozzle with a pulse discharge voltage of 18kV, a frequency of 1000Hz, and a pulse width of 200ns. When the plasma is stable (i.e. no obvious filiform spark), starting a timer, setting the treatment time to be 15s, and collecting the treated cellulase, and sealing and storing at 4 ℃ for later use.
(3) Preparing linseed homogenate: soaking flax seeds subjected to microwave degumming in 1:7 for 2h, removing the soaking solution, adding pure water according to the proportion of 1:7, circulating for 12min by a colloid mill, and collecting the plant milk.
(4) An enzymolysis process: adding 0.1wt% of cellulase treated by CP into the treated solution, performing enzymolysis at 50 deg.C for 30min, inactivating enzyme at 90 deg.C for 15min, and filtering with 200 mesh filter cloth to obtain semen Lini plant milk.
As shown in fig. 2A and 2B, the application of CP was found to increase the efficiency of reducing the viscosity of the release efficiency of total phenols in the linseed plant milk when the CP-assisted cellulase enzymes the linseed plant milk. Thus the complex field selects CP treatment cellulase 60s.
2.3.2 the method for improving the enzymolysis efficiency of the bromelain in the processing of the plant milk by applying low-temperature plasma to the bromelain and optimizing the treatment time comprises the following steps:
(1) Preparing an enzyme solution: 1g of bromelain is weighed, added into 10mL of water and mixed evenly, and stirred for 30min in ice bath.
(2) Treating enzyme liquid by low-temperature plasma: 1.5mL of enzyme solution was pipetted into a clean petri dish (diameter 35 mm) and labeled, and placed under the plasma generator nozzle with a pulse discharge voltage of 18kV, a frequency of 1kHz, and a pulse width of 200ns. When the plasma is stable (i.e. no obvious filiform sparks), a timer is started, the treatment time is set to 45s, and the treated bromelain is collected and stored in a sealed manner at 4 ℃ for later use.
(3) Preparing linseed homogenate: mixing microwave degummed semen Lini with water at 1:7 mass ratio, soaking for 2 hr, removing soaking solution, and colloid milling for 12min to collect plant milk.
(4) An enzymolysis process: adding 0.075wt% of CP-treated bromelain into the above treated solution, performing enzymolysis at 50 deg.C for 1h, inactivating enzyme at 90 deg.C for 15min, and filtering with 200 mesh filter cloth to obtain semen Lini plant milk.
As shown in fig. 3A and 3B, when CP assists bromelain to enzymatically hydrolyze linseed plant milk, it was found that too long or too short a time for CP treatment of bromelain decreased the release efficiency of linseed plant milk protein; and when the CP is treated for 45 or 60 seconds, the content release efficiency of the content of the solid and the protein of the flax seed plant milk with the auxiliary enzymolysis of the CP is improved. Therefore, the composite field selects CP to treat bromelain 60s so as to improve the enzymolysis efficiency of bromelain in the processing of plant milk.
The experimental results show that the CP physical field can not only improve the measured enzyme activity value, but also improve the real enzymolysis effect of the CP physical field in a complex plant milk system.
Example 3 second physical field enhancing Linum usitatissimum plant milk quality
3.1. Materials and reagents
Cellulase CTS, weifang kang Den Biotech Co., ltd; bromelain, beijing solibao science and technology ltd; dry degummed flax seeds, food grade, ministry of agricultural sciences in Gansu province; sea sand, concentrated sulfuric acid, boric acid and the like are purchased from chemical reagents of national drug group, inc.; the Kjeldahl nitrogen-fixing high-efficiency catalytic tablet and the Folin phenol are all from source leaf organisms.
3.2. Main apparatus and equipment
High voltage pulsed electric field generator, THU-PEF4, wuhan New Tianpu laboratory facilities, inc.; ultrasonic cell disruptor, JY92-IIDN, ningbo Xinzhi Biotech GmbH; colloid mill, horizontal 60 model, shenzhen, leitong industries, ltd; digital display constant temperature water bath, HH-6, hongkong instruments and factories of Jintancity; electrothermal blowing dry box, 101-1AB, tester instruments ltd, tianjin; full-automatic kaimen azotometer, K9860, by ocean energy future science and technology group ltd.
3.3. Experimental methods and results
3.3.1 through applying the pulsed electric field to the flaxseed homogenate, optimize electric field intensity, establish high-efficient flaxseed milk enzymolysis process, improve the method of flaxseed plant milk quality, including the following step:
(1) Preparing an enzyme solution: weighing 15g of cellulose CTS, adding 50mL of water, uniformly mixing, and stirring in ice bath for 30min.
(2) Preparing linseed homogenate: mixing microwave degummed semen Lini with water at 1:7 mass ratio, soaking for 2 hr, removing soaking solution, and colloid milling for 12min to collect plant milk.
(3) Pretreatment of linseed milk by using pulsed electric field: performing pulsed electric field and ultrasonic treatment on the prepared linseed homogenate, wherein the electric field intensity of the pulsed electric field treatment is 1.8kV/cm, the pulse frequency is 3 times, the pulse width is 500 mus, the frequency is 1kHz, and the flow rate is 40rpm, so as to obtain a treatment solution;
(4) An enzymolysis process: adding 0.1wt% of cellulose processed by CP into the above treated solution, performing enzymolysis at 50 deg.C for 30min, inactivating enzyme at 90 deg.C for 15min, and filtering with 200 mesh filter cloth to obtain semen Lini plant milk.
As shown in fig. 4, when a low-intensity pulsed electric field is applied to the linseed homogenate, the release effect of the linseed plant milk total phenols is very weak, when the electric field intensity is greater than 2.7Kv/cm, the content of the linseed plant milk total phenols is obviously increased, probably because when the electric field intensity is greater than 2.7Kv/cm, the electric field intensity is increased, the permeability of the plant cell membrane is increased, and the release capacity of the active ingredient is enhanced. And when the electric field intensity is too low, the permeability of the plant cells is not sufficiently influenced. In conclusion, the strength of the pulsed electric field is 3.7Kv/cm, and the linseed plant milk under the process condition has higher total phenols compared with the control group.
3.3.2 second physical field (ultrasound) method for improving milk quality of flaxseed plants, comprising the steps of:
(1) Preparing an enzyme solution: 1g of bromelain is weighed, added into 10mL of water and mixed evenly, and stirred for 30min in ice bath.
(2) Preparing linseed homogenate: mixing microwave degummed semen Lini with water at 1:7 mass ratio, soaking for 2 hr, removing soaking solution, and colloid milling for 4min (12 min) to collect plant milk.
(3) Ultrasonic pretreatment of linseed milk: carrying out ultrasonic pretreatment on the prepared linseed homogenate for 200w,10min (10min, 200w and 400 w) to obtain a treatment solution;
(4) An enzymolysis process: adding 0.075wt% of cellulase subjected to CP treatment or not subjected to CP treatment into the above treated solution, performing enzymolysis at 50 deg.C for 30min, inactivating enzyme at 90 deg.C for 15min, and filtering with 200 mesh filter cloth. Obtaining the linseed plant milk.
As shown in fig. 5A and 5B, after ultrasonic treatment of flaxseed milk for 10min at 200w or 400w, the solids content of flaxseed plant milk was reduced, probably because the power was too high to degrade flavonoids, polysaccharides and soluble proteins. By researching the ultrasonic time, after the ultrasonic-assisted enzymolysis of the linseed plant milk, the total solid content of the linseed plant milk is reduced within 10min of the ultrasonic time; the reason may be that the longer the sonication time, the stronger the cavitation effect, the greater the loss of amino nitrogen and the reduction of the solid content. As can be seen from the comparison of the solid content, the solid content of the linseed plant milk under the process condition is higher and is increased by 5.22 percent, so that the ultrasonic condition is finally selected to be 200w,5min.
The experiment shows that after the plant milk is treated by the second physical field, the enhancement of the enzymolysis effect can be realized indirectly by improving the contact efficiency of enzyme and a substrate, and the effect of improving the quality of the plant milk is achieved.
Example 4 two-field coupling, synergistic enhancement of Linum usitatissimum plant milk quality
4.1. Materials and reagents
Cellulase CTS, weifang kang Den Biotech Co., ltd; bromelain, beijing solibao science and technology ltd; dry degummed flax seeds, food grade, ministry of agricultural sciences in Gansu province; sea sand, concentrated sulfuric acid, boric acid and the like are purchased from chemical reagents of national drug group, inc.; the Kjeldahl nitrogen-fixing high-efficiency catalytic tablet and the Folin phenol are all from source leaf organisms.
4.2. Main apparatus and equipment
A plasma discharge device driven by a pulse power supply; high voltage pulsed electric field generator, THU-PEF4, wuhan New Tianpu laboratory facilities, inc.; ultrasonic cell crusher, JY92-IIDN, ningbo Xinzhi Biotech GmbH; colloid mill, horizontal 60 model, shenzhen, leitong industries, ltd; digital display constant temperature water bath, HH-6, hongkong instruments and factories of Jintancity; electrothermal blowing dry box, 101-1AB, tester instruments ltd, tianjin; full-automatic Kaimen azotometer, K9860, ocean energy future science and technology group, inc.
4.3. Experimental methods and results
4.3.1 double field (low temperature plasma and ultrasound) coupling, synergistic method for improving linseed plant milk quality, comprising the following steps:
(1) Preparing an enzyme solution: 1g of bromelain is weighed, added into 10mL of water and mixed evenly, and stirred for 30min in ice bath.
(2) Treating enzyme liquid by low-temperature plasma: 1.5mL of enzyme solution was pipetted into a clean petri dish (diameter 35 mm) and labeled, and placed under the plasma generator nozzle with a pulse discharge voltage of 18kV, a frequency of 1000Hz, and a pulse width of 200ns. When the plasma is stable (i.e. no obvious filiform spark), starting a timer, setting the treatment time to be 60s, collecting the treated cellulase, and sealing and storing at 4 ℃ for later use.
(3) Preparing linseed homogenate: mixing and soaking the degummed 15 times of microwave flaxseeds and water according to the mass ratio of 1:7 for 2h, discarding the soaking solution, and collecting the plant milk after the colloid mill is circulated for 12 min.
(4) Ultrasonic pretreatment of linseed milk: carrying out ultrasonic pretreatment (200w, 5 min) on the prepared flaxseed homogenate to obtain a treatment solution;
(5) An enzymolysis process: adding 0.075wt% bromelain treated with CP for 60s into the above treated solution, performing enzymolysis at 50 deg.C for 1 hr, inactivating enzyme at 90 deg.C for 15min, and filtering with 200 mesh filter cloth. Obtaining flaxseed plant milk
As shown in fig. 6A and 6B, the effects of the three techniques used alone are limited, but a synergistic effect is exhibited when the three techniques are used in combination. On one hand, the release efficiency of the linseed plant milk solid and the protein is improved through the application of low-temperature plasma, on the other hand, the contact of enzyme and a substrate is promoted through ultrasonic treatment of the plant milk, and the enzymolysis effect is further improved. The linseed milk prepared by low-consumption physical double-field treatment finally contains higher protein and total solid content, and the edible quality of the linseed plant milk is greatly improved.
4.3.2 double field (low temperature plasma and pulse electric field) coupling, method for synergistically improving milk quality of linseed plant comprises the following steps:
(1) Preparing an enzyme solution: weighing 15g of cellulose CTS, adding 50mL of water, uniformly mixing, and stirring in ice bath for 30min.
(2) Treating enzyme liquid by low-temperature plasma: 1mL of enzyme solution was aspirated into a clean petri dish (diameter 35 mm) and labeled, and placed under a plasma generator nozzle with a pulse discharge voltage of 18kV, a frequency of 1000Hz, and a pulse width of 200ns. When the plasma is stable (i.e. no obvious filiform spark), starting a timer, setting the treatment time to be 15s, and collecting the treated cellulase, and sealing and storing at 4 ℃ for later use.
(3) Preparing linseed homogenate: mixing microwave degummed semen Lini with water at 1:7 mass ratio, soaking for 2 hr, removing soaking solution, and colloid milling for 12min to collect plant milk.
(4) Pretreatment of linseed milk by using pulsed electric field: performing pulsed electric field and ultrasonic treatment on the prepared linseed homogenate, wherein the electric field intensity of the pulsed electric field treatment is 3.7kV/cm, the pulse frequency is 3 times, the pulse width is 500 mus, the frequency is 1kHz, and the flow rate is 40rpm, so as to obtain a treatment solution;
(5) An enzymolysis process: adding 0.1wt% of cellulose processed by CP into the above treated solution, performing enzymolysis at 50 deg.C for 30min, inactivating enzyme at 90 deg.C for 15min, and filtering with 200 mesh filter cloth to obtain semen Lini plant milk.
As shown in fig. 7, the three techniques alone have limited effectiveness, but show synergistic effects when the three techniques are used in combination. As shown in fig. 7A, the total phenol content of the flaxseed plant milk after PEF, enzymatic hydrolysis and CP treatment was increased by 21.07%, 12.98% and 28.26%, respectively, while the total phenol content was increased by 65.50% when the two-field complex coupled enzyme method was used. As shown in FIG. 7B, the viscosity also shows a synergistic effect, and finally the viscosity of the two-field complex coupled enzyme method is reduced to 77.47 mPas.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (7)
1. A method for improving the plant milk enzymolysis efficiency by low-consumption physical double-field synergy is characterized by comprising the following steps:
(1) Preparing an enzyme solution: weighing enzyme, adding water for mixing, stirring in an ice bath, and uniformly mixing to obtain an enzyme solution for later use;
(2) Low-temperature plasma treatment: sucking the enzyme liquid into a clean culture dish, placing the culture dish below a nozzle of a plasma generator, treating the enzyme liquid with stable plasma, collecting the treated enzyme liquid, and sealing and storing at 2-8 ℃ for later use;
(3) Preparing linseed homogenate: mixing microwave degummed semen Lini with water, soaking, removing soaking solution, adding water into soaked semen Lini, and colloid milling for 4-12min to collect plant milk;
(4) Pretreating flaxseed homogenate by a pulsed electric field: subjecting the plant milk prepared in the step (3) to a pulse electric field to obtain a treatment solution;
(5) Enzymolysis: and (3) adding 0.1-0.5wt% of the enzyme solution prepared in the step (1) or the step (2) into the treatment solution, inactivating the enzyme after enzymolysis, and filtering to obtain the linseed plant milk with high protein content, high solid content and moderate viscosity.
2. The method for improving the plant milk enzymolysis efficiency through the low consumption physical double-field synergy according to claim 1, characterized in that, in the step (1), the enzyme is one or a mixture of more of cellulase, protease, phytase, hemicellulase, saccharifying enzyme and bromelain;
the mass ratio of the enzyme to the water is 1-5.
3. The method for improving the plant lactogenesis efficiency through low-consumption physical double-field cooperation according to claim 1, wherein the pulse discharge voltage of the low-temperature plasma treatment in the step (2) is 16-20kV, the frequency is 200-2000Hz, the pulse width is 100-500ns, and the treatment time is 15-60s.
4. The method for improving the plant lactogenesis efficiency through the low-consumption physical double-field synergy according to claim 1, wherein the mass ratio of the microwave degummed flaxseeds to the water in the step (3) is 1:5-10, and the soaking time is 0.5-3 hours.
5. The method for improving the plant lactogenesis efficiency through the low-consumption physical double-field cooperation according to claim 1, wherein the electric field intensity of the pulsed electric field treatment in the step (4) is 1.3-3.7kV/cm, the pulse frequency is 3-6 times, the pulse width is 100-500ns, the frequency is 500-1000Hz, and the flow speed is 40-80rpm.
6. The method for improving the enzymolysis efficiency of plant milk through low consumption physical double-field synergy according to claim 1, characterized in that the adding amount of the enzyme solution in the step (5) is 0.1-0.5wt%.
7. The method for improving the enzymolysis efficiency of the plant milk by virtue of the low-consumption physical double-field synergy of the claim 1, wherein in the step (5), the enzymolysis temperature is 45-55 ℃, and the enzymolysis time is 30-75min; inactivating enzyme at 90-100 deg.C for 10-15min; filtering and sieving to obtain 120-200 mesh powder.
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