CN115336666A - Application and method for reducing allergenicity of lactalbumin by nepenthes trapping cyst protease - Google Patents
Application and method for reducing allergenicity of lactalbumin by nepenthes trapping cyst protease Download PDFInfo
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- A—HUMAN NECESSITIES
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- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
- A23J3/343—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
- A23J3/08—Dairy proteins
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Abstract
The invention relates to application and a method of nepenthes trapping cyst protease in reducing lactalbumin allergenicity; the protease in the nepenthes insect-catching sac tissue acts on different types of whey proteins respectively, so that peptide bonds or conformations of whey protein epitopes are broken, even the epitopes are cut off, and the potential of directionally destroying certain peptide bonds is realized; and detecting the peptide fragment distribution condition and immunoreactivity of the product after enzymolysis by using Tricine-SDS-PAGE, indirect competition ELISA and Western Blot.
Description
Technical Field
The invention belongs to the field of biology and immunology, and particularly relates to a method for reducing the allergenicity of whey protein in cow milk by a biological enzymolysis technology.
Technical Field
Cow milk protein allergy is an adverse immune reaction caused by protein in cow milk, and can cause symptoms such as eczema, diarrhea and gastrointestinal bleeding of patients, even shock and death of severe patients; recent epidemiological investigation shows that the allergy rate of infants under 2 years old in China is about 2.69%, and common methods for reducing the allergy of cow milk comprise physical methods (heating treatment, pressurizing treatment, irradiation treatment and ultrasonic treatment), chemical methods (glycosylation and other chemical modifications) and biological methods (enzymolysis, enzyme crosslinking and fermentation); the enzymatic hydrolysis technology can greatly ensure the sensory quality and the nutritional value of the product while reducing the allergenicity; even during enzymatic hydrolysis some functional polypeptides are produced; the method has the advantages of simple process, mild conditions, high safety and mature technology; is currently the most commonly used method.
The first problem of reducing the allergenicity of whey protein by the enzymatic method is generally the selection of enzyme species; the enzymes commonly used at present are proteases of vegetable origin (bromelain, papain and ficin), of animal origin (pepsin, trypsin and chymotrypsin) and of microbial origin (alcalase and subtilisin); the ubiquitous problem of proteases of microbial origin is that their safety is to be further confirmed, and some countries do not even use them for human food; the animal-derived protease has high cost and low efficiency due to factors such as long growth period of animals, limited animal slaughter, animal disease transmission and the like; in contrast, the plant source protease has high safety, strong environmental protection and rich resources, and is suitable for being widely developed and applied to enzymolysis of the whey protein of the cow milk so as to reduce the allergenicity of the cow milk.
As a common insect-feeding plant, nepenthes secrete a large amount of protease firstly, and no research is carried out on reducing cow milk allergenicity by using the protease in the nepenthes insect-catching bag; therefore, the development of hypoallergenic whey protein by using the protease in the nepenthes insect-catching capsule has wide research space and practical research significance.
Disclosure of Invention
The invention aims to separate whey protein and concentrated whey protein by enzymolysis respectively by using novel plant source protease in nepenthes insect-catching sac tissue, and evaluate the molecular weight and IgG immunoreactivity of the product after enzymolysis so as to realize the technology for reducing the allergenicity of whey protein of different cows milk.
The invention is realized by the following technical scheme.
The invention provides application and a method for reducing allergenicity of lactalbumin by nepenthes trapping cyst protease, which comprise initial detection of enzymolysis conditions and sensibility evaluation.
The nepenthes trapping cyst protease is applied to reducing the allergenicity of whey protein, wherein the whey allergen is alpha-lactalbumin or beta-lactoglobulin in whey protein of cow milk;
preferably, nepenthes catching cyst protease and lactalbumin are subjected to enzymolysis reaction; the enzymolysis conditions are as follows: the mass fraction of the grinding powder of the insect catching capsules in the lactalbumin solution is 5 +/-0.5%, the pH value is 5 +/-0.5 or 11 +/-0.5, the reaction time is 2 +/-0.1 h, and the temperature is 50 +/-5 ℃.
The application and the method for reducing the allergenicity of the lactalbumin by the nepenthes trapping cyst protease comprise the following steps:
s1: preparing a whey powder solution: weighing separated whey protein powder or concentrated whey protein powder respectively, preparing a whey protein solution with the mass fraction of 5 +/-0.5% by using distilled water, and sucking 20mL of the whey protein solution into a centrifugal tube respectively;
s2: preparing the insect catching bag grinding powder: cutting off the catching cysts with medium growth time from nepenthes, pouring out digestive juice in the catching cysts, then cleaning, wiping and shearing the catching cysts, adding liquid nitrogen and grinding the liquid nitrogen into powder for later use (wherein after the digestive juice in the catching cysts is poured out, the digestive juice in the catching cysts also needs to be cleaned);
s3: enzymolysis of the isolated whey protein solution or concentrated whey protein solution: and (3) selecting nepenthes trapping cyst tissue powder in the step (S2) and putting the nepenthes trapping cyst tissue powder into a centrifugal tube for storing a lactalbumin solution in the step (S1), wherein the mass fraction of the trapping cyst is 5 +/-0.5%, so as to obtain enzymolysis products under different conditions.
Preferably, the pH of the enzymolysis solution in S3 is 5 +/-0.5 or 11 +/-0.5, the enzymolysis time is 2 +/-0.1 h, and the temperature is set to be 50 +/-5 ℃.
The invention adopts enzymolysis technology to specifically cut proteins in different whey proteins, cuts allergen proteins into small molecular peptide fragments, and breaks peptide bonds in epitopes or destroys the structure of the epitopes.
The invention has the following advantages: the novel plant protease derived from the nepenthes insect-catching sac can destroy epitopes in different kinds of whey proteins; sensitization evaluation the first time, the system evaluates the effect of reducing the sensitization of whey by enzymolysis, the target is accurate, the sensitization evaluation is comprehensive, and the accurate implementation of the enzymolysis technology is ensured.
Drawings
FIG. 1 is a graph showing the effect of protease on WPI and WPC in ground insect-catching capsules under different pH conditions (note: M is an unpigmented small molecule standard protein Marker; lane 1 is a Whey Protein Isolate (WPI); lane 2 is an enzymatic hydrolysate of ground insect-catching capsules against WPI (WPI-5) at pH5 + -0.5; lane 3 is an enzymatic hydrolysate of ground insect-catching capsules against WPI (WPI-11) at pH11 + -0.5; lane 4 is a concentrated whey protein solution (WPC); lane 5 is an enzymatic hydrolysate of ground insect-catching capsules against WPC (WPC-5) at pH5 + -0.5; and lane 6 is an enzymatic hydrolysate of ground insect-catching capsules against WPC (WPC-11) at pH11 + -0.5).
FIG. 2 is a graph showing the change of binding ability of the enzyme-hydrolyzed WPCs-5 and WPI-5 and the enzyme-non-hydrolyzed WPIs and WPCs to rabbit anti-alpha-lactalbumin IgG.
FIG. 3 is a graph showing the change in binding capacity of the enzyme-hydrolyzed WPCs-5 and WPI-5 and the enzyme-non-hydrolyzed WPIs and WPCs to rabbit anti-beta-lactoglobulin IgG.
FIG. 4 is a graph showing the binding capacity of different peptide fragments of enzymatic WPC-5 and WPI-5 and non-enzymatic WPI and WPC to rabbit anti-alpha-lactalbumin IgG (note: from left to right, the samples are WPI, WPI-5, WPC and WPC-5 in sequence; on the left, electrophoresis control corresponding to Western Blot, and on the right, western Blot results).
FIG. 5 is a graph showing the binding capacity of different peptides of enzymolyzed WPC-5 and WPI-5 and non-enzymolyzed WPI and WPC to rabbit anti-beta-lactoglobulin IgG (note: from left to right, the samples are WPI, WPI-5, WPC and WPC-5 in sequence; on the left, the electrophoresis control corresponding to Western Blot, and on the right, the Western Blot result).
Detailed description of the preferred embodiments and results
The invention aims to separate Whey Protein (WPI) and concentrated Whey Protein (WPC) by enzymolysis respectively by using novel plant source protease in nepenthes insect-catching capsule tissues, and evaluate the molecular weight and IgG immunoreactivity of the products after enzymolysis so as to realize the technology for reducing the allergenicity of whey protein of different cows milk.
The invention is further illustrated by the following examples, but is not limited thereto.
Example 1.
And (3) selecting nepenthes catching cyst tissue grinding powder to carry out enzymolysis on the WPI, and carrying out systematic evaluation on an enzymolysis product by technical means such as Tricine-SDS-PAGE, indirect competitive ELISA, western Blot and the like.
The specific implementation process comprises the following steps.
WPI enzymatic hydrolysate preparation scheme:
(1) Preparing a whey powder solution: weighing separated whey protein powder, preparing 5 +/-0.5 mass percent WPI solution by using distilled water, and sucking 20mL of the solution into a centrifugal tube;
(2) Preparing the insect catching bag grinding powder: cutting off the catching cysts with medium growth time from nepenthes, pouring out digestive juice in the catching cysts, then cleaning, wiping and shearing the catching cysts, adding liquid nitrogen and grinding the liquid nitrogen into powder for later use (wherein after the digestive juice in the catching cysts is poured out, the digestive juice in the catching cysts also needs to be cleaned);
(3) And (3) carrying out enzymolysis on WPI: and (3) selecting the nepenthes insect-catching cyst tissue powder in the step (2) and putting the nepenthes insect-catching cyst tissue powder into a centrifugal tube for storing the WPI solution in the step (1), wherein the mass fraction of the insect-catching cyst tissue powder is 5 +/-0.5%.
And (3) selecting two parts of WPI enzymolysis solutions in the step (3), wherein the pH values are respectively 5 +/-0.5 or 11 +/-0.5, the time is 2 +/-0.1 h, and the temperature is 50 +/-5 ℃, so as to obtain WPI enzymolysis products under different conditions.
tricine-SDS-PAGE analysis: comparison of WPI solutions, enzymatic samples at pH5 + -0.5 or pH11 + -0.5, as shown in FIG. 1:
wherein, the WPI solution has thicker bands at the molecular weight ranges of 10-15 kDa and 15-20 kDa, which respectively correspond to the main allergens alpha-lactalbumin (14.2 kDa) and beta-lactoglobulin (18.3 kDa) in WPI.
Under the condition of pH5 +/-0.5, almost all peptide fragments above 20kDa are hydrolyzed, and in a molecular weight section of 15-20 kDa, although partial residues exist, a large amount of protein is subjected to enzymolysis; wherein the residual peptide fragment is located in a part close to 20kDa, which can be presumed that the main allergen beta-lactoglobulin of cow milk may be somewhat residual but most of it is destroyed; only a small band with lighter color appears in the molecular weight range of 10-15 kDa, and the main allergen alpha-lactalbumin in cow milk can be presumed to be damaged to a great extent.
Under the condition of pH11 +/-0.5, the band deepening phenomenon appears in the interval of more than 30kDa, probably because the cross-linking occurs between proteins or between proteins and certain subunits under the alkaline condition; but the peptide segment of about 14-18 kDa is hardly degraded, so that the peptide segment probably has no great degradation effect on alpha-lactalbumin and beta-lactoglobulin; based on this, the immunoreactivity was measured by the IgG binding ability of the enzymatic hydrolysate at pH 5. + -. 0.5.
3, ELISA detection: the IgG binding capacity of the enzymatic products was evaluated by indirect competition ELISA method, as shown in fig. 2 and 3:
the detection is carried out by respectively adopting polyclonal antibodies IgG of rabbit anti-alpha-lactalbumin and rabbit anti-beta-lactoglobulin, and the IgG binding capacity of the alpha-lactalbumin and the beta-lactoglobulin of the WPI is reduced to a certain extent after enzymolysis; the IgG binding capacity of the beta-lactoglobulin is reduced more obviously than that of the alpha-lactalbumin, and the IC50 values of the protease treatment WPI in the nepenthes insect-catching capsule are obtained by fitting the results of the figure 2 and the figure 3 and are respectively shown in the table 1;
TABLE 1 IC50 Fit values of WPI and its enzymatic hydrolysate for rabbit anti-alpha-lactalbumin and beta-lactoglobulin IgG binding capacity
Through enzymolysis, the binding capacity of WPI to rabbit anti-alpha-lactalbumin IgG is reduced to 14.90 times when the enzymolysis is not carried out, and the binding capacity to rabbit anti-beta-lactoglobulin IgG is reduced to 454.55 times when the enzymolysis is not carried out.
Western Blot detection: the immunoreactivity of peptide sections with different molecular weights after enzymolysis is reflected by Western Blot by respectively adopting rabbit anti-alpha-lactalbumin and rabbit anti-beta-lactoglobulin polyclonal antibody IgG;
the WPI subjected to the action of enzymes in the insect-catching cyst tissue has fewer colored regions than the non-enzymolyzed sample, which indicates that the IgG binding ability of different peptide fragments generated by enzymolysis is weak or even none, and the results are shown in fig. 4 and fig. 5.
Example 2.
And (4) selecting nepenthes insect catching sac tissue grinding powder to carry out enzymolysis on the WPC.
WPC enzymatic hydrolysate preparation scheme:
(1) Preparing a whey powder solution: weighing concentrated whey protein powder, preparing 5 +/-0.5 mass percent WPC solution with distilled water, and sucking 20mL of the solution into a centrifugal tube;
(2) Preparing the insect catching bag grinding powder: cutting off the catching capsules with medium growth time from the nepenthes, pouring out digestive juice in the catching capsules, cleaning, wiping, cutting into pieces, adding liquid nitrogen and grinding into powder for later use (wherein after the digestive juice in the catching capsules is poured out, the digestive juice in the catching capsules also needs to be cleaned);
(3) And (3) carrying out enzymolysis on WPC: selecting nepenthes trapping cyst tissue powder in the step 2, putting the nepenthes trapping cyst tissue powder into a centrifugal tube for storing WPC solution, wherein the mass fraction of the trapping cyst tissue powder is 5
±0.5％。
And (3) selecting two parts of WPC enzymolysis solutions in the step (3), wherein the pH values are respectively 5 +/-0.5 or 11 +/-0.5, the time is 2 +/-0.1 h, and the temperature is 50 +/-5 ℃, so as to obtain WPC enzymolysis products under different conditions.
tricine-SDS-PAGE analysis: WPC solutions, enzymatic samples at pH5 ± 0.5 or pH11 ± 0.5 were compared as shown in figure 1:
wherein, the WPC solution has thicker bands at the molecular weight ranges of 10-15 kDa and 15-20 kDa, which respectively correspond to the main allergens alpha-lactalbumin (14.2 kDa) and beta-lactoglobulin (18.3 kDa) in WPC.
Under the condition that the enzymolysis condition is pH5 +/-0.5, almost all peptide fragments above 20kDa are hydrolyzed, and in a molecular weight section of 15-20 kDa, although partial residues exist, a large amount of proteins are subjected to enzymolysis; wherein the residual peptide segment is located at a part close to 15kDa, which can be presumed that the major allergen beta-lactoglobulin of cow milk can be partially destroyed; protein bands appear in the molecular weight range of 10-15 kDa, but the band areas are significantly reduced compared with the WPC solution, and the main allergen alpha-lactalbumin in cow milk is presumed to be destroyed to some extent.
Under the condition of pH11 +/-0.5, the band deepening phenomenon appears in the interval above 25kDa, probably because the cross-linking occurs between proteins or between proteins and certain subunits under the alkaline condition; the peptide segment of about 14-18 kDa is hardly degraded, so that the degradation effect on alpha-lactalbumin and beta-lactoglobulin is probably not too large; based on the above, the immunoreactivity of the enzyme products is detected by the IgG binding capacity of the enzyme products under pH5 +/-0.5
3, ELISA detection: the IgG binding capacity of the enzymatic products was evaluated by indirect competition ELISA method, as shown in fig. 2 and 3;
wherein, polyclonal antibodies IgG of rabbit anti-alpha-lactalbumin and rabbit anti-beta-lactoglobulin are respectively adopted for detection, and the IgG binding capacity of the alpha-lactalbumin and the beta-lactoglobulin of the WPC is reduced to a certain extent after enzymolysis; the IgG binding capacity of the beta-lactoglobulin is reduced more obviously than that of the alpha-lactalbumin;
the IC50 values of the protease-treated WPC in nepenthes trap were obtained by fitting the results of fig. 2 and 3, respectively, as shown in table 2;
TABLE 2 IC50 Fit values of WPC and its enzymatic hydrolysate for rabbit anti-alpha-lactalbumin and beta-lactoglobulin IgG binding Capacity
Through enzymolysis, the binding capacity of the WPC to rabbit anti-alpha-lactalbumin IgG is reduced to 4.71 times when the WPC is not enzymolyzed, and the binding capacity of the WPC to rabbit anti-beta-lactoglobulin IgG is reduced to 172.17 times when the WPC is not enzymolyzed.
Western Blot detection: the immunoreactivity of peptide sections with different molecular weights after enzymolysis is reflected by Western Blot by respectively adopting rabbit anti-alpha-lactalbumin and rabbit anti-beta-lactoglobulin polyclonal antibody IgG;
the WPC subjected to the action of enzyme in the insect-catching cyst tissue has less chromogenic region than the non-enzymolyzed sample, which indicates that the IgG binding capacity of different peptide fragments generated by enzymolysis is weaker or even no, and the results are shown in FIG. 4 and FIG. 5.
The whey protein is widely applied in the food industry, and mainly comprises WPI, WPC and other different types; the main difference between the two is the difference of the manufacturing process, the protein content and the content of other substances; the WPC with lower cost can be applied to frozen foods, baked foods, fermented dairy products and meat foods by virtue of the functional characteristics, and the sensory quality and the physicochemical property are improved in the production process; the WPI can be used for supplementing protein for the body-building people by virtue of the advantages of the nutritional value of the WPI; through the action of enzyme contained in the insect catching cyst tissue, both WPI and WPC can be subjected to enzymolysis, and the allergenicity is remarkably reduced; among them, WPI is slightly more likely to damage the allergen protein than WPC and slightly less likely to reduce the IgG binding ability, but its effect on reduction of WPC sensitization is also apparent.
Based on the method, the WPI and the WPC are subjected to enzymolysis by using the protease contained in the nepenthes trapping cyst tissue, the reduction of the allergenicity of the WPI and the WPC is successfully realized, and the application value is wide.
Claims (5)
1. Application of nepenthes trapping cyst protease in reducing lactalbumin allergenicity.
2. The use of nepenthes cyst-trapping protease of claim 1 to reduce lactalbumin sensitization, characterized by: the lactalbumin is alpha-lactalbumin or beta-lactoglobulin in milk lactalbumin.
3. The use of nepenthes cyst-trapping protease according to claim 1 for reducing allergenicity of whey protein, wherein: carrying out enzymolysis reaction on nepenthes catching cyst protease and lactalbumin; the enzymolysis conditions are as follows: the mass fraction of the grinding powder of the insect catching capsules in the lactalbumin solution is 5 +/-0.5%, the pH value is 5 +/-0.5 or 11 +/-0.5, the reaction time is 2 +/-0.1 h, and the temperature is 50 +/-5 ℃.
4. The application of nepenthes insect-catching cyst protease in reducing the allergenicity of lactalbumin and the method thereof are characterized in that: the method comprises the following steps:
s1: preparing a whey powder solution: weighing separated whey protein powder or concentrated whey protein powder respectively, preparing a whey protein solution with the mass fraction of 5 +/-0.5% by using distilled water, and sucking 20mL of the whey protein solution into a centrifugal tube respectively;
s2: preparing the insect catching bag grinding powder: cutting off the catching capsules with medium growth time from the nepenthes, pouring out digestive juice in the catching capsules, cleaning, wiping, cutting into pieces, adding liquid nitrogen, and grinding into powder for later use;
s3: enzymolysis of isolated whey protein solution or concentrated whey protein solution: and (3) selecting nepenthes trapping cyst tissue powder in the step (S2) and putting the nepenthes trapping cyst tissue powder into a centrifugal tube for storing a lactalbumin solution in the step (S1), wherein the mass fraction of the trapping cyst is 5 +/-0.5%, so as to obtain enzymolysis products under different conditions.
5. The use and method of nepenthes insect-catching cyst protease for reducing the allergenicity of whey protein according to claim 4, characterized in that: in S3, the pH of the enzymolysis solution is 5 +/-0.5 or 11 +/-0.5, the enzymolysis time is 2 +/-0.1 h, and the temperature is set to be 50 +/-5 ℃.
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| US20160022785A1 (en) * | 2014-06-16 | 2016-01-28 | Nepetx, Llc | Compositions and methods for treating gluten intolerance and disorders arising therefrom |
| CN115299523A (en) * | 2022-07-05 | 2022-11-08 | 谭宏凯 | Application and method for reducing allergenicity of lactalbumin by nepenthes digestive juice protease |
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| US20160022785A1 (en) * | 2014-06-16 | 2016-01-28 | Nepetx, Llc | Compositions and methods for treating gluten intolerance and disorders arising therefrom |
| CN115299523A (en) * | 2022-07-05 | 2022-11-08 | 谭宏凯 | Application and method for reducing allergenicity of lactalbumin by nepenthes digestive juice protease |
Non-Patent Citations (2)
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| CAMILA T.M.N. PORFÍRIO ET AL: "Biotechnological potential for the hydrolysis of bovine milk proteins and characterization of three serine carboxyproteases from the digestive fluid of Nepenthes mirabilis carnivorous plant", pages 28 - 61 * |
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