Abstract: Four methods, namely, pH-stat method, formaldehyde titration method, ninhydrin colorimetric method and TCA index method, were used to detect the changes of hydrolysis degree of rice protein by alkaline protease under certain substrate concentration and process conditions, and a more in-depth comparison was carried out. The results showed that, under the fixed hydrolysis conditions, the changing law of protein hydrolysis reflected by the results of the four methods was in line with the enzymatic process of alkaline protease. Among the four methods, the hydrolysis degree of the TCA index method was on the high side, which differed from that of the other three methods. In contrast, the results of the pH-stat method, formaldehyde titration and ninhydrin colorimetry were not much different, and all of them were highly accurate.
Rice protein, with its reasonable amino acid composition, high bioavailability and hypoallergenicity, has been highly regarded by researchers, and a variety of extraction and application methods have been developed for rice protein, as well as related functional products. Among them, the use of papain, alkaline protease and neutral protease to hydrolyze rice proteins into soluble peptides under certain conditions has become one of the mainstream directions of rice protein research [1].
Enzymatic hydrolysis of proteins helps to improve their nutritional value and functional properties, and during the hydrolysis process, the quality control of the products is of great importance in influencing their applied properties. The degree of hydrolysis of rice proteins needs to be strictly controlled in order to obtain the desired special flavor and functional properties. The degree of protein hydrolysis is an important indicator of the degree of protein hydrolysis[2] . Currently, the pH-stat method is used to determine the degree of protein hydrolysis [3]. The pH-stat method [3], the formaldehyde titration method [4-5] and the ninhydrin colorimetric method [6] are the main methods to determine the degree of protein hydrolysis. Different methods lead to large deviations in the final results, and there are few reports on the comparisons and differences between these methods. In this experiment, the hydrolysis degree of rice protein enzymatically digested by alkaline protease under certain technological conditions was investigated and compared, and the variation pattern of hydrolysis degree was investigated through the comparison of four different detection methods, so as to further improve the accuracy of the test.
1 Materials and methods 1.1 Experimental materials
Ground rice: homemade in the laboratory; alkaline protease Alcalase2.4 L FG: Novozymes Enzyme Preparation Co. Bovine serum albumin (biochemical reagent): Institute of Hematology, Chinese Academy of Medical Sciences; the rest of the reagents (analytically pure): Sinopharm Chemical Reagent Co.
1.2 Experimental apparatus
VAPODEST50 Kjeldahl Nitrogen Analyzer: Gerhardt, Germany; SevenMulti S40 pH meter, T70 Potentiometric Titrator: Metallurgy-Toledo Instruments (Shanghai) Co; UV2550 Ultraviolet Visible Spectrophotometer: Shimadzu Enterprise Management (China) Co.
1.3 Determination of conventional composition of crushed rice
Protein content is determined according to GB 5009.5-2016 semi-micro Kjeldahl method with conversion factor F of 5.95; fat content is determined according to GB 5009.6-2016 Soxhlet extraction method; moisture content is determined according to GB 5009.3-2016 105 ℃ constant weight method; ash content is determined according to GB 5009.4-2016 550 ℃ cauterization method; starch content is determined according to GB 5009.9-2016 Fehling's method. The ash content is determined according to GB 5009.4-2016 550 ℃ cauterization method; the starch content is determined according to GB 5009.9-2016 Fehling's method.
1.4 Enzymatic process of rice protein digestion
Crushed rice → pulverized through a 100-mesh sieve → add water → adjust pH9.0 → add alkaline protease ([E]/[S]) 0.6% c. Constant temperature water bath at 50 ℃ for 4 h c. Inactivation of the enzyme (80 ℃, 15 min) c. Centrifugal separation (10,000 r/min, 4 ℃, 3 min) c. Supernatant → freeze-drying → rice protein.
1.5 Methods for determining hydrolysis
Hydrolysis degree is an important indicator of the degree of hydrolysis of proteins and refers to the proportion of peptide bonds in a protein molecule that are broken due to biological or chemical hydrolysis as compared to the total number of peptide bonds in the protein molecule [7]. The degree of hydrolysis is calculated by equation (1):
DH = h/htot×100% (1) where: DH is the degree of hydrolysis, %; h is the amount of hydrolyzed protein per gram of hydrolyzed protein.
The number of millimoles of peptide bonds cleaved, mmol/g; htot is the number of millimoles of peptide bonds per gram of raw protein, mmol/g.
For a particular protein, htot is a constant that can be calculated from the amount of amino acids that make up the protein. In this paper, the htot of rice protein was taken as 6.70 mmol/g [8].
1.5.1 Titration of formaldehyde
Formaldehyde can combine with -NH3+ on amino acids to form hydroxymethyl derivatives such as -NH- CH2OH, -N(CH2-OH)2, etc., freeing the H+ from -NH3+ . The H+ on -NH3+ can be freed, and the free amino acid content can be calculated by titrating the released H+ with alkali and measuring the amino nitrogen content.
1 mL of enzyme solution +10 mL of distilled water +3 drops of phenolphthalein, titrate with 0.01mol/L standard NaOH solution to pH8.2, recorded as V1, add 5 mL of neutral formaldehyde and mix well, titrate with standard NaOH solution to pH9.2, recorded as V2, and use 1 mL of distilled water instead of the enzyme solution to titrate as a blank V0, and the samples and blanks were done in 3 times parallel tests. Amino nitrogen and degree of hydrolysis in the liquid to be measured were calculated according to equations (2) and (3): 2)
× 100% (3)
Where: DH is the degree of hydrolysis, %; M1 is amino nitrogen, g/g; M2 is total nitrogen of raw materials, g/g.
1.5.2 pH-stat method
The process of protein hydrolysis is actually the breaking of the peptide chain of a protein, releasing the amino and carboxyl groups. The dissociation of the carboxyl group and the protonation of the amino group are strictly controlled by pH and temperature. Under certain conditions, the breakage of the peptide chain during enzymatic hydrolysis results in the release of H+ and a decrease in pH, which requires a fixed amount of lye to maintain a constant pH.
Where: DH is the degree of hydrolysis, %; B is the volume of base, mL; Nb is the concentration of base, mol/L; α is the average degree of amino acid dissociation, α=0.99 in this study; MP is the amount of protein in the substrate, g; hhot is the mass molar concentration of peptide bonds in protein, mmol/g.
1.5.3 Ninhydrin colorimetric method
Ninhydrin reacts with α-amino acid to produce blue substance, which has the maximum absorption near 568 nm, and the color of the blue substance is proportional to the content of amino acid [9]. Establishment of standard curve: 100 mg of glycine was dissolved in distilled water and then concentrated to 100 mL to obtain 1,000 mg/L glycine solution, which was diluted to make the final color development of the concentration of the solution in the range of 0.5-6.0 mg/L. 2 mL of the solution to be tested was used for the color development reaction, and the absorbance value was measured at 568 nm, and the sample with the addition of distilled water was taken as the blank. The standard curve was plotted according to the measured values. Prepare a series of different concentrations of glycine standard solution, respectively, take 2 mL of ninhydrin color reaction, through the previously determined standard curve to calculate the content of glycine, the standard curve is shown in Figure 1.
Glycine concentration/mg-L-1
Fig. 1 Standard curve of Glycine
1.5.4 TCA index method
After adding 10% TCA solution to the digest, as the hydrolysis reaction proceeds, the peptide chain of the protein is gradually cut off, and the solubility index of TCA increases. For a specific substrate, the TCA nitrogen solubility index can qualitatively reflect the enzyme digestion of proteins.
Take 5 mL of enzyme solution and add equal volume of 10% TCA solution, mix and shake, let it stand for 30 min, centrifuge at 4000 r/min for 30 min, and take the supernatant to determine the soluble nitrogen (Folinol method). The standard curve of bovine serum albumin is shown in Figure 2.
(5)
Where: DH is the degree of hydrolysis, %; N2 is the total soluble nitrogen of TCA in the supernatant after hydrolysis, μg/mL; N1 is the total soluble nitrogen of TCA in the supernatant before hydrolysis, μg/mL; N3 is the total nitrogen of the raw material, μg/mL.
0.8 - 0.7 - 0.6 - 0.5 - 0.4 - 0.3 - 0.2 - 0.1 -0
Bovine serum albumin concentration/μg-mL-1
Fig. 2 Standard curve of bovine serum albumin
2 Results and analysis
2.1 Results of the four methods determined during enzymatic digestion As can be seen in Figure 3:
(1) After the enzyme starts hydrolysis, the degree of protein hydrolysis increases continuously, the degree of hydrolysis increases greatly in the first 30 min, and as the enzyme hydrolysis process proceeds, the increase in the degree of hydrolysis becomes smaller and smaller, and finally tends to stabilize.
(2) Degree of hydrolysis measured by the TCA index method among the four measurement methods
The pH-stat method is generally high, followed by the pH-stat method, the formaldehyde titration method, and finally the ninhydrin colorimetric method. The high TCA index results are mainly due to the amount of acid soluble proteins prior to enzymatic hydrolysis. pH-stat is highly reproducible and suitable for neutral and alkaline hydrolysis conditions. Formaldehyde titration is affected by the amino acid composition of proteins, and the results of different sources of proteins are not the same. The degree of hydrolysis of enzymatically solubilized rice proteins determined by this method is slightly smaller than that of pH-stat. The ninhydrin colorimetric method is less stable and uses a single amino acid as a standard, which can lead to differences in results.
pH-stat method Ninhydrin colorimetric method Formaldehyde titration method TCA index method
0 0
Time/min
Fig. 3 Results of the four assays in the enzymatic digestion process
2.2 Analysis of the significance of the differences between the results of the four methods 2.2.1 ANOVA chi-square test of the assay methods
As shown in Table 1, the F-values between the four groups of results were less than F0.05(9 ,9), indicating that the differences between the four groups of results were not significant, i.e., the precision of hydrolysis was consistent among the four methods.
Table 1 F-test of the results of the four groups
Methods TCA method pH-stat method Formaldehyde titration Ninhydrin colorimetric method
TCA Act - 2.11 2.84 3.13
pH-stat method 2.11 pH-stat method 2.11 1.35 1.48
Formaldehyde titration 2.84 1.35 1.10
Ninhydrin colorimetric method 3.13 1.48 1.10 -
Note: F0.05(9,9) = 3.18.
2.2.2 Differences in detection methods
As shown in Table 2, the t-value of the TCA index method was greater than t0.05(18) with the other three methods, so there was a significant difference with the other methods; the t-values of pH-stat, formaldehyde titration, and ninhydrin colorimetry were all less than t0.05(18) at the confidence level of α of 0.05, which indicated that the differences among the three methods were not significant.
3 CONCLUSIONS
The results of protein hydrolysis by TCA index, pH-stat, formaldehyde titration and ninhydrin colorimetry could accurately reflect the enzymatic hydrolysis of rice proteins. Among them, the results of TCA index method were relatively high, and there was a significant difference with the other three methods. The formaldehyde titration method, as a classic method for protein hydrolysis, was in good agreement with the pH-stat method, which is more popular in the world. The overall results of the ninhydrin colorimetric method were low due to the amino acid composition of rice proteins, but the differences were within acceptable limits. In the future study of enzymatic hydrolysis of proteins, we should select the test method according to the test object of our own choice, so that we can accurately reflect the experimental process, understand the change rule of the experimental object more deeply, and help to establish the model of the whole process of enzymatic hydrolysis.
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