Does Storage Time Affect Rice Protein Fractions?

 Rice protein consists of four main proteins: serum protein, globulin, alcohol-soluble protein and gluten. Rice protein is an important protein source for human diet. In rice protein, alkali-soluble gluten with high lysine content accounts for 80%, lysine content is higher than that of other cereals, and the amino acid composition is reasonable, close to the optimal amino acid ratio pattern of proteins recognized by the World Health Organization, and the bio-value (BV value) of rice protein is 77, and the protein efficiency ratio (PER value) is 2.2 (wheat is 1.5, corn is 1.5, corn is 1.5, corn is 1.5, and corn is 1.2). (1.5 for wheat and 1.1 for corn), and the digestibility of the protein was 2.2 (1.5 for wheat and 1.1 for corn). The digestibility of rice protein is more than 90%, which is higher than that of other grains, so the nutritional value of rice protein is very high [1]. Rice protein also has an important influence on the eating quality of rice. Several physicochemical properties, such as straight chain starch content, protein content, gel consistency and alkaline dissipation value, are usually used to reflect the quality of cooking flavor. The level of protein content directly affects the water absorption of rice grains during cooking, and rice with higher protein content has lower viscosity, is looser, and produces relatively hard rice[2] .

 

After a long period of storage, due to the effects of temperature and humidity, the processed rice lacks the aroma and has a dull color compared to fresh rice (often called stale rice). After cooking, the eating quality of stale rice decreases. The quality of rice products made from stale rice is also poor. During storage, rice undergoes a series of changes in starch, protein, and fat. Storage time, temperature, humidity and oxygen are the main factors affecting the quality of rice. Currently, studies on the changes in the protein composition of rice grain during storage have focused on the changes in total protein content, amino acid content, glutenin molecular mass, and glutenin sulfhydryl and disulfide bond content [3-7]. Likitwat-tanasadE et al. [8] conducted storage experiments on rice at 60°C and 70% relative humidity, and found that the pasting characteristics and microstructure of rice changed under these conditions.

 

In this study, we investigated the changes in the content of various components and protein subgroups of rice at five different storage times through high temperature and high humidity storage, analyzed the reasons for these changes, and explained the changes in the quality of rice during the storage period from another point of view, so as to provide a certain reference for the theoretical research on the storage of rice, and it is of great significance for the improvement of the nutritive value and the quality of the rice flavor.

 

1 Materials and Methods   

1.1 Materials and reagents

Rice: commercially available Feng Er You medium rice, courtesy of Fuwa Group Rice Company 2014-05, no special treatment.

Non-pre-stained protein molecular mass standard, Thermo Company, USA; Bradford protein quantification kit, BSA standard solution, Tiangen Biochemical Science and Technology (Beijing) Co, TEMED, acrylamide, N,N'-methylenebisacrylamide, tris(hydroxymethyl)aminomethane, n-hexane, sodium dodecyl sulfate, β-mercaptoethanol, and Kaomas Brilliant Blue R-250, were chemically pure.

 

1.2 Major instruments and equipment

BSC-400 Liquid Crystal Display Constant Temperature and Humidity Box; TDZ5 Tabletop Multi-Tube Automatic Balancing Centrifuge; GZX-9070 MBE Digital Display Blast Drying Oven; 90-2 Constant Temperature Magnetic Stirrer; FR96-2 Powder Dissolution Oscillator Gel Doc EZ Imager; Mini-PROTEAN Tetra System Vertical Electrophoresis System with Mini-PROTEAN Compact Vertical Vessel, PowerPac Basic Electrophoresis; Gel Doc EZ Imager Gel Imaging System with White Tray Plate. Gel Doc EZ Imager Gel Imaging System with White Tray Photo Plate.

 

1.3 Methodology

1.3.1 High temperature and high humidity storage of rice

Referring to the general storage methods in the literature [8] and [9], five equal portions of the experimental rice grains were placed in a constant temperature and humidity box and stored at high temperature (45 ℃) and high humidity (95% relative humidity) for 0, 24, 48, 72, and 96 h (which corresponds to about 0, 2, 7, 15, and 28 months of storage under normal storage conditions).

 

1.3.2 Preparation of 4 fractions of rice proteins

According to Osborne's solubility classification, rice proteins were classified into four categories: water-soluble proteins (clear proteins), salt-soluble proteins (globulins), alcohol-soluble proteins, and alkali-soluble proteins (glutenins), and the extraction methods were as follows[10] .

Skimmed rice flour: firstly, crush the raw rice, pass through 80~100 mesh sieve, then dilute it into a mixed solution with mass concentration of 20 g/100 ml with hexane, magnetic stirring for 3 h, centrifugation at 3,570 g for 15 min, starch the rice flour, and then air-dry it in a ventilated kitchen overnight. The subsequent experiments used defatted rice flour as raw material.

 

Water-soluble protein: soak rice powder in double-distilled water, the mass concentration is 20 g/100 ml, magnetic stirring for 4 h, centrifugation at 3,960 g for 15 min, take the supernatant, repeat centrifugation for 1 time, take the supernatant, and then concentrate it to obtain water-soluble protein of rice.

Salt-soluble protein: Soak rice flour in 50 g/L ammonium sulfate solution with a mass concentration of 20 g/100 ml, magnetic stirring for 4 h, centrifuge at 3,960 g for 15 min, take the supernatant, add ammonium sulfate to precipitate the protein, adjust the ammonium sulfate to 50% (solubility) saturation, and settle the protein at 4 ℃ overnight, centrifuge at 3,960 g for 15 min, take the precipitate, and desalinate it with water, then obtain the salty soluble protein of rice. The protein is soluble in water.

 

Alcohol-soluble protein: soak rice flour in 75% volume fraction ethanol, the mass concentration of 20 g/100 ml, magnetic stirring for 4 h, centrifuged at 3,570 g for 15 min, the supernatant was taken and concentrated to obtain rice alcohol-soluble protein.

Alkaline soluble protein: Soak rice flour with 0 smooth 09 mol/L sodium hydroxide, the mass concentration is 20 g/100 ml, stirring magnetically for 4 h, centrifuging at 3,570 g for 15 min, taking the supernatant, adjusting the pH value to 7, centrifuging at 3,570 g for 15 min, taking the precipitate, dissolving it in PBS, and desalting it by dialysis, obtaining the alkaline soluble protein of rice.

 

1.3.3 Preparation of four fractions of proteins from rice with different storage times

In accordance with 1 . 3 . 2 to extract the four fractions of proteins from rice stored for 24, 48, 72, and 96 h. The proteins of the four fractions were extracted from rice stored for 24, 48, 72, and 96 h.

 

1.3.4 Determination of changes in protein content of four fractions of rice during storage

The four proteins of rice after different storage times were prepared according to the above method, and the contents of the four protein fractions were obtained by taking appropriate samples, freezing, drying and weighing.

 

1.3.5 Determination of protein content in rice

The Kjeldahl method was used with reference to GB 5009.5-2010, and the protein coefficient was 5:95. 5 - 2010 streak National Standard for Food Safety Determination of Protein in Foods[11] , and the protein number was 5:95.

 

1.3.6 SDS-PAGE Electrophoresis

Using the Leammli system[12] , the separated gel contained 12 g/100 ml acrylamide, 0.4 g/100 ml N,N'-methylenebisacrylamide, 0.1 g/100 ml sodium dodecyl sulfate, 0.375 mol/L Tris-HCl, pH 8.8. Concentrated gel contains 5 g/100 ml acrylamide, 0.17 g/100 ml N,N'-methylenebisacrylamide, 0.1 g/100 ml sodium dodecyl sulfate, 0.125 mol/L Tris-HCl, pH 6-8. The electrophoresis buffer contained 0 Chang 302 g/100 ml Tris, 1 Chang 88 g/100 ml Glycine, 0 Chang 1 g/100 ml Sodium Dodecyl Sulfate, pH 8 Chang 3.

Sample buffer: 0.06 mol/L Tris-HCl (pH 6 to 8), 25% v/v/v propanetriol, 2 g/100 ml sodium dodecyl sulfate, 5% v/v/v β-mercaptoethanol, 0.1 g/100 ml bromophenol blue.

Constant-voltage electrophoresis was performed at an initial current of 60 V. After the samples entered the separation gel, the current was changed to 100 V. The samples were stained with Cauloblue R-250 overnight, and then decolorized with decolorizing solution for 4~5 h until the background was clear. The data were analyzed using Bó Lè's data processing software.

 

2 Results and Discussion

2.1 Analysis of water-soluble protein content and its fractions in rice storage process

From Figure 1(a), it can be seen that the water-soluble protein in rice slightly decreased during the storage process. Raw rice (0 h), water-soluble protein content of 0.12%, in the storage of 24 h of rice content of 0.11%, when the storage of 96 h, rice in the water-soluble protein content decreased to 0.10%. The water-soluble protein content in rice decreased to 0.10% at 96 h. During the storage process, the water-soluble protein content decreased by about 16.67%. 67%.

For the purpose of analysis, the electrophoretic profiles were divided into three regions according to the molecular mass range. Zone A is for molecular masses greater than 50 ku, zone B is for molecular masses between 25 and 50 ku, and zone C is for molecular masses less than 25 ku (electrophoretic profiles of salt-soluble, alcohol-soluble, and base-soluble proteins).

 

As shown in Fig. 1(b) and analyzed by Borel data, the water-soluble proteins of rice mainly existed in the B and C regions, and the main subunit bands were distributed in the 12-13 ku of the C region. According to the band mobility, the two main protein subunits were named a and b, respectively. At 0 h, the contents of a and b subunits were 73.94% and 22.35%, respectively. At 0 h, the contents of a and b subunits were 73.94% and 22.35%, respectively, and the contents of B subunits were 3.86%; with storage, the contents of a subunit increased to 93.33% at 24 h, b subunit disappeared, and the contents of B subunits were 6.77%; and the contents of a subunit decreased to 69.15% and b subunit increased to 6.77% at 48 h. At 48 h, the content of a subunit decreased to 69.15%, the content of b subunit increased to 26.20%, and the content of B subunit decreased to 4.72%; at 72 h, the content of B subunit decreased to 2.34%, and the content of a subunit decreased to 4.72%. At 72 h of storage, the content of B subunits decreased to 2.34%, the content of a subunit decreased to 61.90%, and the content of b subunit continued to increase. At 72 h of storage, the content of B subunit decreased to 2.34%, the content of a subunit decreased to 61.90% and the content of b subunit continued to increase to 35.88%. 88%; after 96 h of storage, the content of a subunit decreased to 59.50%, the content of b subunit decreased to 29.45%, and the content of B subunit increased to 11.12%. 12%.

 

It was analyzed that the water-soluble proteins underwent the process of decomposition and synthesis among the B subunit, a subunit and b subunit as the storage progressed. At 24 h, the b subunit basically disappeared, the a subunit increased significantly, and the B subunit also increased slightly, indicating that the b subunit mainly decomposed and synthesized the a subunit. With the increase of storage time, the b subunit reappeared at 48 h, and the content of b subunit increased to a maximum of 35.81% at 72 h. The content of b subunit decreased to a maximum of 35.81% at 96 h. 81% at 72 h, and decreased to 29.42% at 96 h. The a subunit, on the other hand, was not present at all. The content of a subunit decreased during storage except for the increase from 0 to 24 h. The content of B subunit had the same trend as that of a subunit from 0 to 72 h, but increased slightly at 96 h. The b subunit was decomposed and then synthesized, while the a subunit, on the other hand, had the same trend of the change of the B subunit and the a subunit. As a more active component in the storage process, rice albumen first consumed the b subunit to synthesize the a subunit, and then the a subunit became the main consuming component to synthesize the b subunit, and the change in the content of the b subunit was not significant at the beginning and the end of the storage process. Similar to the action of the enzyme, the end result is an increase in the polymeric subunits in the B region (1.92-fold) and an increase in the average molecular mass. Similar to the action of the enzyme, the end result is an increase in the macromolecular subunits in the B region (1.92-fold), an increase in the average molecular mass, and a decrease in the total serum protein. Figure 1(c) shows that the results of this electrophoresis experiment are meaningful.

 

2.2 Analysis of salt-soluble protein content and its fractions in rice during storage

From Fig. 2(a), it can be seen that the saline soluble protein of rice showed a decreasing trend during the storage process. The salt soluble protein content of raw rice was 0.58%, and at 24, 48, 72 and 96 hours of storage, the salt soluble protein content was 0.59%, 0.51%, 0.53%, 0.53%, 0.53%, 0.53%, 0.53%, 0.53%, 0.53%, 0.53% and 0.53% respectively. 0.59%, 0.51%, 0.53%, 0.48%, salt soluble protein content At 24, 48, 72 and 96 hours of storage, the salt-soluble protein content was 0.59%, 0.51%, 0.53% and 0.48%, respectively. 24%) during storage, which was generally consistent with the report[1 ].

 

From Figure 2(b), it can be seen that the molecular mass of rice salt-soluble proteins was mainly distributed in the range of 12~105 ku, which was basically consistent with the research report (16130 ku) [13 ]. 12, 17 . 12, 17.5, 22, and 50 ku are the main subunit groups. The presence of subunit proteins with similar molecular mass to water-soluble proteins around 12 ku indicated that the water-soluble proteins and salt-soluble proteins contained similar components.In the A region, there were three obvious subunit bands, named a, b, and c. The content of the a subunit decreased during the storage process, and the content of the a subunit dropped from the initial 1.00% to 0.54% at 96 h. The content of the b subunit did not vary much, and remained constant during the storage process. The content of the b subunit did not change much and remained at 1.98%~2.10% during storage. The content of b-subunit did not change much and remained at 1.98% ~ 2.10% during storage; the content of c-subunit increased from the initial 12.40% to 15.53% at 24 h, and the content of c-subunit increased from the initial 12.40% to 15.53% at 24 h during storage. 53% at 24 h, and then remained at 14.40% ~ 16.15% during the subsequent storage period, with little change. 15% . The contents were 7.57%, 15.60%, 15.56%, 13.28% and 11.19% respectively. The contents were 7.57%, 15.60%, 15.56%, 13.28% and 11.19% respectively. The r subunit content increased slightly (11.43% ~ 19.33%) at 24~48 h. The content of r subunit increased a little (11.43%~19.33%) at 24~48 h; the content of s subunit decreased from 3.10% to 1.61% at 72 h. However, at 96 h, the content of s subunit decreased from 3.10% to 1.61%. The s subunit content decreased from 3.10% to 1.61% at 72 h, but then increased to 2.78% at 96 h. The t-subunit content started to stabilize at around 12.50% from 0 to 48 h, and then increased to 19.40% and 24.80% at 72 h and 96 h, respectively. At 72 h and 96 h, it increased to 19.40% and 24.80%, respectively.

The content of salt-soluble proteins varied considerably during rice storage. The content of α-globulin, the main component, decreased (25%), the a-subunit of the high-mass subunit decreased by almost half (46%), the content of the low-mass t-subunit increased (98%), and the other components did not change much. Figure 2(c) shows that the results of this electrophoresis experiment are meaningful.

 

2.3 Analysis of alcohol-soluble protein content and its fractions in rice storage process

As can be seen from Fig. 3(a), the content of alcohol-soluble proteins in rice remained basically unchanged during the storage process and was maintained at about 0.43%. 43%.

As shown in Figure 3(b), the subunit distribution of rice alcohol-soluble proteins was relatively homogeneous, with the molecular masses of the two main subunit bands around 13 and 15 ku. The contents of the 13 ku subunit at five different storage times were 91.40%, 90.12%, 90.36%, 90.07%, and 96.65%, respectively. The contents of the 13 ku subunit at five different storage times were 91.40%, 90.12%, 90.36%, 90.07% and 96.65%, respectively. The content of 13 ku subunit was 91.40%, 90.12%, 90.36%, 90.07%, and 96.65%, respectively, at five different storage times. After 96 h of storage, the 15 ku subunit content decreased to 3.42%. In general, the alcohol-soluble proteins did not change much during rice storage and were a relatively stable component. Figure 3(c) shows that the results of this electrophoresis experiment are meaningful.

 

2.4 Analysis of alkali-soluble protein content and its fractions during rice storage

From Fig. 4(a), it can be seen that the alkali-soluble protein content of rice decreased slightly during storage. The alkali-soluble protein content of raw rice was 6.18%, which decreased to 6.0% at 24, 48, 72 and 96 h of storage, respectively. 18% . The content decreased to 6.14%, 6.15%, 6.15%, 6.13% and 6.13% at 24, 48, 72 and 96 h, respectively. 13% and 6 . 12%, with a reduction rate of 0.97%. The alkali soluble protein content decreased with storage, but the rate of change was small.

 

From Figure 4(b), it can be seen that the alkali-soluble protein subunits were distributed in A, B and C regions, and the subunit bands around 14, 18 and 31 ku were the main components. For the convenience of analysis, there are five subunit bands in zone A according to the migration rate from small to large, named as a, b, c, d, e, etc. There are fewer subunits in zones B and C, and the subunit bands around 50, 31, 22, 18 and 14 ku are named as α, β, γ, δ, ε, etc. The subunit contents of the different storage times in zone A are 13.00%, 14.33%, 16.58%, 21.58%, 21.58%, 21.58% and 21.58%, respectively. In Zone A, the substituent contents at different storage times were 13.00%, 14.33%, 16.58%, 21.47% and 28.40% respectively. In Zone A, the contents of subunits at different storage times were 13.00%, 14.33%, 16.58%, 21.47% and 28.40% respectively. Among them, the contents of a subunit were 11.79%, 12.79%, 12.58%, 12.47% and 28.40%, respectively. The a-subunit contents were 11.79%, 12.55%, 12.71%, and 28.40%. The content of a subunit was 11.79%, 12.79%, 12.55%, 12.71%, 17.38%, and 19.15% respectively, and the content of this subunit increased significantly with storage (62.43%); b, c, d, and e subunits disappeared in two bands with storage, and then decreased to three subunits at 24 h, and then decreased to two bands at 96 h, and the content of these subunits gradually increased from 1.2% at 0 h to 1.4% at 0 h. The content of the b, c, d, and e subunits increased gradually from 1.2% at 0 h to 1.4% at 0 h. The content gradually increased from 1.2% at 0 h to 1.78%, 1.3% and 1.4%, respectively. The content gradually increased from 1.2% at 0 h to 1.78%, 1.88% and 4.10%. The content of α-subunit gradually increased from 1.2% at 0 h to 1.78%, 1.88%, 4.10%, but decreased to 1.58% at 96 h. The content of the α-subunit was 4.24%, 4.00%, 5.06% and 4.28% at all times, respectively. The alpha subunit content was 4.24%, 4.00%, 5.06%, and 4.28% at all times. The contents of these subunits fluctuated during the process of changing from raw rice to aged rice. In 0-48 h, the contents of α, β, and δ subunits changed similarly, decreasing and then increasing, and the opposite was true for ε subunits; in 48-96 h, the contents of α and δ subunits decreased and then increased with the increase of time, while the contents of β and ε subunits decreased all the time. a subunits (202 ku) and α subunits (50 ku) increased by 62 and 43%, respectively. The contents of a subunit (around 202 ku) and α subunit (around 50 ku) increased by 62.43% and 86.56%, respectively, while the contents of β subunit and ε subunit decreased by 45.87% and 39.70%, respectively.

 

It has been demonstrated that the low-molecular-mass bands decreased, the high-molecular-mass bands increased, and the average molecular mass of glutenin increased in rice glutenins stored for 1-3 a, in agreement with the experimental results[15] . It has also been reported that a class of isoenzymes (LOX) in rice grains may be directly involved in the oxidative cross-linking of intramolecular and intermolecular disulfide bonds of storage proteins and other macromolecules in rice grains, affecting their structure and function [16]. Gluten is the most important storage protein in rice, which can bind more tightly with starch, resulting in a significant increase in the high molecular mass of proteins, and the emergence of the γ subunit after 96 h of storage in this experiment may be due to the breakage of one of the protein bonds, which breaks down and becomes an alkali-soluble protein. Figure 4(c) shows that the results of this electrophoresis experiment are meaningful.

 

3 Conclusion

During the storage process of rice, the total content of water-soluble proteins decreased by 16 Chang 67%, with active changes during the storage process, and a certain low molecular mass subunit underwent an enzyme-like action, decomposing and then synthesizing; the total content of salt-soluble proteins decreased by 17 Chang 24%, and the components of the protein subunits changed greatly, with the content of the main component, α-globulin, decreasing by 25 Chang 30%. The total content of salt-soluble proteins decreased by 17-24%, and the content of α-globulin decreased by 25-30%, while the content of alkali-soluble proteins decreased by 97%, and the content of high-molecular-mass (202 ku) subunits increased significantly to 62-43%, and a subunit with a molecular mass of 22 ku appeared in the protein after 96 h of storage; the alcohol-soluble proteins mainly existed in a low-molecular-mass (13 ku) subunit, and there was basically no change of the protein content and protein fractions in the process of storage.

 

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