Brown rice and whole wheat grains (whole wheat): The effect of oxygen absorber on the storage of free fatty acids during storage and storage

Abstract

The continuous rancidity of fats in whole grain foods such as brown rice and whole wheat pose a problem for wheat producers. Therefore, it is of great importance to remove the spoilage process of existing fats during the time after harvesting the seeds for more profitability.

In this study, the effect of oxygen absorber on reducing the rate of deterioration and spoilage of cereal grains has been investigated. The seeds and flour of brown rice and whole wheat seeds were stored for 8 weeks at 4 degrees Celsius and 26 degrees Celsius, with and without oxygen absorber.

The mass of free fatty acids in bran seeds increased during the storage period by grinding and separating the bran. Flour making (grinding) and storage was done at a relatively high temperature (26 degrees Celsius). The reason for these storage conditions was the simulated activities related to the hydrolysis of fats.

When the oxygen absorber was added and the chamber was properly closed, the mass of free fatty acids FFAs (Free Fatty Acids in unmilled seeds) in both cases Absolute amounts of linoleic acids of lipids And Total tocochromanols in these unmilled seeds reduces and suppresses these variables by adding oxygen absorber to the maintenance system. The reason for this can be explained by the anti-oxidizing property of the oxygen absorber.

These results showed that the addition of oxygen absorber to the storage system of unmilled grains is effective under the conditions that the hydrolytic activities of fats are suppressed.

Introduction

These results showed that the addition of oxygen absorber to the storage system of unmilled grains is effective under the conditions that the hydrolytic activities of fats are suppressed.

In addition, they have more benefits than ground ones, and they also seem to be one of the important raw foods in Asian countries.

However, the spoilage of fats in these unmilled grains is much higher compared to bran-free grains. The reason for this can be attributed to the presence of fats and fat-degrading enzymes in bran and its roots.

Since the breakdown of fats in cereal grains is responsible for food loss, the production and development of techniques to prevent it is of relatively high importance not only for quality control but also for increasing the consumption of cereal grains. Many researchers have studied the storage stability of whole grain rice and wheat flour due to their industrial uses. For example, storage of whole grain rice at high temperatures was investigated. The result was that high temperature causes the hydrolysis of fat repeatedly and thus causes the oxidation and rancidity of fats.

Regarding the study of wheat flour, it was observed that the process of corruption and degradation of fats depends on their flour making conditions and the size of flour particles. However, little is known about the stability and storage stability of fats found in the whole kernels of these whole grains, such as brown rice and whole wheat grains. In addition, comparative studies on the degradation of fats among the kernels and flours of these cereal grains are essential for food industry disciplines.

In recent years, several packaging technologies have been developed alternately in order to increase the shelf life of different food products. Among them, the deoxidizer (called oxygen absorber) is one of the most widespread and widely used methods.

Among chemical oxygen absorbers, processed iron powder that absorbs oxygen in the atmosphere and thus oxidizes itself is widely known as oxygen absorber.

For example, Ageless (Mitsubishi Chemical Gas Company in Japan) in order to reduce the level of oxygen concentration in the atmosphere to less than 0.1% through a series of chemical reactions that lead to the production of ferrous oxide (Fe2þ), of course, when in a leak-proof chamber. be packed

In previous studies, this type of oxygen absorber was successfully used to preserve several types of breakfast cereals and nuts (Doblado-Maldonado et al., 2012; Jensen, Sorensen, Brockhoff, & Bertelsen, 2003; Li-Xin & Fang, 2010; Mexis & Kontominas, 2010;Pastorelli, Valzacchi, Rodriguez, & Simoneau, 2006; Rose et al.,2008; Tarr & Clingeleffer, 2005; Yoshida et al., 2011). Regarding the use of oxygen absorbers for the storage system of cereal grains, the effect of oxygen absorbers on the processes of deterioration and spoilage of their fat should be investigated and tested.

For this purpose, brown rice kernels and flour, rice sprouts and whole wheat grains were studied under different conditions with or without oxygen absorber in this research. The deterioration of fats related to seeds during storage usually starts with the hydrolysis of fats and is done by lipase (lipase is any type of enzyme that accelerates the hydrolysis of fats and acts like a catalyst), this reaction takes place and free fatty acids fatty acids (FFAs) are released.

Because free fatty acids or FFAs are one of the mediators in the process of fat breakdown and were subsequently degraded by different reactions such as chemical and biological oxidation reactions, in this experiment hydraulic activities related to lipase using published amounts of free fatty acids or FFAs It cannot be obtained and calculated directly.

Nevertheless, tracking the mass of free fatty acids is able to express and measure differences between the rate of hydrolysis of lipids and other degradation reactions. A comparison of free fatty acids in kernels and seeds stored with and without oxygen scavenger gives you a good idea of the effect of oxygen scavenger on fat deterioration during this process.

In this study, in addition to the mass of free fatty acids, total tocoramanol content and absolute amounts of unsaturated fatty acids in seeds stored in simulated conditions were investigated in order to estimate the antioxidant effects of oxygen absorbers.

2- Materials and methods

2.1- Materials

Mature and fully grown rice seeds and mature wheat seeds that were investigated in this research were gifts from the National Institute of Harvest Science, National Institute of Agriculture and Food Research Organization. Rice seeds were harvested in Ibaraki, Japan during the fall of 2010. Wheat seeds were obtained in Hokkaido, Japan in spring 2010.

The kernels were removed mechanically (to prepare semi-threshed rice such as sprouted rice), also with an internal milling machine. were milled The rice kernels along with the roots were manually separated and selected to remove the cracked or damaged kernels.

2.2- Solutions

Thin layer chromatography (TLC) related plates (silica gel 60, dimensions 20 x 20 with a thickness of 0.25 mm) were obtained from Merck (Darmstadt, Germany).

The TLC standard composition containing triglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG) and free fatty acids or FFAs were purchased from Nakalai Teske (Kyoto, Japan).

As standards for measurement, tocopherols (alpha, beta, gamma, and sigma tocopherols) and tocotrienols (alpha, beta, gamma, and sigma tocorinols) were kind gifts from Eizai Food Chemicals Co., Ltd. (Tokyo, Japan), and 2, 2, 5 7, and 8-pentamethylhydroxychroman were obtained from Wako Pure Chemical Industries.

To analyze and check the composition of fatty acids, several standards of fatty acid methyl ester (FAME) were purchased from Wako Pure Chemical Industries (Osaka, Japan). For HPLC solvents, analytical grade hexane, acetonitrile, and isopropanol were obtained from Wako Pure Chemical Industries and Kanto Chemical (located in Tokyo, Japan).

Chemical solutions, including potassium hydroxide, anhydrous sodium sulfate, sodium chloride, butylated hydroxytoluene (BHT), were obtained from Wako Pure and Kanto Chemical Industries.

2.3- Storage and storage of seeds

The unground seeds (about 100 grams) were placed in the containers and kept at 4 degrees Celsius or 26 degrees Celsius in the dark in an incubator.

To maintain low oxygen concentration, a commercially available oxygen absorber (Ageless, 25gr Mitsubishi Gas Chemical Company, Tokyo, Japan) was placed in special packaging bags that had very low oxygen permeability. The package (bag) was stored by the heat press method and by the device at a temperature of 4 degrees or 26 degrees Celsius.

The oxygen concentration inside the packaging during different storage periods was less than 0.1%, which was measured by an indicator plate (Mitsubishi Gas Chemical Company, Tokyo, Japan). Its color changed from blue to pink when the oxygen concentration was less than 0.1%. In addition to the samples containing unmilled seed kernels, samples containing flour were also prepared.

Each grain (brown rice, rice with roots and whole wheat grain) was converted into powder and ground with an internal grinder (Miller 800DG, Iwatani Co., Tokyo, Japan) and after some time the grinding process was continued in a mortar. Samples containing flour were also stored under the same conditions as brains.

After 4, 2 and 8 weeks, the kernels and flour of three different types of seeds were removed from the storage containers and their fats and tocochromanols were extracted. Triplicate extractions and measurements were performed on one type of stored samples.

2.4- Extraction of fats

To extract fats from grains (brown rice, rice with root and whole wheat grain), about 20 grams of the kernel of each grain was ground and turned into powder by an internal grinder and in a mortar.

For samples containing flour, they were directly used to extract fats. For samples containing flour, they were directly used to extract fats. or Pressurized Fluid Extraction (PDE) was extracted using a Speed Extractor (E-916, NihonBUCHI K.K., Tokyo, Japan).

Using n-hexane (10 ml) as the extraction solvent, the extraction process was repeated three times at 100 degrees Celsius for 10 minutes under nitrogen gas. Aqueous KCl indicator (0.75%, 2 ml) was added to the combined extraction processes.

After the separation phase, the hexane layer was collected and dried by anhydrous Na2SO4. The solvent was also concentrated using a rotary evaporator (R-210, Nihon BUCHIK.K.) and exposed to nitrogen gas flow.

The weight of the remaining fats was measured in order to determine the content of fats, and then it was kept at minus 30 degrees Celsius until the analysis and further investigations.

In order to compare the quality and quantity of fats extracted by PFE method with those extracted by conventional methods, brown rice fats were also extracted by Folch method (Folch, Lee, & Stoane-Stanley, 1957). In addition, the acid hydrolysis extraction method was performed in order to prepare fats from brown rice according to the JOCS standard method.

2.5- Composition of fatty acids

For the methylation of fats, 20 mg of the fat extracted from the seed was placed in a small vial and its weight was measured. When the absolute amount of each FAME in the extracted fat was measured, margaric acid (C17:0, 1.0 mg ) was added to the said vial.

The two-step methylation of fats to FAMEs was achieved using 0.5 M potassium hydroxide in methanol and boron trifluoride-methanol complex in methanol (Wako Pure Chemicals Industries, Ltd.) according to the JOCS standard method.

The final concentration of FAMEs was changed to 5-10 mg/ml in n-hexanes. According to the JOCS standard method, FAME indicators were determined using a Shi-madzu GC-2010 gas chromatography device, which includes a branch injector (at a temperature of 250 degrees Celsius) and a flame ionization sensor (detector) with a system integration solution (Shimadzu). was placed, were analyzed and reviewed.

An SP-2560 column with the following specifications:

(100 m by 0.25 mm ID, and 0.2 micrometer thickness of its sheet) was optimized.

The column temperature remained at 160°C for 10 minutes, then increased to 230°C at a rate of 2°C/min, and remained at 230°C for 15 minutes.

At a speed of 23.6 cm/s and at a temperature of 180 degrees Celsius, Helium gas (> 99.99995%) was used as the carrier gas. The ratio was set at 50:1.

The peak of each FAME was identified and characterized using FAME standards and previously published information (AOCS, 2005; JOCS, 2003b). This state of investigation and analysis provided sufficient and transparent resolution for each FAME peak in the fats extracted from the seeds to determine the fatty acid composition and absolute amount of each FAME.

2.6- Thin layer chromatography

The process of spoilage and breakdown of fat in seeds during storage was detected using thin layer chromatography (TLC) method.

Each of the extracted fats were dissolved in n-hexane/diethylether solvent (13.87, v/v). The result of this work was the production of a solution of 5 mg/ml. A 20 microliter sample of the solution (0.1 mg of lipids) was separated and placed on a 60-plate silica gel in the n-hexane/diethylether/acetic acid solvent system. After preparation and development, the lipids were visualized and analyzed by pouring concentrated sulfuric acid onto the plates.

2.7- Analysis of free fatty acids

The content of FFA in the fats extracted from seeds was analyzed by HPLC fluorometric method using 9-anthryldiazomethane (ADAM, Funakoshi, Co., Ltd., Tokyo, Japan).

An aliquot of the extracted lipids was accurately weighed in a small tube and dissolved in n-hexane to produce a 10 mg/ml solution.After a 1000-fold dilution, a sample (containing 0.5 g of fat) was transferred to a fresh small tube containing tridecanoic acid (C13:0, 0.01 mg) and used as an internal standard, also solvent under hydrogen gas. came out

The ADAM solution that was dissolved in acetone was added to the tube of extracted lipids and tridecanoic acid, and the obtained mixture was dissolved by the sonication Fluorometric labeling method of FFA carboxyl groups, and ADAM solution made it possible to Operate overnight and at room temperature in the sample tube.

Fluorescence-labeled fatty acid derivatives were separated by HPLC, which consisted of a pump (JASCO model PU-880, Japan Spectroscopic Co., Ltd., Tokyo, Japan), a horizontal column oven (Shimadzu CTO-20AC, Shimadzu, Kyoto) , Japan, hs, fluorescence spectrometer (Shi-madzu RF-10AXL spectrofluorometer), and a chromatography management device (EZChrom, GL-Sciences, Tokyo, Japan). The injector was a Rheodyne Model 7725-022. Separation process on COSMOSIL Cholester C18 column (250 mm 4.6 mm I.D., Nacalai Tesque) was done.

A portion of the sample (10 μL) was injected, then thoroughly washed with acetonitrile (100%) at a flow rate of 10 mg/min. Detection was done by monitoring and observing the fluorescence intensity at 425 nm (excitation at 325 nm).

2.8- Examination of complete tococramanols

Tococramanols (tocopherols and tocotrienols) were extracted from unground seeds based on the previous method and with sample size modification (Peterson & Qureshi, 1992; Shin & Godber, 1993). Briefly, unground seeds were ground and turned into powder, and 0.5 g of each sample was transferred to a test tube. After adding ethanol, sodium chloride NaCl solution (10 g/liter of water) and pyrogallol solution (60 g/liter of ethanol), the samples were turned into foam with potassium hydroxide solution (10 N in water) during one day and night. After the foaming process, cooled sodium chloride solution and n-hexane/ethylacetate (1.9, v/v) or n-hexane/ethyl acetate were added to the sample, then mixed vigorously.After centrifugation at 4800 g for 10 minutes, the solution above the sediment was collected. The extraction process was repeated twice and the solution particles above the sediment (which were combined) were evaporated.The residue was dissolved in 2 mL of isopropyl alcohol (2%) in n-hexane and filtered through a 0.2 μm filter shell.

After adding 2,2,5,7,8-pentamethyl-6-hydroxylchroman or 2,2,5,7,8-pentamethyl-6-hydroxylchroman (abbreviated as PMH) as an internal standard An amount of 10 microliters was injected for analysis and chromatography. Chromatographic separation process of tococramanols was done by normal phase method based on the previous method (Shin & Godber, 1993). A shim-pack with the formula and dimensions of CLC-NH2 (150 mm 4.6 mm i.d., 5 mm particle size) was used and the detection and identification process was performed at an excitation wavelength of 298 nm and an emission wavelength of 325 nm. became. The amount of complete tocoramanols in the seeds was calculated from the total amount of each tocopherol and each tocotrienol.

2.9 Statistical analysis and review

Data were presented as mean +- standard deviation (SD). The results were tested by several different methods to identify significant differences in different groups. Detector values of P​​<​​0.05 were considered as significant values. All investigations and analyzes were performed using StatView software (Abacus Concepts, Berkeley, CA, USA).

3- Discussion and conclusion

3.1 Fat extraction

The extraction of total fat from the seeds is usually done by an acid hydrolysis method or by the Folch method, which is recommended by the Association of Official Analytical Chemists Official Method (AOAC, 1995) and the Standard Society of Petroleum Chemists of Japan. Japan Oil Chemists’ Society Standard Method (JOCS, 2003b) etc. are suggested. However, in this study, the process of extracting fats from seeds was done by pressurized fluid extraction (PFE) method.

First, the effectiveness of fat extraction among these three extraction methods was investigated and compared: the extracted content of fat in brown rice by PFE method, Folch method and acid hydrolysis method were respectively:

(of seeds) 100g/g 0.1+-2.6

100 grams / 0.1 grams + – 2.5

100 grams/0.2+-2.6 grams

These figures show a slight difference in the efficiency and effectiveness of fat extraction among the three mentioned methods. Therefore, the effectiveness of these three methods is almost the same and close to each other. In the next part, the fatty acid compositions of fats extracted from brown rice, sprouted rice and whole wheat by PFE method were summarized in Table 1.

Comparing the composition of fatty acids between brown rice and sprouted rice, there is a slight difference in the amount of oleic acid (18: 1 fatty acid) and linolenic acid (18: 2 fatty acids) showed. This happened while other fatty acid compositions were similar.

In the case of whole wheat, linoleic acid (18: 2 fatty acid) was one of the main ingredients (62.7%). Fatty acid compositions for each of the grains (brown rice, sprouted rice and whole wheat) did not contradict the results obtained from previous studies and standard tables of food composition in Japan (2013) (Deka, Sood, & Gupta, 2000; Maraschin et al., 2008; Takano, 1993; Yoshida, Tomiyama, & Mizushina, 2010).

In order to confirm the quality of fats extracted by PFE method, fats extracted from brown rice by Folch method and its fatty acid composition were also analyzed and investigated. No difference was found in the fatty acid composition of these two extraction methods (data not shown).

These findings suggest that lipids extracted from seeds by PFE method were available for both quantitative and qualitative lipid analysis. The following table shows the composition of fatty acids related to brown rice, sprouted rice and whole wheat:

FA : Fatty acids

Brown rice : Brown rice

Rice with sprouts: Rice with germ

Whole wheat: Whole grain wheat

In previous studies, the dominant components of total fat particles extracted from brown rice and whole wheat grains were FFAs, phospholipids, and other lipid compounds such as natural fat.

In the case of extraction by PFE method using n-hexane, whole brown rice fat particles not only contain natural fats (tri-acylglycerols (TAGs, diacylglycerols, diacylglycerols (DAGs) and monoacylglycerols, monoacylglycerols (MAGs)). but included small lipolytic components such as FFAs, polar lipids, etc.

As far as the extraction efficiency of FFAs is concerned, the amount of FFAs in fats extracted by the PFE method using hexane was similar to that extracted by the Folch method, as confirmed by fluorescent HPLC using 9-anthryldiazomethane (data not shown). . On the other hand, the presence of fats or polar lipids in whole fat particles extracted by PFE method was detected in TLC. As described in the above section, the extraction efficiency of complete fats from rice bran by PFE method was also equal to those extracted by conventional Folch method. These events show that the fats extracted from seeds by PFE method will be suitable for further analysis and investigation of fats.

3.2 storage of free fatty acids or FFA during the storage period

To elucidate the spoilage processes of fats in stored seeds, the fat (0.1 mg) extracted from each seed was applied and spread on TLC. Figure 1 shows the TLC models of total fats extracted from brown rice flour, sprouted rice and whole wheat grain samples stored for 8 weeks under different conditions. As shown in Figure 1, the result of 8-month storage of these flours was an increase in their hydrolyzed compounds, FFAs, DAGs, MAGs and other spoilage products and compounds. And this TLC showed that the amount of hydrolyzed compounds in each flour stored at 26 degrees Celsius was higher than the corresponding (similar) amount of flour stored at 4 degrees Celsius. In addition, in both cases at temperatures of 4 and 26 degrees Celsius, it seemed that the addition of oxygen absorber accelerates the accumulation of FFA in the seed.

In order to identify the FFAs accumulated in these unmilled seeds during the storage period, the FFAs in the extracted lipids were separately labeled with ADAM solution and their fluorescent derivatives were analyzed and investigated by an HPLC method using fluorescence detection and inspection. In this analysis, the absolute amount of each FFA present in the total fat (1.0 g) extracted from the seed was determined using an internal HPLC standard (13:0 fatty acid). Figure 2 showed that the chromatograms showing fluorescence-labeled FFAs remained in the extracted fats from brown rice, sprouted rice and whole wheat flours.

The composition of stored FFAs in the lipid of each seed before storage was generally not consistent with the fatty acid composition of the corresponding seed lipid (Table 1). Although the amount of each FFA in the seeds before storage was small (Figure 2(c), each FFA was found to have accumulated in the floured seeds after 8 weeks of storage at 26°C (Figure 2(c)). ((a) and (b)). The amount of total FFAs was calculated based on the summation of the content of each FFA. As shown in Figure 3, the total FFAs in fats increased depending on their storage time. For samples containing the kernel, total FFA values in lipids from brown rice, sprouted rice, and whole wheat (or whole wheat) ranged from 15 mg/g (g of extracted fats) to 24–29 mg/g, from 12 mg/g, respectively. to 34-15 mg/g, and from 11 mg/g to 25-35 mg/g depending on the storage conditions. In kernel samples, FFA accumulation of sprouted rice was more and stronger than that of brown rice affected by storage conditions. Also, adding an oxygen absorber to the storage system simulated the accumulation of FFAs in these three types of brains with statistical differences compared to those stored without oxygen absorbers.

As for the flour samples, the FFA mass reserves in fats during the storage period at 26°C were significantly higher than those stored at 4°C (as shown in Figure 3). . When these flours were stored with oxygen scavengers, the FFA reserves in these flours were again higher than those stored without oxygen scavengers. For example, the amounts of total FFA in fats obtained from brown rice, sprouted rice and whole wheat during the storage period at 26°C for 8 weeks with oxygen absorbers ranged from 15mg/g to 57mg/g, respectively, from 12 mg/g increased to 60 mg/g and from 11 mg/g to 75 mg/g. The effects of oxygen scavenging on FFA reserves (masses) in flour-containing samples were high compared to those in the corresponding brain-containing samples. These results were in good agreement with those obtained from TLC (Figure 1).

Figure 1. Thin layer chromatography of a lipid (fat) extracted from samples containing brown rice flour (A), sprouted rice (B), and whole wheat (C) that were stored for 8 weeks under different conditions. 0.1 mg of the extracted fat was placed on each TLC column and spread. Fat extracted from each grain sample before storage (0), from samples containing flour stored at 4 degrees Celsius with oxygen absorber (OA+4C), stored at 26 degrees Celsius with oxygen absorber (OA+26C), Stored at 4 degrees Celsius without oxygen absorber (4C), stored at 26 degrees Celsius without oxygen absorber (26 C). Fat particles: triglycerides (TAGs), free fatty acids (FFAs), diacylglycerols (DAGs), monoacylglycerols (MAGs).

Figure 2. Fluorescence-tagged HPLC of FFA derivatives in brown rice flour samples (A), Rice with sprouts (B), and whole wheat (C) kept under different conditions for 8 weeks. Fat extracted from each seed before storage (c), from flour samples stored at 26 degrees Celsius with oxygen absorber (a), from flour samples stored at 26 degrees Celsius without oxygen absorber (b). It is provided as an internal standard (13:0 fatty acid) for HPLC analysis.

Figure 3. Absolute amounts of total FFAs accumulated in brain samples (upper part) and in flour samples (lower part) of brown rice (A), Rice with sprouts (B), and whole wheat (C) kept under different conditions for a period of 8 weeks. Lipid (fat) of each seed before storage (0), from flour samples stored at 4 degrees Celsius with an oxygen absorber (4C+OA), stored at 26 degrees Celsius with an oxygen absorber (26C+OA), stored at 4 degrees Celsius without oxygen absorber (4C), and stored at 26 degrees Celsius without oxygen absorber were extracted.

It is possible that the addition of oxygen scavengers to the storage system may have led to a slight accumulation of FFA in these unmilled grains, especially in the flour containing samples.

Previous studies showed that the accumulation of FFA aggregates in seeds during the storage period was accelerated by simulating several hydrolytic enzymes (Zhou, Robards, Helliwella, & Blanchard, 2002). In this study, it was also revealed that the accumulation of FFA masses in seeds stored at 26°C was higher than those stored at 4°C. High storage temperature promotes (intensifies) the activities of hydrolytic enzymes, so the regulation of storage temperature will be one of the most important parameters in order to control the accumulation of FFA masses in unmilled seeds. In addition, in this study, the accumulation of FFA masses in the samples Brown rice flour, sprouted rice and whole wheat flour were greater than the corresponding values in samples containing kernels Was. Regarding milling, the accumulation of FFA masses in semi-milled grains, sprouted rice, was statistically higher than that in unmilled grains, brown rice, under similar storage conditions (Figure 3). These results showed that the procedures of flour making and milling will intensify the interactions between hydrolytic enzymes and lipids, which results in the increase of hydrolytic activities and related to the release of FFAs. Therefore, the results showed that flour making and grinding early during the food processing period will not be desirable.

3.3 Changes in 18:2 fatty acids during the storage period

In the next part, the effects of storage temperature and oxygen absorber on the progress of oxidation of fats in stored seeds were investigated. When the double bonds of unsaturated fatty acids were subjected to oxidation, they were converted to the corresponding hydroperoxides, which would not be detectable in conventional GC analysis. As a result, the oxidation of unsaturated fatty acids can be estimated by the decrease in the absolute amount of methyl ester corresponding to the main unsaturated fatty acid in the GC chromatogram (estimated as an estimate). In this study, using brown rice flour, sprouted rice and whole wheat, changes in the absolute amount of one of the main unsaturated fatty acids, linoleic acid (18:2 fatty acid), which was present in lipids during the storage period, were observed. It was monitored by GC analysis using internal standard (margaric acid 17:0 fatty acid).

As shown in Figure 4, the absolute amounts of 18:2 fatty acids in the lipids extracted from these three types of flour decreased slightly, and the amount of this decrease was dependent on the storage periods. In addition, the storage conditions (storage temperature or oxygen absorber) affected the oxidative degradation progress (oxidative spoilage process) of 18:2 fatty acid, as shown in Figure 4. The decrease in the absolute amount of 18:2 fatty acid in the seed stored at 26 degrees Celsius was statistically greater than the same seed stored at 4 degrees Celsius. Previous studies showed that the oxidation of unsaturated fatty acids in rice was dependent on the storage temperature (Liu, 2011; Maraschin et al., 2008), which was also confirmed by the results of this study. When these seeds were stored with oxygen scavengers, the reductions in 2:18 fatty acids were statistically smaller than the corresponding values in seeds stored without oxygen scavengers.

As a result, when these flours were stored at 26 degrees Celsius without oxygen absorber, the amount of 18:2 fatty acid decreased with the highest rates among those stored under the other three types of test conditions. Thus, it was shown that the low concentration of oxygen induced by the oxygen absorber will slow down the process of spoilage and oxidative damage of unsaturated fatty acid.

3.4 Changes in the content of Tukur Emanuel during the maintenance period

Because the oxygen absorber lowers the oxygen concentration in the closed storage system (by heat press), it is expected that the oxidation reaction using atmospheric oxygen

Do it slowly. As a result, the consumption of antioxidants, which originally remained in the seeds themselves, was expected to be reduced. Thus, the antioxidant effects of oxygen absorbers were evaluated by measuring the content of total tocopherols (tocopherols + tocotrienols) in stored seeds. Under the analysis conditions by HPLC in this study, 8 types of tocopherols such as alpha, beta, gamma and sigma tocopherol and alpha, beta, gamma and sigma tocotrienols could be determined separately. A representative HPLC chromatogram of tococramanols is shown in Figure 5. Before storage, alpha-tocopherol, alpha-tocotrienol, and gamma-tocotrienol were the main tocochromanols in brown rice, sprouted rice, and whole wheat. In brown rice and sprouted rice, beta-tocopherol was barely detectable. On the other hand, a small amount of beta-tocopherol, beta-tocotrienol and sigma-tocopherol remained in whole wheat. After storage under different conditions, the amount of each tocopherol and tocotrienol remaining in the seed decreased, indicating a decrease in total tocoramanols. The loss of total tococramanols in unmilled seeds during an 8-week storage period is shown in Table 2. The primary contents of total tococramanols in brown rice, sprouted rice and whole wheat are equal to +1.3-26.6 μg/g, +1.2-22.8 μg/g, respectively.

and were +1.2-23.0 μg/g. Regarding storage at 4 degrees Celsius and with oxygen absorber, we were not able to identify and find statistical reductions in total tocoramanols in these three types of brain samples. But in the case of storage at a temperature of 4 degrees Celsius and without an oxygen absorber, the remaining amounts of total tococramanols in sprouted rice and whole wheat are statistically similar to those samples that were stored at a temperature of 4 degrees Celsius and with an oxygen absorber, or It was even different from those that had been examined before storage. While the total tococramanols in these three types of samples containing brains stored at 26 degrees Celsius even with oxygen absorbers were reduced exactly compared to the samples before storage. 8-week storage at 26 degrees Celsius without oxygen absorber caused a significant decrease in total tocochromanols in three types of brain samples.

Storage of the flours of these three types of seeds at 26 degrees Celsius compared to those in the brain samples stored in the same conditions caused more loss of total tococharomanols. Especially, storage of brown rice flour, sprouted rice and whole wheat flour at 26 degrees Celsius without oxygen absorber resulted in the loss of about 75%, 58% and 53% of total primary tocopherols contents, respectively. These results showed that adding oxygen absorber to the storage system can slow down the consumption of total tocoramanols during storage. Although the oxygen scavenger itself is not antioxidant (in the sense that it has no antioxidant properties), its antioxidant effect can be evaluated by observing and measuring changes in the amounts of total tocochromanols remaining in these stored seeds.

One of the main and important results in this study was that the accumulation of free fatty acids (FFA accumulation) was alternately enhanced and increased by the addition of oxygen absorbers, although the intrinsic and main purpose of adding oxygen absorbers to the storage system was delayed. Dropping was the oxidation reactions associated with unmilled grains.

Figure 4: Absolute amount of 18:2 fatty acids in lipids of kernels and flour samples of brown rice (A), sprouted rice (B) and whole wheat (C) stored under different conditions for periods of 4 and 8 weeks.

Amounts of 18:2 fatty acids from lipid extracted from samples stored at 4 degrees Celsius with oxygen absorber (white circle), stored at 26 degrees Celsius with oxygen absorber (white triangle), stored at 4 degrees Celsius without absorber Oxygen (black circle), kept at 26 degrees Celsius without oxygen absorber (black triangle).

The enzymatic activity of fats and antioxidants in unmilled grains such as brown rice and whole wheat is higher than that of milled grains and thus affects their final consumption and storage properties (Adom, Sorrells, & Liu, 2005; Every, Simmons, & Ross, 2006). To control the quality of unground seeds during the storage period, it is better and recommended to keep the storage temperature low. The reason for this is to reduce enzyme activities. Other strategies and solutions to reduce the corruption of lipids (fats) in these seeds include controlling the concentration of atmospheric oxygen in the storage system by means of oxygen absorbers. Enzymatic lipid degradation occurs through lipase and lipoxygenase activities, which are mainly located in the germ and bran of these seeds (Galliard, 1994; Loiseau, Vu, Macherel, & Le Deunff, 2001). The spoilage of fats in seeds begins with hydrolytic rancidity, which is an enzymatic reaction by lipases. Oxygen absorber will be less effective on this enzymatic hydrolysis.

On the other hand, lipids in unmilled seeds can be oxidized enzymatically by lipoxygenase or by auto-oxidation by heat or exposure to light (Brash, 1999; Galliard, 1986a, 1986b; Robards & Kerr, 1988).

Although lipid oxidation is thus a much slower process than hydrolytic fat spoilage (Galliard, 1994), lipid oxidation reactions contribute significantly to product quality loss. In oxidation reactions, lipoxygenases to methylene groups between double bonds in unsaturated fatty acids, preferably Non-esterified unsaturated fatty acids attack (Galliard, 1986a). Whereas, auto-oxidation to lipids can occur by non-enzymatic reaction with atmospheric oxygen. Under both oxidation mechanisms, lipid oxidation involves the addition of oxygen to the double bonds of unsaturated fatty acids, forming hydroperoxides followed by cleavage of the carbon chain to smaller volatile compounds, such as epoxyaldehydes, ketones, lactones, and furans (Robards & Kerr, 1988). As a result, these oxidation processes will progress slowly under low oxygen concentration.

In this study, it was revealed that the addition of oxygen scavengers in the storage system accelerated the accumulation of free fatty acids, or FFAs, in brown rice, sprouted rice, and whole wheat.

Because the oxygen scavenger has no effect on the hydrolytic rancidity of lipids by lipase, increases in the accumulation of FFA aggregates stimulated by the oxygen scavenger

It was somehow caused by the prevention of subsequent oxidative spoilage processes of released FFA.

However, since the storage system with the oxygen absorber is completely closed (by heat pressing or any other method that completely closes the chamber), the behavior of the water in these grains during the storage period and the atmospheric humidity in the storage system It may affect the progress of lipid hydrolysis processes in seeds. More information on the water content of these stored seeds will be necessary to understand the accumulation of excess FFA masses by oxygen scavengers. We are currently studying the relationship between storage stability of fats and water content of these unmilled grains during storage.

Accumulation of FFA mass in the grain is thought to play a vital role for the colorability of rice and for the increase in amylograph viscosity during the storage period and helps to maintain them (Deka et al., 2000; Noomhorm, Kongseree, & Apintanapong, 1997). ; Okabe, 1979; Takano, 1989). The introduction of oxygen absorber to the storage system of milled seeds will be effective under the conditions that hydrolytic activities of lipids are suppressed.

Figure 5. Representative HPLC chromatograms of tocogramanols extracted from brown rice kernels before storage (A) and after storage without oxygen absorber at 26°C for a period of 8 weeks (B).

Table number 2

4. Conclusion

In order to investigate the strength (stability and stability) of storage of unmilled grains, brown rice, sprouted rice and whole wheat, the accumulation of free fatty acids, the absolute values of linoleic acid and also the amount of total tocoramanol content under different storage conditions as indicators of lipid spoilage, under were monitored and investigated. It was found that storage at high temperature and flouring and milling processes promoted the deterioration process of fat (lipid) in stored seeds. Storage and storage with an oxygen absorber caused more accumulation of free fatty acids in the lipid of these unmilled seeds, but it caused a decrease in both the absolute amount of linoleic acid in the lipid and in total tocoramanol content in these seeds.

In the case of storage of unmilled seeds, introducing oxygen absorbers into the storage system will be effective in conditions where the hydrolytic activities related to their lipids are suppressed.

Baste Raz Salamat Paya Company was established in 2012 with the aim of producing technological products that help health, reduce waste and increase the shelf life of foodstuffs, and the Bihava oxygen absorber is its first product. In this direction, we are trying to make our products an alternative to the traditional methods of preserving food, which are often harmful to human health, such as the use of rice tablets or toxic gases such as methyl bromide. Oxygen absorbers are produced in various capacities from 30 cc to 3000 cc. Currently, only the 3000 cc product is produced and the product portfolio will be completed soon. Contact us for more information.

References

2. Adom, K. K., Sorrells, M. E., & Liu, R. H. (2005). Phytochemicals and antioxidant activity of milled fractions of different wheat varieties. Journal of Agricultural and Food Chemistry, 53, 2297e2306.
3. AOCS, American Oil Chemist’s Society. (2005). Official Method Ce 1h-05: Determination of cis-, trans-, saturated, monounsaturated and polyunsaturated fatty acids in vegetable or non-ruminant animal oils and fats by capillary GLC.
4. AOCS, Association of Official Agricultural Chemists. (1995). Official method 922.06 fat-acid hydrolysis.
5. Bender, D. A., & Mayer, P. A. (2003). Vitamins and minerals. In R. K. Murray, K. Granner, P. A. Maycs, & V. W. Rodwell (Eds.), Harper’s illustrated biochemistry (26th ed.). New York: Lange Medical Books/McGraw-Hill (45 pp.).
6. Brash, A. R. (1999). Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate. The Journal of Biological Chemistry, 274, 23679e23682.
7. Deka, S. C., Sood, D. R., & Gupta, S. K. (2000). Effect of storage on fatty acid profiles of basmati rice (Oryza sativa L.) genotypes. Journal of Food Science and Technology
8. Mysore, 37, 217e221. Doblado-Maldonado, A. F., Pike, O. A., Sweley, J. C., & Rose, D. J. (2012). Key issues
9. and challenges in whole grain wheat flour milling and storage. Journal of Cereal Science, 56, 119e128.
10. Every, D., Simmons, L. D., & Ross, M. P. (2006). Distribution of redox enzymes in mill-streams and relationships to chemical and baking properties of flour. Cereal Chemistry, 83, 57e61.
11. Folch, J., Lee, M., & Stoane-Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. The Journal of Biological Chemistry, 226, 497e509.
12. Galliard, T. (1986a). Oxygen consumption of aqueous suspensions of wheat whole- meal, bran and germ: involvement of lipase and lipoxygenase. Journal of Cereal Science, 4, 33e50.
13. Galliard, T. (1986b). Hydrolytic and oxidative degradation of lipids during storage of whole-meal flour: effects of bran and germ components. Journal of Cereal Science, 4, 179e192.
14. Galliard, T. (1994). Rancidity in cereal products. In J. Allen, & R. Hamilton (Eds.), Rancidity in foods (3rd ed.) (pp. 141e159). London: Blackoe Academic & Professional.
15. Goffman, F. D., & Bergman, C. (2003). Hydrolytic degradation of triacylglycerols and changes in fatty acid composition in rice bran during storage. Cereal Chemistry, 80, 459e461.
16. Jensen, P. N., Sorensen, G. B., Brockhoff, P., & Bertelsen, G. (2003). Investigation of packaging systems for shelled walnuts based on oxygen absorbers. Journal of Agricultural and Food Chemistry, 51, 4941e4947.
17. JOCS. (2003a). Japan Oil Chemist’s Society, Standard method for the analysis of fats, oils and related materials. Reference3-1-3-1996, Extraction by Acid Hydrolysis.
18. JOCS. (2003b). Japan Oil Chemist’s Society, Standard method for the analysis of fats, oils and related materials, 2.4.2.2e1996 Fatty acids composition (FID Temperative Programmed Gas Chromatography).
19. Labuza, T. P. (1987). Oxygen scavenger sachets. Food Research, 32, 276e277. Liu, K. (2011). Comparison of lipid content and fatty acid composition and their distribution within seeds of 5 small grain species. Journal of Food Science, 76, C334eC342.
20. Li-Xin, L., & Fang, X. (2010). Effect of initial oxygen concentration and oxygen- barrier property of film on fat oxidation rate of packed cookies. European Food Research Technology, 231, 347e351.
21. Loiseau, J., Vu, B. L., Macherel, M. H., & Le Deunff, Y. (2001). Seed lipoxygenases: occurrence and functions. Seed Science Research, 11, 199e211. Maraschin, C., Robert, H., Boussard, A., Potus, J., Baret, J.-L., & Nicolas, J. (2008).
22. Effect of storage temperature and flour water content on lipids, lip- oxygenase activity, and oxygen uptake during dough mixing. Cereal Chemistry, 85, 372e378.
23. Mexis, S. F., & Kontominas, M. G. (2010). Effect of oxygen absorber, nitrogen flushing, packaging material oxygen transmission rate and storage conditions on quality retention of raw whole unpeeled almond kernels (Prunuts dulcis). LWT e Food Science and Technology, 43, 1e11.
24. Noomhorm, A., Kongseree, N., & Apintanapong, N. (1997). Effect of aging on the quality of glutinous rice crackers. Cereal Chemistry, 74, 12e15. Okabe, M. (1979). Texture measurement of cooked rice and its relationship to the eating quality. Journal of Texture Studies, 10, 131e152.
25. Pastorelli, S., Valzacchi, S., Rodriguez, A., & Simoneau, C. (2006). Solid-phase microextraction method for the determination of hexanal in hazelnuts as an indicator of the interaction of active packaging materials with food aroma compounds. Food Additives and Contaminants, 23, 1236e1241.
26. Peterson, D., & Qureshi, A. A. (1992). Genotype and environment effects on tocol of barley and oats. Cereal Chemistry, 70, 157e162. Prabhakar, J. V., & Venkatesh, K. V. (1986). A simple chemical method for sta-
27. bilization of rice bran. Journal of the American Oil Chemists’ Society, 63, 644e 646.
28. Ramezanzadeh, F. M., Rao, R. M., Windhauser, M., Prinyawiwatkul, W., & Marshall, W. E. (1999). Prevention of oxidative rancidity in rice bran during storage. Journal of Agricultural and Food Chemistry, 47, 2997e3000. Robards, K., & Kerr, A. F. (1988). Rancidity and its measurement in edible oils and snack food: a review. Analyst, 113, 213e224.
29. Rose, D. J., Ogden, L. V., Dunn, M. L., & Pike, O. A. (2008). Enhanced lipid stability in whole grain wheat flour by lipase inactivation and antioxidant retention. Cereal Chemistry, 2, 218e223.
30. Shin, T. S., & Godber, J. (1993). Improved high-performance liquid chromatography of vitamin E vitamers on normal-phase columns. Journal of American Oil Chemists’ Society, 70, 1289e1291.
31. Suzuki, Y. (2011). Isolation and characterization of a rice (Oryza sativa L.) mutant deficient in seed phospholipase D, an enzyme involved in the degradation of oil-body membranes. Crop Science, 51, 567e573.
32. Takano, K. (1989). Studies on the mechanism of lipid-hydrolysing in rice brans. Journal of Japanese Society of Food Science and Technology, 36, 519e524.
33. Takano, K. (1993). Advances in cereal chemistry and technology in Japan. Cereal Foods World, 38, 695e698.
34. Tarr, C. R., & Clingeleffer, P. R. (2005). Use of an oxygen absorber for disinfestation of consumer packages of dried vine fruit and its effect on fruit colour. Journal of Stored Products Research, 41(1), 77e89.
35. Yoshida, H., Tanigawa, T., Yoshida, N., Kuriyama, I., Tomiyama, Y., & Mizushina, Y. (2011). Lipid components, fatty acid distributions of triacylglycerols and phospholipids in rice brans. Food Chemistry, 129, 479e484.
36. Yoshida, H., Tomiyama, Y., & Mizushina, Y. (2010). Lipid components, fatty acid and triacylglycerol molecular species of black and red rices. Food Chemistry, 123, 210e215.
37. Yoshida, T., Uetake, A., Yamaguchi, H., Nimura, N., & Kinoshita, T. (1988). New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent la- beling reagent for fatty acids and derivatives. Analytical Biochemistry, 173, 70e
38. Zhou, Z., Robards, K., Helliwella, S., & Blanchard, C. (2002). Aging of stored rice: changes in chemical and physical attributes. Journal of Cereal Science,
39. 35, 65e78.