COMPARISON OF THE RESPONSIBILITY TO EXTRACT AND PURIFY β-GLUCAN OF SOME CEREAL GRAINS FOR SPECIFIC METHOD

1238 COMPARISON OF THE RESPONSIBILITY TO EXTRACT AND PURIFY β GLUCAN OF SOME CEREAL GRAINS FOR SPECIFIC METHOD H.S. Karim M.W. Zainulabidden D.A.Sabir Assist. Lecturer Prof. Assist. Prof. Dep. Food Sci. & Quality Control -Coll. Agric. Engine. Sci. University of Sulaimani E-mail: hiran.sarwar@univsul.edu.iq ABSTRACT The current study prioritized certain popular local cereal species like Wheat, Oat, Rye and two local Barley varieties known as Aksad and Amal to extract their βglucan using alchemical methods. The results of this study indicated that rye contained the lower percentage of β-glucans (0.756%) whereas, both types of barley Aksad and Amal contained highest amount (9.489%) and (7.395%) respectively. However, the results showed that the crude extracted of βglucans from wheat, oat, rye, barley (Aksad) and barley (Amal) were 0.373,1.409, 0.26, 2.287 and 1.855 g/100g flour respectively which was reflected the efficiency of extraction process which ranged between 8.83  0.01for wheat to 34.39  0.02 for rye, the precipitating by ammonium sulfate were useful for purifying of oat and barley βglucan which their purity increased to about 1015% to reach their purity to 84.88 for barley (Amal) to 95.18% for oat. All the bands of Ft-IR Spectra showed to take place absorbance at these regions by βglucan are present in the all scanned studied samples with some differences in their areas and absorbance. A robust and sharp peak at 1304.37 cm -1 appeared only with the control sample while it is absent in the studied β-glucan. Results of HPLC illustrated that all purified cereal βglucan appeared only one peak at the same Retention time RT of standard oat βglucan which ranged between 17.429 to 17.453 min.


INTRODUCTION
Cereal β -glucan (1  3) (1 4)--d-glucans are defined as fibrous structures found in the aleurone, Cell walls of cereals sub-aleurone and endosperm. In general, the composition of b-glucan in barley, oats, rye and wheat is 3-11%, 3-7%, 1-2%, and,1%, consistently (3). There are variations in the composition of βglucan across cereals, with a more significant percentage identified in resources of barley, after the resource of oat, and a reduced proportion in sources of rice and wheat. Widely, β -glucan constituents that have been collected comprise more than 50 per cent of the fiber composition. Nonetheless, most aggregate contains significant concentrations of starch and proteins tied to the molecules with which enzymatic therapies are recommended to help eliminate these extracts' impurities (5). Extraction utilizing wet approaches requires inactivation of the endogenic enzymes and acid or alkaline solvent processing. Contaminant reduction (proteins and starch) is achieved with hydrolytic enzymes or by direct adsorption. Bglucan precipitation is achieved with alcohol (99 %), and eventually, the -glucan extract procedure is done with dehydration (by freezing or spray drying)(4,7). By using wet strategies for the derivation of -glucan, the temperature, pH, exposure time, and molecular weight should be monitored. However, even at low concentrations of β-glucan wet extraction process are characterized by a high cohesiveness of the aqueous extracts. This contributes to greater fluid quantities, and high concentrate drying expenses. Outside this, low pH and endogenic enzymes can decrease the β-glucan molar volume when subjected to humidity (6). Purification procedures for the removal of starch and proteins after extraction of β-glucan are often not conducted. Such pollutants in the extraction may inhibit proper analysis and cause considerable difficulties in food application. Enzymatic -amylase and protease intervention (7). The overall -glucan amount of oats was measured by using Megazyme Mixed-Linkage--glucan Assay Package method (Megazyme method) has been successfully evaluated by AOAC International (Method 995.16), AACC (Method 32-23.01) and ICC (Method No. 166, approved 1998), along with improvements. Through the practice of α-amylase, lichenase, and β-glucoseoxidase in the implementation of enzymatic methods (15). The aim of this research is to study the responsibility of different cereal crops to extraction and purification of -glucan by restricted method and effect it on their characteristics.

MATERIALS AND METHODS Materials
Cereal grains samples of Wheat (Aras), Rye, Oat, Barley (Aksad and Amal) from local cultivar were used in this study.Megazyme kit was obtained from (megazyme company, USA) to determine glucan. Standard oat βglucan was generated from megazyme with purity >94 per cent and low viscosity, Protenaise-k-(3000u / g) was acquired from Bio-lab.

Methods
Cereal grains samples were washed twice at room temperature, then cured inside the (oven,memmert,Germany)at 50°C. Eventually, a 40-mesh panel was used to mill the wholegrains. potassium phosphate buffer (20 mM, pH 6.5) was obtained by dispersing KH2 PO4.2H2o in 900 ml of distilled water, accompanied by pH adjustment with (4g/L) NaOH, adjusting volume to 1L.

Proximate Analysis
Each cereals flour was analyzed for moisture (Oven at 102°Cº) and protein (Kjeldhal process with protein N factor of 5.7) according to (AACC 44-15,46-10) procedures respectively, lipid (Soxhlet; hexane) using (AOAC2000), and β-glucan (AOAC 995.16) content, ash (combustion at 525°C) contents, total carbohydrates by differences were also determined. For each study, repeated, measurements were conducted. Extraction of grains -glucan -glucan of grains were extracted using alkaline extraction method (21) as shown in Figure 1, and crude -glucan percent was determined according to: crude -glucan %= weight of extracted  glucan (g)/ weight of cereal grain (g) X 100

Figure 1. Extraction of grains β-glucan Purification of β-glucan
The crude β-glucans were purified by pretreated them with the addition of Potassium phosphate buffer 1g/25ml (20mM, pH7.5), then samples were mixed with proteinase-k-5.8ml(3000u/g) placed into water bath at 40ᵒC, 3h, then 60% ethanol was added and Centrifuged, 2000g for 10 min. The precipitate dried by hot air 60ᵒC,6h which was done to extract free sugars and flush off specific proteins. By precipitation with aqueous ammonium sulfate (30% w/v) at 5ᵒC for 72h as described previously by YOUNG-TACK, et al. (5) with little modification. The precipitate was redissolved in potassium phosphate buffer (20mM, pH6.5), and the solution was cooled. Dialysis membrane was used to remove the ammonium sulfate. The percentage of Purified -glucan was calculated as follow: Purified -glucan% = weight of purified  glucan (g) /weight of cereal grain (g) X100 β-glucan content analysis Total b-glucan content was determined by using a β-glucan assay kit for mixed linkage (β-glucan Assay Kit [Mixed Linkage (AACC method 32-23.01AOAC method 995. 16, and ICC standard method no.168)], Megazyme, Ireland), with some alterations the β-glucan during pretreatment process the material was analyzed. Flour samples which are dry homogenized, 100 mg (80-120 mg) of each flour were taken as shown in figure (2)

Fig2. β-glucan content analysis Detection of ß-glucan
High-Performance liquid chromatography with detection of refractive index (RID) (Waters 2695 alliance HPLC) was used to determine the purity extracted -glucan compared with standard medium molecular weight of oat βglucan (imported from Megazyme.) as described previously (22).

FT-IR Spectrophotometer analysis
Fourier transform infrared spectrophotometer (FTIR), as defined formerly by Ji, et al. (23) used for the isolated polysaccharides and compared with standards to examine the structural characteristics of the -d-glucan specimens collected. The spectra were measured from 4000 to 400 cm 21 in absorbance mode (mid-infrared region).

RESUITS AND DISCUSSION Proximate analysis of cereal crops
As shown in Table 1. moisture content of cereals flour were between (12.064-12.748%), the higher protein content was in wheat flour (12.12%) however, oat flour reported as the highest lipid content (3.905) but, also the lowest protein content related to oat flour (7.81 %). Abid &Naser's experiment (24)reported that the chemical composition of two classes of barley (IPA99 and IPA265) Flour was as following.The percentage of moisture, protein and fat in class IPA265 was higher (12.60%, 13.20%, 2.8%) respectively, than that for class IPA99 (12.05%, 11.70%, 1.50%) correspondingly. While the percentages of fiber, carbohydrates and ash were lower in the flour of class IPA265, being 5.9, 66.5 and 4.9% respectively, as compared to that of class IPA99 flour, being 6.15%, 69.4% and 5.14% respectively. Kunkulberga, et al. (25) have also shown a clear negative association (r= −0.981) between starch content and protein in grains of most common rye cultivars.

Table1.Proximate analysis of cereal grains
The extractability of β-glucan from varying cereal flours: Table 2. illustrates the percentage content of βglucans in cereal samples which indicated that rye contained the lower percentage of βglucans (0.7560.043%) while, both types of barley Aksad and Amal contained highest amount (9.489  0.7330%) and (7.395  1.0204) respectively. On the other hand, wheat and oat contained about (4.220.2121%) and (6.450.3167) respectively. Showing that there was a noteworthy alteration was shown between all the studied samples of cereal grains in their content of β-glucans. The literature reported a wide range of β-glucans content among the cereal crops. HozoVá, et al. (26) found that insoluble βglucans, which represent about 3/4 -2/3 of the total β-glucans, ranged between 15.34 to 18.67, 5.18 to 28.2 and 8.48 to 16.09 g/100 g dry matter for barley, oat, wheat varieties respectively. These values were remarkably higher than that they reported in their research attributing these differences mainly to the enzymatic methods which determine only the water-soluble βglucans. However, although there is a vast difference between the researchers about the content of cereal crops of β-glucans there is an agreement that barley and oat contain higher amount compared with rye and wheat which contain a lower amount of β -glucans (27). compared with the content of each cereal crop. These obtained amount of β-glucans reflected the efficiency of extraction process which ranged between 8.83  0.01for wheat to 34.39  0.02 for rye, but, in general, the efficiency of extraction was higher in those cereal grains have high percentage of β-glucans such as barley and oat compared to wheat. Zhang, et al. (27) obtained a yield of 4.0% (w/w) as a crude β-glucans with 80% purity and usually, the β-glucan recovery was 82.1% (w/w) from barley flour. The purity of extracted βglucans is important but although,Sampson et al. (28) reached to efficiency of extraction about 19.1 to 41.9% of crude β-glucans according to the genotypes of corn and isolation method but the purity of these crude extraction not exceeded more than 6.108 -7.59%. However, the results showed there is a negative correlation between the purity and the efficiency of extraction according to the availability of isolation methods. That is mean, there are a lot of βglucans will be lost during its isolation and purification attributing to the strong bond with other materials, especially in the cell wall (29)

Table3. Efficiency of crude b-glucans extraction
Data in the table (4) represent % of βglucan obtained from the purification of extracted β-The results showed that these purification processes especially, the precipitating by glucan using three steps including three steps of purification.ammonium sulfate were useful for purifying of oat and barley βglucan which their purity increased to about 10-15% to reacht 84.88 for barley (Amal) to 95.18% for oat. Although, using ammonium sulfate increased the purity about 3-7% in wheat and rye but in the same time the final amount of purified βglucan was minimal since most of the crude extracted of β-glucan was lost during the process of salt precipitating and dialysis (see Table 4). However, the results of this work suggest using ammonium sulfite and dialysis useless for wheat and rye β-glucan purification. Zhang, et al. (27) used alkali extraction and repeated-precipitation with ethanol to isolate β-D-glucan had high purity of (80.8%) from hull-less barley (Hordeum vulgare L. var. nudum Hook. f.). followed by precipitating the barley β-glucans from their solution by using 37% ammonium sulfate to obtain high purity of βglucan (93%). As a result of AL-Esawi, et al. (10) study neutralization on the protein particles, a decrease in protein solubility and exude of water layer coated the protein particles may occur and hence particles is deposited. A relationship is found between the concentration of sulfate used in precipitation and the quantity and distribution of loads and the number of non-ionic and water-damaging aggregates on the surface of the protein molecule. Shamurad.et.,al (8) stated that the enzymatic modification which considered the most safety method to get a good functional an nutritional characteristic of wheat protein. Table 5. showed that there were some differences and similarities between the control sample (oat's glucan) and purified studied cereal glucans samples in their values of Ft-infrared wave and maximum absorption. A board peak was recorded at a range of wave numbers from 3392.89 for rye to 3434.96 cm -1 for aksad glucan for all samples which it is ascribed to the alcoholic group O-H stretching vibration of carbohydrate. Also, Du (27) reported that the influential band at 3386 cm -1 was indicated to the hydroxyl stretching vibration of the extracted polysaccharide. However, the waves number 3386.67, 3402, 3417, 3430 cm −1 were allocated to O-H stretching vibration intermolecularly and intramolecularly in glucan from different sources (27). However, all the samples showed a broad curve at these wave nos. Nevertheless, they differ in their maximum absorption, which ranged from 0.4 for wheat to 1.69 for rye glucans. The fingerprints (1400-500 cm -1 ) emphasized this results by another curve ranges from 1330-1430 and 650-770. All studied samples absorb IR wave about at 1350, 1383.95 and 1421.40 cm -1 also, an absorption was noticed in all studied sample at 765.04 and 660 cm -1 with some differences among the samples but this range of wavelengths attributes to alcoholic O-H indicating to the presence of polysaccharides. Du (27) claimed that the precise, strong points at 2934 cm -1 were due to the C-H stretching vibrations. The findings of this research showed that all the samples contained a sharp curve ranged from 2926.32 to 2933.93 cm -1 which attributed to CH 2 stretching vibration. Also, in all studied samples, an absorbance for CH 2 was showed at 1421.40 and 1383.95 cm -1 a week bending vibration. The result also, showed that the contaminated protein of all  -glucans samples including the control (96% oat glucans) absorbed the IR waves at (2147.24-2435.40 cm -1 ) which was attributed to the protein presence by Du (27) who identified a tiny signal at 2362 cm −1 as indicated to the protein contamination (N-H absorbing groups). On the other hand, all samples showed absorbance at 1653.26, 1525,1450 cm -1 presenting amide I, amide II and amide II respectively may due to the remaining of insoluble protein with the. Although, the hydroxyl group O-H interrupted with amide II wavelengths region, generally this group belongs to the same amino acid. The IR curve of control sample (oat glucans) identified by contains a strong sharp peak at 1304.37 cm -1 in addition to not appear any other curve until 1078.97cm -1 while the studied samples of βglucans did not contain peak at 1304.37 cm -1 but contained different peaks at this region of wavelengths (from 1304.37 to 1078.97 cm -1 ). However, the absorption at 1304.37 may vary due to the presence of amide group that may explain the disappearing of this peak which may attribute to the protease treatment that used in the purification the studied βglucans. The results showed an identified peak in all studied samples, except control sample at about 1230-1250 cm -1 which may attribute to the polysaccharide bonds especially C-O bending . The results also showed another peak that was absented in control sample at 1158.71-1155.85 cm-1. Zhang,et al. (27) reported that besides, sharp points at 1154 cm −1 and β-D-glucopyranose ring vibrations overlapping with stretching vibrations of (C-OH) side groups and (C-O-C) glycosidic bond vibrations of β-(1 → 4) and β-(1 → 3), respectively, were ascribed to 1070 cm −1 . Also, the peak that appears at 1080.75 cm -1 in all samples except control which was 1078.97 cm -1 may both are similar since Du (27) attributed these bands at 1082 and 1070 cm -1 respectively to the C-O-C and C-O-H link band in glucopyranose ring. An identical board peak at 1019.31 to1024.00 cm -1 appeared in all studied samples except control (oat βglucan) which was a small peak at about 1030 cm -1 and overlapped with the peak at 1078.97 cm -1 . It can conclude that the differences between the two methods to produce the βglucan is the leading cause this difference since the studied oat βglucan has differed from the control βglucan (imported from Megazyme co.). The results showed that there were four bands of IR absorbance appeared clearly and sharply in all samples except control and barley var. Aksad. These bands were at about 931.63, 860.75, 765.22 and 68020 cm -1 to the vibration of (COC), (CC), (CO), (CCH) (CO)ring, (CC)ring, (COC), (CCO), (OCO) of glyosidic bonds. The results also showed that there were two peaks at 574.86 and 527.48 cm -1 in all studied samples, although there is a difference among the samples in their peaks areas and absorbency. This result is with the agreement of JÄRVAN, Lukme, Tupits and Akk (6) who attributed the bands at 573 cm -1 to the bending vibration of polysaccharide while 523 cm -1 to pyranose ring C=O a symmetric deformation. Finally, it can conclude from the IR results the following: There is shifting between the wavenumber of control (high purity of oat β-glucan) and the studied samples. This shifting may attribute to the method of extraction and purification more than on the type of cereal since this shifting was between the studied oat and oat control. Using different method may affect the intensity of hydrogen bonds, degree of crystallinity, the type of binding between βglucan and other impurities, especially protein and pentosans. All the bands that the researches indicated to take place absorbance at these regions by polysaccharides are present in the all scanned studied samples with some differences in their areas and absorbance. A robust and sharp peak at 1304.37 cm -1 appeared only with the control sample while it is absent in the studied β-glucan. This band did not determine by researches as a region for βglucan absorption but, Wiercigroch,et al. (13) claimed that the band at 1306 cm -1 found only in raffinose or trisaccharide as a result of CH 2 twisting vibration. On the other word, did not contain the studied samples such this band not mean there is a defect on purified β-glucan. There are somewhat differences between the two types of barley β-glucan (Aksad and Amal), especially at the region between 400-1000 cm -1 in, which, Aksad bands were very short and not identified compared with Amal. However, in this region where the structure of pyranose units is effective, may some changes take place due to its ability to rearrange its structure caused decreasing of its capability to absorb IR wave at these bands. Comparison between purified glucan and standard oat glucan In this study, HPLC was used for comparing the purity of purified cereal -glucan with the standard (Oat -glucan) and detection of quantification of -glucan hydrolysis with lichenase. Table 6. and Figure 2. showed that all purified cereal glucan appeared only one peak at the same Retention time RT of standard oat glucan which ranged between 17.429 to 17.453 min that may give somewhat an indication about the purity of the prepared cereal glucan. Also, Abd El Ghany, et al. (14) identified peak of (1→3)(1→6)-β-Dglucan extracted from Pediococcus parvulus by using HPLC, had retention time at 9.044 min. The results also showed that all the purified extracted cereal -glucan have the same RT either between the tested -glucan or with standard -glucan was about 17.45 min. This may mean that the extraction and purified method caused producing -glucan has the same molecular weight as standard -glucan, which has moderate molecular weight. The results indicated that there is a difference among the tested -glucans in their resistance to lichinase hydrolysis depending on the time of break down may occur due to their difference in structure. -glucan of barley (type Amal) had a higher resistance to hydrolysis until 15 min. but it was decline completely at 20min. (as the result of the determination of -glucan showed that.) Also, -glucan of rye firstly at 7 min profoundly analyzed then resisted the lichinase hydrolysis for 15 min or may more time. On the other hand, -glucan of barley (type Aksad) oat and wheat completely hydrolysis during 15 min although there was some retardation of glucan of oat and wheat at 7min. compared with Aksad type.  Although, there is a difference among the studied cereal grains in their content of glucan but the differences of their responsibility forward extraction and purification were wide. The efficiency of beta glucan extraction depended on the content of cereal grains of it but the purity may be independed characteristic. Therefore, using a modified method of -glucan extraction and purification which are suitable for each cereal crop may increase the efficiency of the extraction and purification. However, using Ft infrared and HPLC are very suitable to determine the beta glucan properties included the purity.