Starch Irradiation Physicochemical Properties Biology Essay

Abstract

V. ervilia starches were exposed to electron beam irradiation (EB-irradiation) (10, 20 and 30 kGy) and moisture conditions, structure, physicochemical properties, antinutritional factors and subsequent effects on in vitro and in vivo digestibility of starch in cockerels were investigated. Irradiation had no significant effect (p > 0.05) on chemical compositions. The results showed that Pasting properties (peak, trough, final, and setback viscosities), swelling properties and Apparent Amylose Content (AAC) were significantly decreased in a dose-dependent manner, whereas the solubility was increased following irradiation. In addition, irradiation decreased percentage syneresis of the unmodified starch. Seed flours showed a significant dose-dependent increase in water absorption capacity. Electron beam irradiation of seeds resulted in significant dose-dependent elevation of antinutritional factors (total phenols, tannins, condensed tannins, canavanine and trypsin inhibitor). EB-irradiation (10–30 kGy) decreased the average molecular size of vicia starches, but increased the proportions of enzyme-resistant starch (RS). The increase in RS content indicates that the irradiation induced the structural modification besides the chain degradation. Irradiation improved (p < 0.05) in vivo digestibilities of dry matter, crude protein, true protein and gross energy, but decreased starch digestibility.

Key Words: EB-irradiation; V. ervilia starch; Pasting properties; Solubility; Swelling properties; rapidly digestible and resistant starch

1. Introduction

Throughout the world, many countries are producing seed crops that were adapted to their specific environment and are used as sources of protein and starch in feed for humans, animal or poultry. Some species of leguminous family are sources of cheap protein for animals (Lopez Bellido, 1994). Bitter vetch (Vicia ervilia) is known for its high nutritional value, capacity of nitrogen fixation, and ability to grow in poor soils (Lopeze Bellido, 1994). Its seeds contain about 22.8% CP (Farran et al., 2001a, b). Bitter vetch seeds has been used in animal feeds and, when treated, as an alternative source of protein in poultry diet (Fernandez­Figares et al., 1995; Farran et al., 2001b). Raw bitter vetch, however, is detrimental to monogastric animals, especially chickens. The adverse effects arise from the presence of some antinutritional factors in the raw seeds including L-canavanine (0.035-0.11%, Berger et al., 2003), trypsin inhibitor (2.14mgg1 DM, Berger et al., 2003), and Catechin (2.01gkg 1 DM, Aletor et al., 1994). Feeding a diet with 60% raw bitter vetch has decreased weight gain and reduced feed intake in broilers and resulted in cessation of egg production of laying hens within 2 weeks post feeding. Several detoxification methods have been evaluated for leguminous seeds, including soaking in water (Barbour et al., 2001; Farran el al., 2001a; Farran el al., 2001b), acetic acid (Farran et al., 2001a), sodium bicarbonate solutions (Farran el al., 2001a) and potassium bicarbonate solution (D'Mello and Walker, 1991), boiling (Dhurandhar and Chang, 1990; Udedie, 1991; Belmar and Morris, 1994; Farran el al., 1995), autoclaving (Barbour el al, 2001; D'Mello and Walker, 1991), urea treatment (Udedie, 1991), and Alkaline extraction (Oioghobo et al., 1993). However, one or more antinutritional substance, representing a relatively high proportion of little-known or unconventional legumes could not be eliminated completely or even partially by the application of the earlier mentioned processing methods.

Additional techniques are the application of ionizing irradiation (molins, 2001). Compared to the physical and rheological changes, the effect of electron beam irradiation on starch and protein structure and consequent digestibility has been rarely investigated.

Electron beam irradiation is often applied for the modification of food materials to change their physical properties (Waje & Kwon, 2007). The irradiation may generate active radicals which readily react with food components to change their molecular structure (Yu &Wang, 2007). It has also been suggested as rapid and convenient modification technique which breaks large molecules into smaller fragments and is capable of cleaving glycosidic linkages (Yu & Wang, 2007).

Therefore, in this study, it was aimed to evaluate effects of electron beam irradiation on structure and physicochemical properties and subsequent effects on in vitro and in vivo digestibility of bitter vetch starch in cockerels.

3. Results and Discussion

3.1. Proximal features

The moisture content of raw V. ervilia seed was 6.07% and irradiation caused significant loss of moisture (p < 0.05) (Table 1). Low moisture content will be advantageous in maintenance and improvement of shelf life. The crude protein and crude fibre content in raw seed flour were 24.4% and 3.15%, respectively. The high protein content in V. ervilia seeds emphasizes their value as a vital source of nutrients. EB irradiation decreased the crude protein and crude fibre of seeds, but not significantly at any of the doses delivered. It would be interesting to determine total, soluble and insoluble dietary fibre fractions in raw and EB-irradiated Vicia seeds, to gain a better insight into the fibre contents. Crude lipid was significantly reduced on EB irradiation (control, 3.02%; 30 kGy, 2.11%) (P < 0.05), while ash significantly increased at 10 kGy onwards (control, 5.95%; 30 kGy, 6.49 %) (P < 0.05). The quantity of ash in any seed sample assumes importance, as it determines the nutritionally important minerals (Vadivel & Janardhanan, 2004). V. ervilia seeds contained a high amount of carbohydrates (63%), which might be due to low lipid content. However, EB irradiation increased the crude carbohydrates, which was significant only at 30 kGy (30 kGy, 70.54%) (p < 0.05). The increase in carbohydrates might be attributed to radiation-induced breakdown of complex sugars (polysaccharides) into simple extractable forms (e.g., free sugars) (Narasinga Rao, Deosthale, & Pant, 1989).

Table 1

Chemical composition of irradiated bitter vetch seed (as g/100g dry matter).

NFE

Crude fiber

Ether extract

Crude protein

Ash

Moisture

Irradiation Dose (KGy)

63c

5.23a

3.02a

22.8a

5.95c

6.07a

Control

67.25b

4.28b

2.12b

19.6c

6.75a

5.22b

10KGy

67.17b

4c

2.06b

20.4b

6.37b

5.26b

20KGy

68.24a

3.86c

2.11b

19.3c

6.49b

5.27b

30KGy

0.01

0.001

0.001

0.005

0.001

0.005

SEM

Means in the same column without superscripts are not significantly different (p > 0.05); SEM: standard error of the means

3.2. Antinutritional features

The highest impediment to consume any wild or underutilised seeds is the presence of antinutritional factors, particularly those which are heat-stable and difficult to eliminate on processing (Liener, 1994; Nowacki, 1980). These antinutritional factors decrease the digestibility and bioavailability of nutrients in the intestine. Most of the methods used to deactivate the antinutrients (e.g., dry heating, cooking, roasting, germination, fermentation) need not necessarily reduce or completely eliminate antinutrients, instead some methods reduce the nutritive value of plant produce.

3.2.1. Phenolics and tannins

Total phenolics, tannins and condensed tannins of EB-irradiated V. ervilia seeds revealed a significant dose-dependent decrease (p < 0.05) compared to the control (Table 2). Decrease in total phenolics, tannins and condensed tannins were significant at all of the irradiation doses (control, 202, 188 and 230 mg 100-1g DM; 30 kGy, 46, 94 and 94 mg 100-1 g DM) respectively. About 18%, 32% and 50% of the tannin content of V. ervilia was reduced at irradiation dose levels of 10, 20 and 30 kGy, respectively. Reduction of phenolics in V. ervilia was 52%, 66% and 77%, respectively. These reductions for condensed tannins were 22%, 38% and 59%, respectively. The variation in tannin concentration in legumes has been reported to range between 3.0 and 15.6 g/kg (Barampama & Simard, 1994). Although the effects of electron beam and gamma irradiation on phenolics and tannin contents of some materials have been reported, there is no information available in literature on the effect of ionizing irradiation on tannin contents of V. ervilia. Mechi et al. (2005) and Villavicencio et al. (2000) found that gamma radiation promoted reduction in the tannin contents as the radiation dose increased until a limited dose. This reduction in the tannin contents is very favorable, once this antinutritional factor presents the capacity of decreasing the protein digestibility. When this antinutritional factor is found at the proportion of 5:1 tannin/ protein, all protein is precipitated due to the tannin action (Pino and Lajolo, 2003). De Toledo et al. (2007) observed that gamma radiation (up to 4 kGy) significantly decreased phenolic compounds. Reduction of phenolics and tannin by EB irradiation in the present study is consistent with some previous studies using gamma irradiation (El-Niely, 2007; De Toledo et al., 2007) and electron-beam (Bhat and Sridhar, 2008; Shawrang et al., 2011), but Stajner et al. (2007) observed tannin content of soybean seeds to increase (21.6%) by gamma irradiation at the dose of 1 kGy. In some of the studies using ionizing irradiation a decrease in the level of tannin was accompanied with an increase in total phenolic compounds (Stajner et al., 2007; Harrison and Were, 2007; Kim et al., 2008). In contrast, some studies reported an increase in the amount of tannin (Stajner et al., 2007; Harrison and Were, 2007) or decrease in the amount of total phenolic content (Bhat and Sridhar, 2008) by irradiation. Such differences may be attributed to the differential response, variability in the genetic constituents (strains and varieties), geographical origin and other biological factors of legumes.

3.2.2. Canavanine

Canavanine is a water soluble non amino acid antinutritional factor (D'mello and Walker, 1991). Raw V. ervilia seeds contained an average of 0.078 percent canavanine. Berger el al. (2003) and Sadeghi et al. (2004) had reported that vicia ervilia seeds contained 0.035 to 0.11 (mean: 0.083) percent canavanine, which is quiet comparable to the levels detected in the present study. The potent antimetabolic properties of canavanine result primarily from its ability to function as a highly effective antagonist of arginine metabolism due to its structural similarity to this protein amino acid. It is also believed to function in maintaining nitrogen requirements of developing plants and to contribute significantly to plant chemical defence. The arginine-like structure enables canavanine to bind many enzymes that usually interact with arginine, and it is incorporated into polypeptide chains, resulting in structurally aberrant canavanine-containing proteins (Siddhuraju et al, 2002). Canavanine is subject to hydrolytic cleavage by arginase, yielding urea and L-canaline (Rosenthal, 1977). Although canavanine was only slightly toxic to adult and neonatal rats following single subcutaneous. Injections, multiple injections caused growth inhibition, alopecia, appetite depression and weight loss. The pancreas was affected more than other organs that were evaluated (Thomas & Rosenthal, 1987a, 1987b). Monkeys fed alfalfa sprouts developed a systemiclupus erythematosus-like syndrome, which was attributed to canavanine toxicity (Montanaro & Bardana, 1991). Human consumption of alfalfa seeds gives an initial reduction in serum cholesterol levels, but prolonged ingestion of alfalfa seeds has been associated with pancytopenia, anemia, leukopenia, and with the development of antinuclear antibodies (Montanaro & Bardana, 1991). Canavanine-sensitive organisms, mainly insects, metabolize it by incorporating it into protein macromolecules. Synthesis and utilization of such aberrant and impaired canavanine containing protein, adversely affects various developmental processes in insects, manifesting itself as toxic to these organisms. In the present study, the level of canavanine in the V. ervilia was found to be 0.078% and this value is lower than the value (2.65%) reported in the Jack bean (Canavalia ensiformis) by Natelson (1985). There was significant change in the canavanine content of the raw Vicia seeds following the irradiation treatments. Based on the above observations, it may be concluded that the toxic amino acid concentration could be reduced by irradiation.

3.2.3. Trypsin inhibitor activity

EB-irradiation had a substantial effect on the antitrypsin activity naturally present in V.ervilia. Controls presented the highest values, followed by doses of 10 and 20 kGy and dose of 30 kGy presented the lowest value. Trypsin inhibitor may decrease CP digestibility of feed particularly in monogastric animals and can depress their growth (Liener and Kakade, 1980). Reduction of trypsin inhibitor activity by irradiation was proportional to the dose. According to Rackins et al. (1975), only 50–60% of reduction on the trypsin inhibitory activity is required to avoid pancreatic hypertrophy in rats, and the inactivation of 70–80% resulted in a maximum value of protein efficiency rate (PER) of diet containing soybean flour. Abu-Tarboush (1998) found reduction of 34.9% on the trypsin inhibitor activity in soybean flour irradiated by 10 kGy. Farag (1998), found increase in the inactivation level with increase in the doses used (41.8%, 56.3%, 62.7% and 72.5% of loss in the trypsin inhibitory activity) for doses of 5, 15, 30 and 60 kGy, respectively. In the present work, radiation with dose of 10 kGy promoted reduction of 19.21% in average on the trypsin inhibitory activity, dose of 20 kGy reduced 48.76%, and 30 kGy reduced 83.25%. Published studies about the effects of EB-irradiation on the trypsin inhibitor activity of V. ervilia are scarce, but the reduction in antitrypsin activity is due to the breakage of the trypsin inhibitor structure by irradiation.

Table 2

Effect of radiation on the tannin concentration, Canavanine (mg100g-1 DM) and trypsin inhibitor (mg g-1 DM) in the bitter vetch seeds.

Irradiation dose (KGy)

I

II

III

IV

V

Control

202a

188a

230a

78a

2.03a

10

96b

155b

180b

77.1a

1.64b

20

68c

128c

142c

76.3a

1.04c

30

46d

94d

94d

71.5b

0.34d

SEM

2.5

1.2

1.5

0.35

0.003

I: total phenols; II: tannins; III: condensed tannins; IV: canavanine; V: trypsin inhibitor.

3.3. Englyst classification of starch

Starch nutritional fractions (RDS, SDS and RS) of irradiated V. ervilia seeds starches are presented in Table 3. RDS and SDS content of V. ervilia starch were decreased with increasing irradiation dose. It was assumed that the proportion of SDS might be partially transformed to RS, since the RS content was increased by irradiation. The amounts of RDS, SDS and RS fractions in pulse starches have been determined by the Englyst et al. (1992) and AACC (2000) methods. The above two methods differ with respect to time of hydrolysis and enzyme source. Only a few pulse starches have been analyzed for their RDS, SDS and RS contents and No data were found by authors regarding the effect of irradiation on the in vitro digestibility of V. ervilia. Furthermore, due to the different methods used for this analysis, it is difficult to make a meaningful comparison of the levels of RDS, SDS and RS among pulse starches. SDS and RS levels have been shown to be influenced by factors such as amylose content, crystallinity, crystalline perfection, and amylopectin structure (Benmoussa, Moldenhauer, & Hamaker, 2007; Chung et al., 2009a; Chung et al., 2008; Miao, Zhang, & Jiang, 2009; Zhang, Ao, & Hamaker, 2008). Our result is in accordance with other reports which observed a reduction in starch digestibility (Rombo et al, 2004; Chung and Liu, 2009b; Chung and liu, 2010). Rombo et al. (2004) suggested that an increase in the proportion of β-bonded starch after irradiation as a result of transglucosidation might induce decreased starch digestibility. Chung and Liu (2009b) claimed that the increase in carboxyl groups by irradiation resulted in inhibition of enzyme attack. This assumption might be supported by the present study, in which the content of carboxyl groups was considerably increased by irradiation of v. ervilia starches. The increase of RS by gamma-irradiation of vicia starches in the present study may indicate that changes in molecular structure such as the production of β-bonded starch, the increase in carboxyl groups, and the formation of physically less accessible packed structure occurred, which might lead to decreased starch hydrolysis. The reduced digestibility of pulse starches has been attributed to the absence of pores on the granule surface (Hoover & Sosulski, 1985), the high amount of amylose (Chung et al., 2009b; Hoover & Zhou, 2003), B-type crystallites (Hoover & Zhou, 2003), and strong interactions between amylose chains (Hoover & Sosulski, 1985). The susceptibilities of pulse starches towards hydrolysis by a-amylase reported in the literature cannot be compared due to differences in a-amylase source (bacterial, fungal, pancreatic), enzyme concentration, time of hydrolysis, and enzyme purity. From the present and previous studies (Chung and liu, 2009b; 2010), it could be suggested that irradiation increased the proportion of RS content and decreased the proportion of SDS content, regardless of crystalline type, but no consistent trends in the proportion of RDS were observed.

Table 3

The amounts of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) of EB-irradiated bitter vetch seed starches.

RS (%)

SDS (%)

RDS (%)

Irradiation dose (KGy)

55.1d

34.6a

10.3a

Control

59.7c

31.8b

8.5b

10KGy

64.5b

27.6c

7.9bc

20KGy

68.9a

24.3d

6.8c

30KGy

0.08

0.11

0.08

SEM

Values followed by the different superscripts letter within a column differ significantly (P < 0.05) from each other.

3.4. In vitro starch digestibility

In Fig. 1, the enzymatic digestibility of V. ervilia starch and its irradiation derivatives are depicted. The results indicate pronounced decreases in enzymatic digestibility as the level of irradiation dose increased. On the other hand, enzymatic digestibility increased progressively as the period of incubation increased. A reasonable explanation to substantiate decreases in enzymatic digestibility is the increase of β-bonded starch after irradiation as a result of transglucosidation (Rombo et al, 2004). Result obtained from this method is supported by Englyst method and in vivo starch digestibility.

In vitro starch digestion (total starch basis)

Incubation time (hour)

Fig. 1. Time course of in vitro starch digestion (proportion of total starch) of vicia ervilia.

3.4. Effects on in vivo digestibility

The results of in vivo digestibility of untreated and irradiated V. ervilia grains are shown in table 4. With increase in doses, digestibility of dry matter, crude protein, true protein and gross energy increased significantly compared to control, but digestibility of starch decreased significantly.

No data were found by authors regarding the effect of irradiation on in vivo digestibility of V. ervilia, however there are some data regarding in vitro digestibility of irradiated legume starches and proteins. Duodu, Taylor, Belton, and Hamaker (2003) reported that tannins have a detrimental effect on the ileal digestibility of proteins. Due to their hydroxyl groups, tannins may interact with and form complexes with proteins, which may lead to precipitation because of the large size of the tannins. In addition to possibly causing a change in protein conformation, study of Siddhuraju et al. (2002) showed that the tannins may also exert steric effects (due to their large size) and prevent enzymes access to the proteins. Similarly, Rehman and Shah (2001) showed that the partial removal of tannin probably created a large space within the matrix, which increased the susceptibility to enzymatic attack and consequently improve the digestibility of protein after irradiation treatment. Moreover, this effect could also be due to inactivation of proteinaceous antinutritional factors (Van der Poel., 1990). The amelioration in protein digestibility can be attributed to destruction of antinutritional factors, such as protease inhibitors and lectin which lead to an improvement in the digestibility of the protein (Liener, 1978). The in vitro apparent digestibility of protein data indicated a beneficial effect for radiation when the in vitro digestibility of the studied legume seeds was considered. These results are in good agreement with those obtained by Rady et al. (1987) who reported that the nutritional value of all varieties of beans, based on chick growth, was significantly improved by irradiation. Arora (1983) and Bressani et al. (1987) attributed the poor nutritive value of legumes to the presence of some forms of proteins which inhibit the digestive enzymes such as trypsin, chymotrypsin inhibitors. The apparent improvement in vitro digestibility that being ensured through radiation treatment, may be attributed to appreciable effect of irradiation on the antinutritional factors present naturally in non-irradiated studied legumes which are more sensitive to enzyme action. Another possible reason for increasing in protein digestibility is modification in the three dimensional structure of proteins due to irradiation. Studies of Shawrang et al. (2007) and Shawrang et al. (2008) illustrated that protein denaturation occur by irradiation that lead to improvement in intestinal protein digestion. Sreenivasan et al. (1974) showed that Irradiation at 10 kGy caused slight degradation of proteins, which increases the susceptibility of this product to proteolytic action. Therefore, it could be concluded that the irradiation process offers a good treatment for legumes to reduce or eliminate their antinutritional factor(s) with consequent increase in their digestibility and thereby increase the utilization of their proteins.

Existence of Non Starch Polysaccharides (NSPs) in seeds causes the intestinal contents to become viscous and interfere with nutrient assimilation and the general well-being of the chick (Smits & Annison, 1996). Irradiation of grains containing NSPs, fed to chicks, improves the apparent absorption of fat, amino acids and starch (Campbell et al., 1983; Classen et al., 1985). It has been suggested that this increase was induced by structural degradations in NSPs which allowed easy access of the digestive enzymes to starch (Yoon et al., 2010). Results obtained from this study regarding Viscosity of V. ervilia seeds confirm this claim that irradiation causes reduction of Viscosity. But, results achieved from in vivo and in vitro digestibility of V. ervilia showed that starch digestibility decreased by irradiation. Results obtained from some in vitro studies suggested that an increase in the proportion of β-bonded starch after irradiation as a result of transglucosidation might induce decreased starch digestibility (Rombo et al, 2004). Chung and Liu (2009c) claimed that the increase in carboxyl groups by irradiation resulted in inhibition of enzyme attack. This assumption might be supported by the present study, in which the content of carboxyl groups was considerably increased by irradiation of v. ervilia starches. The reduced digestibility of legume starches has been attributed to the absence of pores on the granule surface (Hoover & Sosulski, 1985), the high amount of amylose (Chung et al., 2009b; Hoover & Zhou, 2003), B-type crystallites (Hoover & Zhou, 2003), and strong interactions between amylose chains (Hoover & Sosulski, 1985). Compare of results achieved regarding viscosity, in vitro and in vivo V. ervilia starch digestibility may show that the effect of irradiation on starch granules was stronger than effects on NSPs that finally caused decrease of starch digestibility.

Table 4

Effects of electron beam irradiation on bitter vetch seed in vivo digestibility.

In vivo digestibility (%)

Irradiation dose (Kgy)

True protein

Crude protein

Gross energy

Dry matter

starch

81.43d

76.22

37.8c

61.42d

43.5a

Control

83.83c

76.98

38.4c

63.5c

42.2b

10KGy

86.3b

79.2

40.6b

64.7b

39.7c

20KGy

90.4a

82.6

44.5a

67.3a

37.6d

30KGy

0.03

0.04

0.04

0.04

0.07

SEM

Values followed by the different superscripts letter within a column differ significantly (P < 0.05) from each other; SEM: standard error of the means.

3.5. Water absorption capacity

Significant increase in water absorption capacity (WAC) of V. ervilia starch was observed at an irradiation dose of 10 kGy (Table 5). The increase in WAC with irradiation could be attributed to irradiation induced damage or degradation of vicia starch to simpler molecules such as dextrins, maltose and other sugars that have higher affinity for water than starch (Rayas-Duarte & Rupnow, 1993; Whistler & Daniel, 1985). WAC at 10, 20 and 30 kGy further showed little increase which may be due to cross linking occurring simultaneously with degradation phenomenon. The extent of cross linked starch might have increased at higher irradiation doses & counteracted WAC at those doses. Elevated levels of cross linked starch in maize and bean starch have been reported at higher irradiation doses (Rombo, Taylor, & Minnaar, 2004; Adil Gani et al, 2012).

3.6. Swelling Index

Irradiation, at all the doses caused significant reduction in swelling index of V. ervilia starch (Table 5). Similar results have been reported by several researchers with various starches (Abu et al, 2006; Rayas-Duarte and Rupnow, 1993; Ezekiel et al, 2007; Chung and Liu, 2010). Swelling results from the ability of starch to trap and retain water within its structure (Whistler & Daniel, 1985). This capability may be diminished markedly once starch degradation occurs with irradiation (De kerf et al, 2001). Amylopectin fraction of starch is considered to be primarily responsible for swelling (Tester and Morrison, 1990) and a significant reduction in molecular size of amylopectin fraction of various starches with irradiation was found (De kerf et al, 2001). It indicates that amylopectin is sensitive to irradiation. De Kerf et al (2001) reported a significant reduction in amylopectin fraction of various starches with irradiation. Chung and Liu (2010) stated that the decrease in swelling power could be beneficial to improve the textural quality upon cooking as the bursting of starch could be prevented.

3.7. Carboxyl Content

The carboxyl content increased as the irradiation dose was increased in V. ervilia starches (Table 5). Abd Allah, Foda, and El Saadany (1974) observed substantial increase in the production of formic acid in irradiated wheat starch. The radiation degradation of starch is initiated by the generation and transformation of free radicals and follows the low-molecular products with the number of carboxylic acids and aldehydes (Sharpatyi, 2003). Ghali et al. (1979) reported that formic, acetic, pyruvic and glucuronic acids were formed during irradiation of starch. Therefore, the main degradation products formed during irradiation of native starch were carboxylic acids, which resulted in an increase in carboxyl content and a decrease in pH value of all starch samples. Similar findings were reported by Chung, Hoover, and Liu (2009b) and Chung and Liu (2010) with irradiated corn and bean starch respectively.

3.8. Viscosity

Levels of viscosity in raw and irradiated V. ervilia seeds at dose levels of 10, 20 and 30 kGy are shown in Table 5. EB irradiation at 10-30 kGy significantly reduced the viscosity of V. ervilia when compared with the control samples. The high viscosity in V. ervilia is due to the presence of NSPs, i.e. galactomannan, and it has also been reported to interfere in the nutrient metabolism of monogastrics (Smits & Annison, 1996). These carbohydrate polymers cause the intestinal contents to become viscous and interfere with nutrient assimilation and general well-being in chicken (Smits & Annison, 1996). The depolymerisation of such non-starch polysaccharides in oats by irradiation significantly improved the growth parameters in chicks (Campbell et al., 1986). In this regard, further extensive studies of the effect of higher doses of irradiation on the viscosity nature of NSP, its solubility parameters and nutrient utilisation studies through in vivo approaches are needed on V. ervilia samples.

Table 5

Water Absorption, Swelling index, Carboxyl content and Viscosity of V. ervilia seeds.

Irradiation dose (KGy)

WAC (g/g)

SI

(g/g)

CC (g/100g)

Viscosity (mPa.s)

Control

2.00b

11.68a

0.00d

7.72a

10

2.29a

7.32b

0.06c

6.28b

20

2.32a

5.34c

0.09b

4.36c

30

2.35a

3.14d

0.13a

3.85d

SEM

0.003

0.02

0.001

0.01

WAC: Water Absorption Capacity; SI: Swelling Index; CC: Carboxyl Content.

3.9. Apparent and Absolute amylose content

Both apparent and absolute amylose contents of the granule starch of V. ervilia were analyzed and the results are shown in Table 6. Apparent and Absolute amylose content of non-irradiated vicia was 43.5 ± 0.06% and 40.21 ± 0.09%, respectively. Apparent amylose contents were reduced by 4.59%, 6.89% and 11.49% and Absolute amylose content reduced by 5.27%, 7.08% and 11.46% at 10, 20 and 30 kGy doses, respectively (Table 6). This effect of EB- irradiation pretreatment on AAC was associated with the structure of starch. The values of the apparent amylose contents were larger than that of the absolute amylose content (43.5%, 41.5%, 40.5% and 38.5% for the apparent amylose content and 40.21%, 38.09%, 37.36 and 35.6% for absolute amylose content at10, 20 and 30 kGy doses, respectively). The difference between the apparent and the absolute amylose contents was attributed to that the long branch-chains of amylopectin bound iodine during the iodine affinity analysis and inflated the value of the amylose content. The amylose content of starch is, in general, proportional to the granule size and maturity of starch (Jane & Shen, 1993; Kulp, 1973; Meredith et al., 1978; Pan & Jane, 2000). Amylose is an essentially linear polymer of α-(1–4)-linked-D-glucopyranosyl units with up to 0.1% α-(1–6) linkages, with degree of polymerization (DPn) of 800–4920. Amylopectin consist of α-(1–4)-linked-D-glucosyl chains and is highly branched with 5–6% α-(1–6)-bonds, with degree of polymerisation of 8200–12,800. Evidently, the content and degree of polymerization of amylopectin were so higher than that of amylose. So the amylopectin had higher probability to be broken and cleaved during irradiation (Yu and Wang, 2007). The current concept of AAC described in this paper is actually composed of two components: amylose and partly branched long chains (b chains) of amylopectin (Vandeputte & Delcour, 2004). Based on this background, it can be assumed that the decrease of AAC largely originated from the breakage or cleavage of long chains in amylopectin caused by EB-irradiation (Chiang & Yeh, 2002; Vandeputte & Delcour, 2004; Yu and Wang, 2007). According to the starch biosynthesis (Martin and Smith, 1995), the amylopectin molecules are arranged radially and adjacent branches within the branch clusters may form double helices that can be packed regularly, giving crystallinity to the starch granule. The starch granule is not uniformly crystalline; however, it contains relatively amorphous regions. Amylose molecules form single helical structures and are thought to be packed into these amorphous regions, and the percentage of amorphous regions occupied by amylose varies obviously depending on the amylose amounts that different cultivar have. Current results basically concurred with observation of Descherider (1960); Dianxing et al (2002) and Chung and Liu (2009) that the degraded starch resulted from the shortening of polysaccharide chains.

Table 6

Amylose contents of V. ervilia starches.

Iodine Affinity (%)a

Amylose content (%)

Irradiation

dose (KGy)

starch

Amylopectin

Apparentb

Totalc

Control

8.7a

1.1a

43.5a

40.21a

10

8.3b

1.1a

41.5b

38.09b

20

8.1b

1.00a

40.5b

37.36b

30

7.7c

0.9a

38.5c

35.6c

SEM

0.005

0.002

0.15

0.17

a Averaged from at least two analyses; Means in the same column without superscripts are not significantly different (p > 0.05); SEM: standard error of the means.

3.10. Pasting properties

Pasting properties of EB-irradiated millet starches with different doses are presented in Table 7. The pasting temperature was considerably decreased with increasing irradiation dose (table. 7). This result was in agreement with those reported by Yu and Wang (2007); Chung and Liu (2009) and Chung and Liu (2010). The degradation of molecular structure induced by irradiation might cause the swelling to occur at a lower temperature and to reach a maximum earlier. The low pasting temperature of starch indicated their lower resistance towards swelling (Sandhu & Singh, 2007). The value of PV decreased considerably with the increase in irradiation dose. The decrease in peak viscosity (PV) may be attributed to the breakdown of inter- and intra-molecular physical unions due to damage to the ordered structure of starch granules and consequent reduction in their swelling (Karim et al., 2008; Sandeep Singh et al., 2011). The decrease in viscosity of starch with an increase in the level of irradiation has also been reported by other researchers (Sabularse et al., 1992; Chung & Liu., 2010; Adil Gani et al, 2012). Kang et al. (1999) reported that increase in doses of irradiation was the reason for decrease of viscosity of starch paste while Yu and Wang (2007) attributed decrease in peak-, trough- and breakdown-viscosity to the decrease in size of the starch granules caused by irradiation. The decrease in viscosity with irradiation may also be attributed to the degradation of starch to simpler molecules such as dextrins and sugars (Sandeep Singh et al., 2011). Other workers have also reported depolymerization of various starches following irradiation (Sabularse et al., 1992). Other values of starch pasting properties, breakdown viscosity (BV), trough viscosity (TV), final viscosity (FV), and setback viscosity (SV) all decreased following the irradiation. With the increase in irradiation dose from 0 to 30 kGy, the values of BV, TV, FV, and SV decreased by 450 cP , 1681 cP, 3141 cP and 1460 cP, respectively. The results were consistent with the statement that values of PV, TV, FV, and SV for starch were considerably reduced with increasing dose (Abu et al., 2006; Liu et al., 2012; Adil Gani et al, 2012). SV, which was the difference between FV and TV, was an index of the tendency of a starch paste to retrograde during cooling (Tianyi et al., 2011). The results of decreased FV and SV showed that the starch gel was softer and showed fewer tendencies to retrograde after higher EB-irradiation dose. This was attributed to the decrease in degree of polymerization of EB-irradiated starch, due to the breakage of starch chains, inducing the reduction in paste viscosity (Sandeep et al., 2011; Adil Gani et al, 2012). Similar results have been reported by Abu et al. (2006) and Chung and Liu (2010). Breakdown viscosity, which was the difference between PV and TV, was a measure of fragility of the swollen granules, indicating the stability of the starch pastes and the tendency of starch granules to resist shear force during the heating process (Liu et al., 2012; Luo et al, 2009). Decreased trend in breakdown was observed as the irradiation dose increased. The results were in agreement with those reported by Abu et al. (2006) and Yu and Wang (2007).

Table 7

Pasting properties of V. ervilia starches under different EB-irradiation doses.

Irradiation dose (KGy)

PT (ºC)a

PV (ºC)a

TV (ºC)a

FV (ºC)a

BV (ºC)a

SV (ºC)a

Control

443a

2217a

1723a

3226a

494a

1503a

10

184b

887b

691b

985b

196b

294b

20

80c

288c

137c

281c

151c

144c

30

29d

96d

42d

85d

44d

43d

SEM

0.6

0.4

1.05

1.02

0.8

1

Values are means ± SD of three determinations (n = 3); Values followed by the different superscripts letter within a column differ significantly (P < 0.05) from each other; a PT = pasting temperature; PV = peak viscosity; TV = trough viscosity; FV = final viscosity; BV = breakdown viscosity; SV = setback viscosity.

3.11. Solubility index

The solubilities of different EB-irradiated V. ervilia starches in different temperatures are presented in Table 8. Non-irradiated vicia starch appeared less soluble at temperatures close to ambient, and showed a slight increase with increasing temperature. Under same testing temperatures, the solubilities of irradiated vicia starches were all increased with the increase in irradiation dose. Furthermore, the solubilities of irradiated starches were all increased following the testing temperature. The effect of irradiation on solubilities of starches was completely different in the range of lower temperature and higher temperature. It was clear that rising of temperature and elevation of irradiation dose all induced the increase of solubility of starch samples. When heated in the presence of excess water, starch granules were considerably swollen and water molecules become linked by hydrogen bonding to the exposed hydroxyl groups of amylose and amylopectin (Tianyi Liu et al., 2011). When the testing temperature were raised from 30 ºC to 90 ºC, it was clear that heating increased the solubility of non-irradiated by 10.67 g/100 g, however, solubilities of the treated starch increased by 32.05 g/100 g, 32.4 g/100 g and 42.59 g/100 g, after 10 kGy, 20 kGy and 30 kGy, irradiation dose, respectively. It was concluded that effect of irradiation on solubility was bigger than the effect of heating on solubility. The increase in polarity due to chain scission (breakage of glucosidic bonds) under the effect of EB irradiation (Roushdi et al., 1983; De Kerf et al., 2001; Tianyi Liu et al., 2011) and the decrease in inter-chain hydrogen bonds explain the increase in solubility. In fact, it has been stated in the literature (Merlin and Fouassier., 1981; Bertolini et al., 2001; Raffi and Agnel., 1983; Henry et al., 2010) that the amylose molecules grow shorter while the branched amylopectin molecules are cut and stretched to a more linear form. Irradiation decreased inter-chain hydrogen bonds and increased the hydrogen bonds with water, which improved the solubility of starch processed by EB-irradiation (Liu, Ying Ma, Xue, & Shi, 2012). Similar results have been reported in maize, rice, corn and potato starches with EB- and gamma irradiation treatment (Hebeish et al., 1992; De Kerf et al., 2001; Tianyi Liu et al., 2011).

Table 8

Solubility (%) of EB-irradiated V. ervilia starches under different temperatures.

Irradiation dose (KGy)

Solubility (g/100g)

30 (ºC)

40 (ºC)

50 (ºC)

60

(ºC)

70 (ºC)

80 (ºC)

90 (ºC)

Control

5.83c

7.05d

8.13d

10.89c

12.38d

15.06d

16.5d

10

6.15b

8.13c

10.45c

13.9b

23.1c

27.6c

38.2c

20

12.6ab

14.16b

16.31b

19.88a

23.46b

33.44b

45.00b

30

15.01a

16.11a

17.32a

19.96a

28.22a

39.91a

57.6a

SEM

0.04

0.11

0.05

0.15

0.09

0.06

0.08

Values are means ± SD of three determinations (n=3); Values followed by the different superscripts letter within a column differ significantly (P < 0.05) from each other.

3.12. Rapid Visco-Analyser (RVA)

Syneresis was expressed as the volume of water that separated out as the result of gel shrinkage (Hoover & Manuel., 1996). When a starch paste or gel is frozen, phase separation occurs with the formation of ice crystals. On thawing, the paste or gel will continue to be composed of a starch-rich and starch-deficient aqueous phase. The extent of phase separation increases with an increase in the number of freeze-thaw cycles due to an increase in amylopectin retrogradation in the starch-rich phase (Yuan & Thompson, 1998). Upon thawing, the water can be easily expressed from the dense network, a phenomenon known as syneresis. The amount of syneresis is directly related to the tendency of a starch to retrograde. EB-irradiation decreased percentage syneresis of the unmodified starch (Table 9). In native starch, percentage syneresis increased progressively as the number of freeze thaw cycles increased. The gels from irradiated V. ervilia starches discharged less water in comparison to native millet starches. Syneresis in freeze–thawed gels is attributed to the increase of molecular association between starch chains at reduced temperature, exuding (resulting from inter- and intra-molecular hydrogen bonding due to associations between the separated amylase chains during frozen storage) water from the gel structure (Hoover & Manuel., 1996). Drastically decreased syneresis after modification could in part be attributed to decreased apparent amylose content at higher irradiation doses (Srichuwong et al., 2011). Decrease in apparent amylose content after irradiation treatment has been reported by Chung and Liu (2010) and Yu and Wang (2007) and Adil Gani et al (2012). It could partly be related to amylopectin chain ratio which has higher water holding capacity, highly branched structure & shorter chains would retrograde in a slower rate (Srichuwong & Jane, 2007). Syneresis was relatively higher which might be attributed to higher amylose content, the interaction between leached out amylose and amylopectin chains leading to gel shrinkage (Hermansson & Svegmark,1996;Wani, Sogi,Wani, Gill, & Shivhare, 2010), degree of polymerization of amylose, amylopectin chain length, proportion of short chains or shrink back of partially disintegrated granules to their original size during cooling.

Table 9

Freeze–thaw stability of EB-irradiated bitter vetch seed starches.

Ir dose (KGy)

Percentage syneresisa

Cl 1

Cl 2

Cl 3

Cl 4

Cl 5

Cl 6

Cl 7

Cl 8

Crl

78.4d

79.3d

81.15d

81.98d

82.91d

83.67d

85.12c

86.15c

10

73.5c

74.8c

75.92c

76.81c

77.67c

78.73c

79.83b

81.14b

20

69.6b

70.4b

71.86b

72.62b

73.81b

74.61b

76.1b

76.92a

30

64.5a

65.9a

67.2a

67.95a

69.15a

71.18a

72.25a

73.44a

SEM

0.09

0.03

0.15

0.11

0.07

0.1

0.06

0.09

Ir dose = Irradiation dose; Crl = Control; Cl = Cycle; the conditions of synthesis are given in Table 4; a Values are means ± S.D.s of three replicate determinations; Values followed by the different superscripts letter within a column differ significantly (P < 0.05) from each other.

3.13. Pearson’s correlation analyses between digestibility and different physicochemical properties

The Pearson’s correlation was used commonly to reflect the degree of linear relationship between two variables. The Pearson’s correlation coefficients for the relationships between various properties of vicia seeds with different irradiation doses are shown in Table 10 and 11. Irradiation dose was positively correlated to RS, DMD, GED, CPD and TPD (r = 0.995, 0.978, 0.942, 0.950 and 0.986, respectively, P < 0.05), and negatively correlated to RDS and SDS (r = -0.911 and -0.987, respectively, P < 0.05). RS were positively correlated to in vivo digestibility and RDS and SDS was negatively correlated to in vivo digestibility.

Irradiation dose was positively correlated to solubility on 30 ºC, 90 ºC and Water Absoption Capacity (r = 0.895, 0.975 and 0.818 respectively, P < 0.05). Irradiation dose was positively correlated to PT and PV (r = -0.942 and -0.938, respectively, P < 0.01) and also negatively correlated to BV, TV, FV, and SV (r = -0.933 -0.936, -0.907 and -0.861, respectively, P < 0.05). Irradiation dose was negatively correlated to swelling capacity on 90 ºC, Carboxyl content, Viscosity, Apparent and Absolute content of amylose (r = -0.980, -0.979, -0.976, -0.936 and -0.920, P < 0.05). In the interrelationship between the various pasting parameters, PT was positively correlated to BV, (r = 0.989, p < 0.05). PV was positively correlated to TV, FV, SV and BV (r = 0.999, 0.996, 0.997 and 0.983, respectively, p < 0.05). Different properties were observed to be related to each other. Solubility was negatively correlated to all properties except Water Absorption Capacity and Carboxyl content. Pasting properties were positively correlated to swelling capacity, freeze-thaw stability, Viscosity and Amylose content. Similar results were reported by Tianyi Liu et al. (2011); Yu and wang, (2007) and Abu et al. (2006).

Table 10

Pearson’s correlation coefficient among irradiation dose, in vitro and in vivo digestibility of millet starches.

In vitro digestibility

In vivo digestibility

Ir d

RDS

SDS

RS

DMD

GED

CPD

TPD

Ir da

1

-0.911a

-0.987*

0.995*

0.978*

0.942*

0.950*

0.986*

RDSa

1

0.905*

-0.915*

-0.916a

-0.794a

-0.796a

-0.882a

SDSa

1

-0.978*

-0.978*

-0.930*

-0.943*

-0.969*

RSa

1

0.968*

0.937*

0.938*

0.984a

DMDa

1

0.939*

0.945*

0.977*

GEDa

1

0.986*

0.974*

CPDa

1

0.973*

TPDa

1

*, means the correlations are significant at P < 0.05 levels, respectively; a Ir d = Irradiation dose; RDS = Rapid Digestible Starch; SDS = Slowly Digestible Starch; RS = Resistance Starch; DMD = Dry Matter Digestibility; GED = Gross Energy Digestibility; CPD = Crude Protein Digestibility; TPD = True Protein Digestibility.

Table 11

Pearson’s correlation coefficient among different physicochemical and functional properties of millet starches.

Ir d

S30

S90

PT

PV

TV

FV

BV

SV

Cy 1

Cy 8

SW

WAC

Carboxyl

Viscosity

AAC1

AAC2

Ir da

1

0.895*

0.975*

-0.942*

-0.938*

-0.936*

-0.907*

-0.933*

-0.861*

-0.995*

-0.996*

-0.980*

0.818*

-0.979*

-0.976a

-0.936*

-0.920a

S30a

1

0.907*

-0.904

-0.894*

-0.888*

-0.880*

-0.903*

-0.855*

-0.879*

-0.908*

-0.910*

0.795*

0.869*

-0.894a

-0.819*

-0.778a

S90a

1

-0.980*

-0.975*

-0.968*

-0.962*

-0.987*

-0.942*

-0.976*

-0.984*

-0.997*

0.894*

0.985*

-0.956a

-0.931*

-0.922a

PTa

1

0.998*

0.996*

0.995*

0.989

0.978*

0.933*

0.964*

0.985*

-0.930*

-0.960*

0.961a

0.885*

0.879a

PVa

1

0.999*

0.996*

0.983*

0.977*

0.930*

0.961*

0.982*

-0.929*

-0.958*

0.964a

0.883*

0.880a

TVa

1

0.993*

0.975*

0.972*

0.927*

0.960*

0.978*

-0.924*

-0.954*

0.968a

0.877*

0.872a

FVa

1

0.984*

0.992*

0.901*

0.936*

0.968*

-0.941*

-0.939*

0.937a

0.860*

0.859a

BVa

1

0.979*

0.932*

0.954*

0.983*

-0.931*

-0.959*

0.930a

0.890*

0.885a

SVa

1

0.859*

0.895*

0.942*

-0.947*

-0.911*

0.888a

0.827*

0.831a

Cy1a

1

0.988*

0.979*

-0.808*

-0.982*

0.960a

0.960*

0.946a

Cy8a

1

0.989*

-0.856*

-0.982*

0.984a

0.923*

0.908a

SW

1

-0.892*

-0.986*

0.971a

0.939*

0.929a

WAC

1

0.867*

-0.842a

-0.734*

-0.763a

Carboxyl

1

-0.952a

-0.938a

-0.943a

Viscosity

1

0.897a

0.877a

AAC1

1

0.987a

AAC2

1

*, means the correlations are significant at P < 0.05 levels, respectively; a Ir d = Irradiation dose; S30 and S90 means the solubility at 30 and 90 ºC, respectively; PT = pasting temperature; PV = peak viscosity; TV = trough viscosity; FV = final viscosity; BV = breakdown viscosity; SV = setback viscosity; Cy 1 and Cy 8 = Cycle 1 and Cycle 8 at Freeze-Thaw stability; SW= Swelling Factor at 90 ºC; WAC= Water Absorption Capacity; AAC1= Apparent Amylose Content; AAC2= Absolute Amylose Content.

4. Conclusion

Irradiation processing has been used as a means to inactivate antinutritional factors and increase of nutritional quality of V. ervilia seeds. The present work clearly shows that irradiation processing of vicia at dose levels of 10-30 kgy can improve its nutritional quality. Maximum improvement in protein quality (i.e. in vivo protein digestibility) was observed at the higher radiation (30 kGy). EB irradiation decreased tannin, cannavanine, trypsin inhibitore activity and starch digestibility (approximately 50%, 9%, 83% and 13% reduction, respectively) of V. ervilia seeds. EB-Irradiation induced the degradation of amylose and amylopectin and consequently altered the physicochemical properties, including an increase in carboxyl content, solubility and water absorption capacity and a decrease in swelling index, apparent amylose content and syneresis. The pasting properties decreased drastically with increasing irradiation dose. The decrease in SV and BV indicated that higher irradiation doses reduced the stability of starch pastes and their tendency to retrogradation. Further studies are needed to evaluate the definite effect of EB radiation on antinutritional factors using pure molecules. Unlike chemical treatments, which are time consuming, irradiation can be a quick and efficient method for modifying the properties of different seeds.