J Sci Food Agric 1996,70,197-203

The Effects of Fungal Infection on the Chemical and Functional Properties of ChickDeas (Cicer arietinum) and Faba Beans (Vicia f i ba ) ‘ James Sarantinos”, Tran V Hung, Robert G Black*

Australian Food Research Institute, Agriculture Victoria, Sneydes Road, Werribee, Victoria, 3030, Australia

and Jan Bert Brouwer

Victorian Institute for Dryland Agriculture, Natimuk Road, Private Bag 260, Horsham, Victoria, 3400, Australia

(Received 9 January 1995; revised version received 19 June 1995; accepted 18 August 1995)

Abstract: We investigated the effects of fungal infection on the chemical composi- tion and functional properties of ‘healthy’ and ‘diseased’ chickpeas and faba beans which were classified on the basis of visual contamination. Following fungal testing, the ‘diseased’ seeds were shown to possess relatively low levels of potentially toxigenic fungi as a proportion of the total number of colonies, which was also lower than expected. Mycotoxins could not be detected in either crop. There were no appreciable differences in the average moisture, ash, crude fat, crude protein and acid detergent fibre contents between the ‘healthy’ and ‘dis- eased grain of both chickpeas and faba beans. Hydration capacity of the whole seeds did not differ between ‘healthy’ and ‘diseased’ grain samples of both chick- peas and faba beans. However, hard seed coatedness was greater in both ‘dis- eased’ chickpeas and faba beans compared with the ‘healthy’ samples. Water and oil absorption capacities were affected by disease in the chickpea and faba bean flours, although the effects were inconsistent between the two seed types. The foaming characteristics of the ‘diseased‘ chickpea and faba bean flours were severely affected.

Key words : fungal infection, chickpeas, faba beans, chemical and functional properties.

INTRODUCTION not only cause significant reductions in crop yields but also cause unsightly discolouration of the seeds. Cur-

During the 1992-1993 grain legume harvest in Victo- rently, seeds damaged in this way are considered to be ria’s Wimmera region (Australia), both the chickpea unsuitable for utilisation and are therefore discarded (Cicer arietinum) and faba bean (Vicia faba) crops were because of their lack of marketability. adversely affected by fungal disease favoured by Grain legumes, like other crops, are susceptible to unseasonally high rainfall. Most prevalent were foliar fungal attack during adverse weather conditions and diseases such as Ascochyta blight in faba beans and during prolonged storage (Deo and Gupta 1980). Botrytis grey mould in kabuli chickpeas. These diseases Fungal contamination has been observed to result in

both structural and chemical changes in infected chick- * To whom correspondence should be addressed. pea (Williams and Singh 1987). Ahmad and Singh

197 J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain

198 J Sarantinos et a1

(1991) observed that high seed moisture levels, environ- mental temperatures and relatively high humidity after heavy rainfall enhanced the proliferation of potentially toxigenic fungal species, particularly Aspergillus flauus and Aspergillus parasiticus, thus favouring aflatoxin production. Other frequently observed moulds in chick- peas are species of the Botrytis, Nigrospora, Phoma, Stemphylium and Penicillium genera.

Fungal infection causes structural and chemical changes in diseased grain legumes. In one study Geno- vese and Lajolo (1992, 1993) investigated the physi- cochemical changes that occurred in protein isolates extracted from soya beans which had been damaged by breakage caused by storage and other factors such as rotting, shrivelling and discolouration which were attributed to fungal contamination. Certain functional properties, especially emulsification capacity and stabil- ity, protein solubility, viscosity and gel forming ability were found to be compromised when the proteins derived from damaged soya beans were compared with those of healthy seeds. There is a paucity of information on the effects of fungal contamination on chickpeas and faba beans.

The aims of this study are, firstly, to characterise the fungal infection of each seed type, and, secondly, to determine if the contamination has affected the chemical composition and functional properties of the seeds and their flours.

EXPERIMENTAL

Sample collection and preparation

Three grain samples of kabuli chickpeas (cv Kaniva) and two samples of faba beans (cv Fiord) grown in separate fields in the Wimmera region (Western Victo- ria, Australia) were pooled and manually sorted into ‘diseased’ and ‘healthy’ fractions on the basis of dis- colouration usually associated with fungal disease.

Fungal analyses

These assays were conducted by the Division of Food Science and Technology, Commonwealth Scientific and Industrial Research Organisation (Sydney, Australia).

Microbiological analyses were performed according to previously published methods (Hocking 1982). One hundred seeds from each of the ‘diseased’ fractions of the chickpea and faba bean samples were surface disin- fected for 2 min in 0.4% sodium hypochlorite. Since it was subsequently found that only a relatively small pro- portion of the seeds were infected, it was considered unnecessary to analyse the ‘healthy’ fractions of the two crops.

Duplicate samples were plated out onto the following media: Aspergillus flauus and Aspergillus parasiticus agar (AFPA), Dichloran Rose Bengal Chloramphenicol agar (DRBC), Dichoran 18% Glycerol agar (DG18), and Czapek Iprodione Dichoran agar (CZID) (Hocking 1981).

AFPA plates were inoculated with sample and incu- bated at 30°C for 2-3 days. The other media were incu- bated at 25°C for 5-7 days. After the incubation period, the plates were assessed for total fungal infection and differential counts of various fungal colony types. Rep- resentative isolates were subcultured onto identification media for genus and, where possible, species identifica- tion.

Infection rates were calculated as the average number of seeds showing microbial growth on the broad spec- trum agar, ie DRBC and CZID. In addition, infection rates on the media specific for certain species were also determined. Aspergillus flavus infection was calculated solely from the AFPA medium, while Eurotium species infection was calculated from the DG18 medium only.

Mycotoxic analyses

The assays were performed at the Academy of Grain Technology (Melbourne, Australia).

The levels of aflatoxin,, B,, G , and G, were deter- mined using the method of Paulsch et al(1988). Deoxy- nivalenol was determined according to the method of Trucksess et al (1984). The determination of T, toxin, fusarenon-x, zearalenone, alternariol and alternariol monoethyl ether followed the method of Blaney et a1 (1984).

Chemical composition

Seeds were ground into flours (to pass through a sieve with an aperture of 300 pm in diameter) on setting ‘0’ (finest setting) on a Falling Number Laboratory Mill (Model 3303) for the determination of chemical com- position and functional property tests.

Ash (method 08.01), moisture (method 44-19) and crude fat (method 30-25) were determined in duplicate using official AACC (1983) methods. Acid detergent fibre (ADF) content was determined by the Van Soest method (1963). Crude protein (nitrogen x 6.25) was measured by a Leco Nitrogen Analyzer (Model FP228). Amino acid composition was determined according to the methods of Rayner (1985) and Rayner et al (1991). Carbohydrates were estimated by difference.

Functional and physical properties

All tests were conducted in duplicate. Seed size was expressed as the mass of 100 whole intact seeds (Williams et a1 1988). Hydration capacity of the whole

Fungal infection in chickpeas and faba beans 199

seeds was measured by soaking duplicate 10 g samples of seeds in deionised water at 25°C for a period of 24 h according to the methods described by Williams et a1 (1988) and Hung et a1 (1993). Measurements were taken at the following times: 15 min, 30 min, 45 min, 60 min, 2 h, 3 h, 4 h, 6 h, 12 h, 18 h and 24 h. Data are ex- pressed as the amount of water absorbed (g) per 10 g seeds.

Hard seed coatedness (number of seeds which did not swell during the hydration capacity test) was also deter- mined.

Water absorption of the flours was measured follow- ing the method of Sosulski (1962). Oil absorption capac- ity of the flours was determined by the methods of Sosulski and McCurdy (1987) and Lin et a1 (1974).

Whipping and foaming characteristics of a 3% (w/v) aqueous dispersion of the flours were assessed using the method of Mohanty et a1 (1988) and Sathe et a1 (1982). The foam and liquid volumes were recorded at 0, 30, 60 and 120 min. Foam expansion (FE) was calculated as the difference between the initial sample volume and the total volume after whipping. Foam liquid stability (FLS) was measured as the volume of liquid released from the foam at a given time and expressed as a pro- portion of the initial sample volume. Foam volume sta- bility (FVS) was calculated by measuring the foam volume at a given time and expressing it as a ratio of the foam volume immediately after whipping.

Emulsification capacity of aqueous dispersions of the samples (0.1%, w/v) was determined by the methods of Vuillemard et al(l990) and Webb et a1 (1970).

RESULTS AND DISCUSSION

Fungal and mycotoxic analysis

The average proportion of fungal infection in the ‘dis- eased’ chickpea fraction was 38 seeds per 100 seeds and 22 seeds per 100 seeds in the faba beans (Table 1). These figures are considered to be relatively low since the samples were manually ‘concentrated’ into ‘diseased’ fractions. This observation suggests that although many seeds were discoloured by dark lesions, it did not neces- sarily confirm the presence of fungal contamination. The fungal species isolated in the chickpeas were pri- marily Stemphylium botryosum (average infection = 14 seeds per 100 seeds) and Botrytis cinerea (average infection = 10 seeds per 100 seeds) (Table l), neither of which are known to be toxigenic (Hocking 1982; Blaney et al 1984). The potentially toxigenic species observed were Alternaria alternata (average infection = 4 seeds per 100 seeds) Fusarium acuminatum (average infection = 0.5 seeds per 100 seeds) and Aspergillus flauus (average infection = 6 seeds per 100 seeds) were detected in small amounts in relative to other species.

None of the mycotoxins tested were detected in either crop.

TABLE 1 Microbiological profile of the ‘diseased’ fractions of chickpeas and faba beans (contamination is expressed as the number of

infected seeds per 100 seeds on an ‘as is’ basis)

Seed Fungus Contamination

Chickpeas Stemphylium botryosum Botrytis cinerea Aspergillus flauus Alternaria alternata Eurotium amstelodami Eurotium chevalieri Pleospora herbarum Chaetemonium spp Chaetomium globosum Cladosporium spp Fusarium acuminatum Nigrospora oryzae Phoma spp Penicilhm brevicompactum Rhizopus oryzae Average total infection

Faba beans Coelomycete spp Eurotium amstelodami Alternaria alternata Aspergillus jlauus Phoma spp Rhizopus oryzae Penicillium spp Eurotium rubrum Fusarium semitectum Rhizopus stonifer Chaetomiurn spp Eurotium chevalieri Fusarium acuminatum Penicillium aurantiogrieseum Penicillium chrysogenum Pen ic ihm corylophilum Penicillium solitum Penicillium thomii Average total infection

14 10 5 4 2 2 2 1 0.5 0.5 0.5 0.5 0.5 0.3 0.3

38 14

1.3 1 1 1 1 0.8 0.5 0.5 0.5 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3

22

The microbiological profile of the chickpeas is similar to data published by Bretag and Mebalds (1987). They reported that ten samples of chickpea, also from Victo- ria’s Wimmera region, contained substantial levels of Cladosporium spp, Stemphylium spp, Botrytis cinerea, Alternaria alternata, Penicillium spp, Phoma spp and Fusarium spp as a proportion of the total number of colonies. Ahmad and Singh (1991) reported substantial levels of Alternaria spp, Cladosporium spp and Fusarium spp in chickpeas stored after harvest. These species were also observed in the present samples. They also report- ed that prolonged storage of seeds (up to 12 months) resulted in a shift of the fungal profile to one which is higher in certain aflatoxigenic species, particularly Aspergillus spp. The fact that the chickpea samples in this study were analysed at least 1 year post-harvest might partially account for the comparatively high

200 .I Sarantinos et a1

levels of Aspergillus Jlaous and Alternaria alternata con- tamination. The fungal spectrum of the ‘diseased’ faba beans differed from that of the chickpeas in the present study. The major contaminants belonged to the class of fungi known as Coelomycetes spp (infection rate = 14 seeds per 100 seeds), and most probably (identification could not be confirmed) included large numbers of Ascochyta pisi. Coelomycetes are exclusively plant pathogens and are not known to be harmful to humans (Table 1). A small number of Alternaria alternata and Aspergillus j a m s colonies were isolated from the faba beans, but the next most common fungal specimens were Penicillium spp.

Chemical composition

The chemical compositions of the chickpeas and faba beans are given in Table 2. The data generally show that fungal contamination of the seeds did not have any detrimental effects on the nutrient composition of the seeds. The mean moisture (0.91-0.99 g kg-’), ash

(0.24-0.28 g kg- ’) and crude fat (0.10-0.52 g kg- ’) contents of the chickpeas and faba beans displayed little variability and are consistent with published values (Sosulski and Youngs 1979; Geervani and Theophilus 1980; Chavan et al 1988; Hung et a1 1993). The mean crude protein contents of the ‘diseased’ chickpeas (2.18 g kg- ’) were approximately 5% higher compared with the levels found in the ‘healthy’ seeds (2.07 g kg-’). However, no difference was observed in the mean crude protein contents of the ‘healthy’ and ‘diseased’ faba beans (2.50-2.51 g kg-’). These observ- ations are mirrored in the differences in amino acid composition of both crops (Table 3).

Functional and physical properties

The hydration capacities of the whole chickpeas and faba beans are shown in Figs 1 and 2. The amount of water absorbed by the ‘healthy’ chickpeas rose exponen- tially before reaching a saturation point at 8 h (11.6 g and 12.2 g water per 10 g seed, for the ‘healthy’ and

TABLE 2 Chemical composition of chickpea and faba bean flours (data are given as mean f SEM (g kg-’ flour ‘as is’ basis))

Sample Moisture Ash Crude fa t Crude Acid detergent Carbohydrate protein fibre

Healthy chickpeas (n = 3) 0.91 f 0.01 0.24 f 0.01 0.39 f 0.01 2.07 f 0.08 0.57 & 0.01 5.23 _+ 0.03 Diseased chickpeas (n = 3) 0.90 f 0.03 0.27 f 0.02 0.52 & 0.01 2.18 f 0.05 0.53 & 0.01 5.23 5 0.05 Healthy faba beans ( n = 2) 0.89 f 0.03 0.27 & 0.01 0.10 f 0.01 2.50 5 0.11 1.11 & 0.01 4.30 f 0.05 Diseased faba beans (n = 2) 0.89 f 0.03 0.28 f 0.01 0.10 0.01 2.51 5 0.03 1.11 0.03 4.29 & 0-05

TABLE 3 Amino acid composition of chickpea and faba bean flours (data are given as means (g kg-’ protein

‘as is’ basis where protein = N x 6.25))

Amino acid Healthy Diseased Percent Healthy Diseased Percent chickpeas chickpeas change faba beans faba beans change

(n = 3 ) (n = 2) (n = 2) (n = 2)

Alanine Arginine Aspartic acid Cystine/cysteine Glutamic acid G 1 y c i n e His tidine Isoleucine Leucine Lysine Methionine Phen ylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

0.84 2.02 2.24 0.37 3.32 0.79 0.59 0.90 1.49 1.36 0.34 1.17 0.90 1.13 0.77 0.24 0.57 0.96

0.91 2.17 2.35 0.37 3.43 0.82 0.64 0.94 1.58 1 *42 0.35 1.23 0.88 1.20 0.83 0.22 0.62 1 a03

+ 8.3 + 7.4 + 4.9

0.0 + 3.3 + 3.8 + 8.5 + 4.4 + 6.0 + 4.4 + 2.9 + 5.1 - 2.2

+ 15.0 + 7.8 - 8.3 + 8.8 + 7.3

1.10 2.47 2.72 0.34 4.20 1.1 1 0.72 1.17 1.94 1.71 0.22 1.11 1.14 1-37 0.99 0.25 0.88 1.27

1.09 2.60 2.72 0.33 4.15 1.10 0.71 1.14 1.91 1.72 0.22 1.14 1.09 1.37 0.98 0.25 0.88 1 *29

- 0.9 + 5.3

0.0 - 2.9 - 1.2 -0.1 - 1.4 - 2.6 - 1.5 + 0.6

0.0 + 2.7 - 2.6 0.0

- 1.0 0.0 0.0

+ 1.6

Fungal infection in chickpeas and faba beans 20 1

0 ‘Healthy’ seeds ‘Diseased’ seeds

2 -

Pooled data for duplicate analyses of each fraction

0 ~ ~ 1 ~ 1 ’ 1 ’ 1 ~ 1 ’ 1 ’ 1 ~ 1 ’ 1 ’ 1 ’

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (hours)

Fig 1. Rate of water absorption in chickpeas at 25°C.

‘diseased’ fractions, respectively). Thereafter, no further 7% of the total seeds number in the chickpea and faba increases in the amount of water absorbed occurred. beans, respectively), and therefore had little overall The ‘diseased’ faba beans absorbed water at a greater impact on the amount of water absorbed by each rate compared with the ‘healthy’ fraction for the first sample as a whole. 8 h, although the amount of water absorbed was lower Fungal attack resulted in a small reduction in the size than that of the chickpeas after a similar period of chickpeas, 43.2 and 40.8 g per 100 seeds in the (appruxirriateiy o g water per i u g seeuj. riitnuugn itit:

curves began to plateau after 8 h, the saturation point had not been reached by 24 h. The initial hydration capacities indicate that fungal contamination had inter- rupted the integrity of the coat in the faba beans, allow- ing water to penetrate more easily.

Hard seed coatedness was higher in both ‘diseased’ chickpeas and faba beans (Table 4). This is expected since seeds that were in the advanced stages of disease were hard and shrivelled. However, these seeds only represented a small proportion of the sample size (4 and

neaitny anu uiseaseu iraciiuns, respectively \ 1 awe 4). This rzduction was not apparent in faba beans since both fractions had a seed size of approximately 48 g per 100 seeds.

The water, and oil absorption capacities of the flours and emulsification capacity of aqueous solutions of the flours did not reveal any differences between the ‘healthy’ and ‘diseased’ fractions of either crop (Table 4).

The foaming characteristics of the seed flours clearly show that the faba bean flours possess a greater FE than the chickpea flour (Table 5). Fungal contamination

14 I I

‘Healthy’ seeds ‘Diseased‘ seeds

Pooled data for duplicate analyses of each Fraction

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (hours) Fig 2. Rate of water absorption in faba beans at 25°C.

202 J Sarantinos et a1

TABLE 4 Functional properties of chickpeas and faba beans (data are given as mean f SEM)

Sample Seed size Hard seed coatedness Water absorption Oil absorption Emulsijication (mass of 100 (number of unswollen capacity capacity capacity (ml oil added

seeds) (9) seeds 100 per 100 (ml g- flour) (ml g- ’ flour) to 0.1% aqueous seeds) solution ofpour)

Healthy chickpeas (n = 3) 43.15 f 1.60 0.67 f 0.14 1.52 f 0.06 1.80 0.08 128.17 f 2.50 Diseased chickpeas ( n = 3) 40.81 f 0.32 4.15 f 1.18 1.40 f 0.01 1.85 f 0.06 128.75 f 2.56 Healthy faba beans (n = 3) 47.96 k 1.29 1.75 f 1.18 1.45 & 0.03 1.88 & 0.10 125.75 f 0.75 Diseased faba beans (n = 3) 48.13 f 2.18 7.00 f 2.38 1.53 f 0.01 1.75 f 0.03 125.00 f 2.55

considerably reduced the FE in both seed types; by 92% in the chickpea flours and 43% in the faba bean flours.

The FLS and FVS data indicate that neither of the flours possessed good foaming properties (Table 5) . The foams produced from the ‘healthy’ chickpea flours suf- fered a 97% collapse within 30 min.

Although the ‘healthy’ faba bean flours produced more foam after whipping (FE = 30%), it was extremely unstable and collapsed after only 30 min. The foams produced from the ‘diseased‘ faba bean flour yielded much less foam than the ‘healthy’ flour, but they appeared to be more stable.

In all cases, the foams produced from these flours col- lapsed within 2 h, indicating that both chickpea and faba bean flours have poor foaming properties regard- less of whether they were derived form ‘diseased’ seeds or not. Since the foaming properties of grain legumes are related to protein quality, other studies have exam- ined the foaming characteristics of protein isolates in addition to the flours of the seeds (Sosulski and Youngs 1979; Sosulski and McCurdy 1987; Genovese and Lajolo 1992).

To the best of our knowledge, this is the first study that has extensively examined the physicochemical changes that occur in chickpeas associated with fungal attack following heavy rainfall. However, two other reports investigated functional properties of proteins extracted from ‘diseased’ (also classified on a visual basis) soya beans (Genovese and Lajolo 1992, 1993). These studies reported that the gel-forming properties, apparent viscosity, water absorption and emulsification

capacity and stability of the acid precipitated proteins extracted from ‘diseased’ seeds were significantly differ- ent from those extracted from ‘healthy’ seeds.

During the present study, functional property tests were conducted on flour dispersions rather than protein isolates. This may partially explain the general lack of difference between the functionality of the ‘healthy’ and ‘diseased’ fractions of each crop observed in other studies, with the exception of foaming properties. Alter- natively, the present data may simply reflect differences in the effects of fungal infection on other grain legumes.

CONCLUSIONS

This study has shown that the fungal contamination of chickpeas and faba beans harvested during the 1992- 1993 season is unlikely to be associated with any adverse effects in humans if ingested. Analysis of nutri- ent composition and functional properties generally revealed minor differences between the ‘diseased and ‘healthy’ seeds. This suggests that the diseased seeds may potentially have certain food applications after various treatments to reduce or remove fungal contami- nation have been implemented. The use of such seeds and their flours in preparing certain food products will be further examined.

Preliminary work has already yielded promising observations. Flours produced from seeds of each type were used to produce a deep-fried low water activity snack food which was well accepted in a taste panel. Moreover, panellists could not distinguish between the products prepared from ‘healthy’ or ‘diseased’ seeds.

TABLE 5 Foaming characteristics of chickpeas and faba beans (data are given as mean k SEM)

Sample Foam expansion Foam liquid stability (%) Foam volume stability (%)

30 min 60 min 120 min 30 min 60 min 120 min

Healthy chickpeas ( n = 3) 12.0 f 2.6 30.0 & 19.0 30.0 k 19.0 Nil“ 3.3 k 2.1 3.3 k 2.1 Nil Diseased chickpeas (n = 3) 1.0 & 0.5 Nil Nil Nil 88.5 f 0.3 81.5 & 1.4 Nil Healthy faba beans ( n = 2) 30.0 f 2.3 88.0 k 0.0 89.0 & 0.3 Nil Nil Nil Nil Diseased faba beans (n = 2) 17.0 k 4.0 88.0 & 0.0 88.0 k 0.3 nil 85.0 k 0.0 85.0 f 0.0 Nil

Nil denotes lack of foam formation or foam collapse.

Fungal infection in chickpeas and faba beans 203

Dips and canned products were also prepared and, once again, panellists could not distinguish between ‘healthy’ and ‘diseased’ seeds on a number of organoleptic parameters. Despite the successful incorporation of ‘dis- eased’ chickpeas and faba beans into food products intended for human consumption, the issue of the long- term microbiological safety of these foods during storage remains to be addressed.

ACKNOWLEDGEMENTS

This work was supported by a grant from the Grain Research and Development Corporation of Australia (Project Code DAV 228M). The authors gratefully acknowledge the technical assistance of Ms Gail Di Gregorio, Ms Lorraine Ruddick and Ms Daphne Womersley from the Grains Group, Australian Food Research Institute, Agriculture Victoria, Werribee, Vic- toria, Australia. Thanks also to Dr Ailsa Hocking (Food Mycologist at CSIRO-Division of Food Science and Technology, North Ryde, New South Wales, Australia) and her technical staff for performing the microbiological analyses and Dr Ron Wills (Academy of Grain Technology, Werribee, Victoria, Australia) and his staff for performing the mycotoxin determinations. The authors also thank Mr Carl Rayner (State Chem- istry Laboratory, Agriculture Victoria, Australia) and his laboratory staff for performing the amino acid analyses.

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