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Effect of fermentation on the starch digestibility, resistant starch and some physicochemical properties of sorghum flour Abd Elmoneim O. Elkhalifa, Burkhard Schiffler and Rita Bernhard The effect of natural fermentation of Tabat sorghum cultivar (Sorghum bicolor L. Moench) at 37 8C for up to 36 h on pH, titratable acidity, starch digestibility, resistant starch and total starch was studied. The pH of the fermenting dough decreased sharply with a concomitant increase in the titratable acidity. In vitro starch digestibility markedly

increased as a result of fermentation, while resistant starch and total starch decreased. Results showed that iodine absorption capacity increased during fermentation. Fermented sorghum had more soluble starch and swelling power at 100 8C than at 85 8C.

1 Introduction

the changes in some chemical and biochemical properties which occur as a result of the Sudanese traditional fermentation of sorghum.

Fermented foods contribute to about one-third of the diet world-wide [1]. Cereals are particularly important substrates for fermented foods in all parts of the world and are staples in Africa and Asia where the lack of resources limits the use of energy and capital intensive processes for food production and preservation. Sorghum (Sorghum bicolor L. Moench) is a major cereal crop in the semiarid tropics of Africa and Asia. It is the main staple food for the world’s poorest people. It is the most important cereal crop in the Sudan where it is consumed in fermented forms, mainly as kisra (local thin bread), aceda (thick porridge) and nasha (thin porridge). The nutritional characteristics of fermented sorghum has been examined by many workers [2–4]. Kazans and Fields [5] reported that natural fermentation of sorghum produced significant increase in available lysine, leucine, isoleucine and methionine. Elkhalifa and El Tinay [6] found appreciable changes in sorghum protein fractions during fermentation. Moreover, Hassan and El Tinay [7] reported that fermented sorghum flour had higher in vitro protein and starch digestibilities (IVPD; IVSD) than sorghum flour. Recently, Elkhalifa and El Tinay [8] found an increase in the IVPD and IVSD of the fermented sorghum pretreated with papain or cysteine. The physicochemical properties of sorghum flour affects the textural characteristics of the food preparations made from it. The effect of fermentation on the physicochemical properties of sorghum flour has not been studied until now. As pointed out by Wood [9], there is a possible backlash if consumers in developing countries abandon traditional fermented foods for smart, sophisticated products popularised in Europe and America. This could have an impact on the nutritional status of consumers in these countries. Therefore, basic research of indigenous cereal fermentation will lead to technology transfer. The present investigation was aimed to study

Correspondence: Dr. A. O. Elkhalifa, Universitt des Saarlandes, FR 8.8 Biochemie, D-66123 Saarbrcken, Germany E-mail: aoelkhalifa@hotmail.com Fax: +49-6813024739 Abbreviations: IAC, iodine absorption capacity; IVPD, in vitro protein digestibility; IVSD, in vitro starch digestibility; RS, resistant starch; TA, titratable acidity; TS, total starch Keywords: Fermentation / Physicochemical properties / Resistant starch / Sorghum / Starch digestibility / i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2 Materials and methods 2.1 Material A low-tannin sorghum cultivar (Tabat), obtained from the Food Research Centre, Shambat, Sudan, was used in this study. The seeds were carefully cleaned and ground in a hammer mill to pass through a 0.4 mm screen, and the flour was stored in polyethylene bags at 4 8C. 2.2 Fermentation of sorghum Sorghum flour was fermented in the Sudan according to the traditional method as practised by the Sudanese housewife and as described by El Tinay et al. [10]. Fermentation was carried out by mixing sorghum flour (uncooked, 1 kg) with 2 L of water in a round earthenware container. Previously fermented dried flour dough (300 g) was then added to the mixture of flour and water to act as starter. After thorough mixing, samples were taken at 4 h intervals until the end of fermentation, which was terminated after 36 h at 37 8C. After a distinct incubation period, the samples were dried in a hot air oven (Heraeus UT 5042, Germany) at 60 8C for 16 h. Dried samples were ground to pass a 0.4 mm screen and stored in polyethylene bags at 4 8C prior to be transferred to Germany for analysis. 2.3 pH determination and titratable acidity The pH of the fermenting dough was monitored directly in the dough and every 4 h for the 36 h by using a glass electrode pH meter (PUSL Mnchen, Germany). Titratable acidity, expressed as lactic acid, was determined with 0.1 M NaOH [11]. 2.4 Starch digestibility In vitro starch digestibility was determined according to the method of Dahlqvist [12]. Sorghum flour samples (100 mg), suspended in 4 mL of 0.1 M phosphate buffer, pH 7, were gelatinised, cooled to 40 8C and incubated with 5 mg a-amylase (Bacillus species, EC 3.2.1.1, 538 U/mg; Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The enzyme was inactivated by heating in a boiling water bath for 10 min. The mixture was centrifuged (5000 6 g, 10 min) and the residue was washed

DOI: 10.1002/food.200300322

Nahrung/Food 48 (2004) No. 2, pp. 91 – 94

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with water. The supernatant was made up to 15 mL with all washings and analysed for released reducing sugars by the dinitrosalicylic acid (DNS) method [13] with slight modification. An aliquot of 0.2 mL of the above supernatant was added to 0.8 mL water and 3 mL of DNS reagent were added and the contents were incubated in a boiling water bath for 20 min then after cooling the solution was diluted two times by adding 8 mL of water and the optical density was taken at 550 nm using a double beam spectrophotometer (UV2100 Shimadzu, Kyoto, Japan). Standard curve was prepared using D-glucose and the released reducing sugars were calculated from the standard curve as mg glucose 6 0.9.

620 nm as follows. A weighed amount (100 mg) of each sorghum sample was transferred into a 100 mL volumetric flask and combined with 1 mL ethanol (95%) and 9 mL NaOH (0.1 N). The mixture was heated on a boiling water bath for 10 min, cooled and brought up to 100 mL with distilled water. Five millilitres of the last mixture were pipetted into a 100 mL volumetric flask, combined with 1 ml acetic acid (0.1 N) and 2 mL iodine solution (0.2 g I2 and 2 g KI in 100 mL aqueous solution), made up to volume with distilled water, shaken and then allowed to stand for 20 min. Absorbance of final solution was measured at 620 nm using distilled water as blank and was taken as an index for iodine affinity.

2.5 Resistant starch

2.8 Water retention and starch solubility

In vitro resistant starch (RS) was estimated according to the method of Champ et al. [14]. Its principle is that RS is defined as that starch which is not hydrolysed by incubation with a-amylase. Amyloglucosidase (EC 3.2.1.3, 22570 U/g; SigmaAldrich) is added to avoid inhibition by by-products of amylase digestion. Hydrolysis products are extracted with 80% ethanol and discarded. The RS is then solubilised with 2 N KOH and hydrolysed with amyloglucosidase. The released glucose was determined by the DNS method (as above).

Water retention and starch solubility were determined according to the method reported by Zhang and Hamaker [16]. Water was added to flour slurries and incubated at 85 8C and 100 8C for 30 min with shaking every 5 min. After the samples were cooled at room temperature for 15 min, they were centrifuged at 4900 6 g for 10 min. Supernatants were collected to measure total soluble starch, and the precipitate was used to determine the water retention. Total soluble starch was precipitated by adding 80% ethanol and dried at 40 8C for two days. The water retention was the ratio of sample weight after incubation to original sample weight.

2.6 Total starch Sorghum flour (1 g) was added to distilled water (50 mL) and kept in a boiling water bath for 30 min for gelatinisation. Then 200 lL Termamyl were added and kept for 60 min in a boiling water bath and then cooled to room temperature. Acetate buffer was added so as to make the concentration to 0.05 M and 100 mg of glucoamylase (Rhizopus, EC 3.2.1.3, 12 000 units/g of solid, Sigma Chemical, St. Louis, MO, USA) were added and incubated at 60 8C for 2 h. The sample was brought back to room temperature and filtered. The filtrate after suitable dilution, was mixed with 2 mL of glucose oxidase/peroxidase reagent (Sigma-Aldrich) and incubated in a shaker water bath for 30 min at 37 8C. Absorbance of the resultant solution was read at 540 nm against reagent blank. Starch was calculated from glucose standard curve as mg glucose 6 0.9. 2.7 Iodine affinity (iodine absorption capacity) The iodine affinity of sorghum flour samples was determined according to the procedure of Juliano [15] by the reaction with iodine solution (0.2 g I2 and 2 g KI in 100 mL aqueous solution) and measuring the resulting absorbance at

Table 1.

2.9 Statistical analysis Each sample was analysed in duplicate and the figures were then averaged. Data was assessed by analysis of variance (ANOVA) [17] and by Duncan’s multiple range test with a probability of p F 0.05 [18].

3 Results and discussion 3.1 Changes in pH, titratable acidity, starch digestibility, resistant starch and total starch The changes in pH, titratable acidity, starch digestibility, resistant starch and total starch are shown in Table 1. The pH of the fermented dough dropped from 5.9 to 3.7 during the first 20 h and then it remained unchanged up to 28 h. Concomitant with the drop in pH there was a rise in titratable acidity (TA) of the fermentation medium throughout the process. The TA increased from 10.15 to 116.25 mg/100 g lactic acid. This finding was in agreement with the work of El Tinay et al. [10],

Effect of fermentation of sorghum on pH, TA, IVSD, RS, TS and IAC

Fermentation time (h)

pH

TA (mg/100 g)

IVSD (%)

RS (%)

TS (%)

IAC (OD at 620 nm)

0 4 8 12 16 20 24 28 36

5.90 l 0.00a 5.55 l 0.07b 4.30 l 0.00c 3.95 l 0.07d 3.80 l 0.00e 3.70 l 0.00f 3.70 l 0.00f 3.70 l 0.00f 3.60 l 0.00g

10.15 l 3.04h 14.25 l 0.07hg 19.20 l 1.13g 28.30 l 1.70f 39.85 l 4.45 e 42.90 l 0.57e 55.70 l 0.85d 94.35 l 1.06b 116.25 l 5.30a

34.55 l 0.38f 35.09 l 0.38f 39.41 l 0.38e 41.03 l 0.38e 45.08 l 0.76d 48.05 l 2.67c 53.18 l 2.29b 42.11 l 0.38e 36.36 l 0.76f

42.30 l 0.10a 33.57 l 1.24b 26.82 l 0.81c 26.03 l 0.56cd 22.18 l 2.31 e 17.06 l 2.10f 11.47 l 2.15g 23.30 l 0.03de 28.27 l 1.41c

74.45 l 0.07a 65.38 l 1.38b 65.20 l 0.67b 64.13 l 0.08c 63.85 l 0.43c 62.21 l 1.07cd 62.17 l 0.38d 63.06 l 0.76 c 61.93 l 0.21d

0.325 l 0.03d 0.340 l 0.02d 0.383 l 0.04c 0.420 l 0.01abc 0.427 l 0.01 ab 0.432 l 0.00a 0.437 l 0.00 a 0.444 l 0.01a 0.388 l 0.00bc

Values are means of two replicates l SD. Values with the same superscript letter in a column are not significantly different (p f 0.05). 92


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Effect of fermentation on properties of sorghum flour

Mohammed et al. [19] and Yousif and El Tinay [20]. According to El Hidai [21], natural sorghum fermentation occurs by the action of lactic acid produced by Lactobacillus spp. Yeast and acetic acid fermentation occurs to a lesser extent during the later stages of fermentation. This could explain the apparent increase in lactic acid towards the end of fermentation period accompanied by lack of changes in pH. The effect of fermentation on the IVSD is shown in Table 1; unfermented sorghum flour had the lowest IVSD (34.55%); this may be due to the restriction in accessibility of starch caused by endosperm proteins [22, 23]. As endosperm proteins may restrict the starch granules from fully gelating, thereby resulting in lower digestibility [24]. Fermentation of sorghum flour led to an increase in the IVSD from 34.55 to 56.69% after 28 h. This increase may be due to the fact that fermentation led to changes in the endosperm protein fractions [6, 20] and this makes starch more accessible to the digestive enzymes. Hassan and El Tinay [7] also found an increase in IVSD from 32.3 to 45.2% during fermentation of sorghum cultivar Dabar. Natural fermentation of sorghum had a significant effect on the reduction of resistant starch (Table 1). RS decreased significantly (p f 0.05) during the fermentation period and 77% of the RS was eliminated after 28 h of fermentation. The increase in RS after 28 h of fermentation may be due to changes in the chemical nature of the starch in some way by the effect of high concentration of lactic acid after this time of fermentation and corresponding decreasing the enzyme action and thus increase of RS. Bach Knudsen et al. [25] also reported a significant decrease in RS of sorghum cultivar Dabar dough with a pH 3.9. Total starch (TS) of the fermented sorghum flour was significantly (p f 0.05) decreased throughout the fermentation process (Table 1). Starch tends to decrease during fermentation of sorghum [2]. 3.2 Physicochemical properties Amylose has a strong affinity for iodine with which it forms a deep blue complex, whereas amylopectin has little affinity to iodine (iodine absorption capacity) giving a purple-reddish coloration [26]. It is shown in Table 1 that fermentation increased the iodine absorption capacity of sorghum flour by 35% after 32 h. This indicates an increase in accessibility of amylose, as iodine binding takes only place if iodine has access to the amylose. Numfor et al. [27] found an apparent increase in amylose content of fermented cassava starch compared to native samples. Sorghum had lower swelling power (water retention) at 85 8C than at 100 8C (Table 2); and fermentation had not much effect on sorghum swelling power at 85 8C but at 100 8C the swelling power of sorghum flour increased as the fermentation time increased to reach its maximum value after 28 h. This indicates that sorghum requires higher temperatures to reach full granule swelling. The amount of soluble starch increased due to the effect of fermentation at 85 8C compared to the control but it decreased at 100 8C during the first 28 h of fermentation and increased thereafter. Generally, more starch is soluble at 100 8C than at 85 8C (Table 2).

4 Concluding remarks The traditional Sudanese fermentation method of sorghum has important nutritional advantages as it helped to overcome the formation of resistant starch and therefore increased the digestibility of the starch. Additionally, it affects the biochemi 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Table 2. Effect of fermentation of sorghum on water retention and starch solubility Fermentation time (h) 0 4 8 12 16 20 24 28 32 36

Temperature ( 8C)

Water retention (g/g)

Starch solubilitya)

85 100 85 100 85 100 85 100 85 100 85 100 85 100 85 100 85 100 85 100

7.19 l 0.04 8.81 l 0.02 7.80 l 0.13 12.11 l 0.07 7.95 l 0.16 12.51 l 0.06 7.91 l 0.11 12.56 l 0.21 7.85 l 0.35 12.85 l 0.74 7.75 l 0.04 12.87 l 0.02 7.63 l 0.06 12.90 l 0.07 7.56 l 0.45 13.30 l 0.04 7.68 l 0.18 12.79 l 0.29 7.68 l 0.04 11.75 l 0.37

13.30 l 0.07 36.25 l 0.49 13.90 l 0.14 34.45 l 2.33 14.40 l 0.00 31.90 l 0.14 14.61 l 0.41 31.05 l 2.90 14.83 l 0.04 32.50 l 0.71 16.10 l 0.14 33.25 l 0.35 13.95 l 0.07 33.45 l 0.07 14.70 l 0.14 33.60 l 0.84 14.30 l 0.07 38.25 l 1.06 12.23 l 0.04 42.00 l 1.41

Values are means of two replicates l SD. a) Measured as mg/200 mg sorghum flour

ical and physicochemical properties of sorghum flour by increasing the iodine affinity and starch solubility. These results can give an idea about the possibility of using fermented sorghum flour or starch isolated from it in food industry. In addition, results indicate that there is no need for long fermentation time (36 h) as most of the important changes occur before that time of fermentation, the optimum fermentation time being 28 h. This work was supported by Alexander von Humboldt Foundation through a Fellowship to A. O. Elkhalifa.

5 References [1] Campbell-Platt, G., Food Res. Int. 1994, 27, 253–261. [2] El Tinay, A. H., Abdel Gadir, A. M., El Hidai, M., J. Sci. Food Agric. 1979, 30, 859–865. [3] Axtel, J. D., Kirlies, A.W., Hassen, M. M., D’Cooz Manson, N., Mertz, E. T., Munck, L., Proc. Natl. Acad. Sci. USA 1981, 78, 1333–1335. [4] Eggum, B. O., Monowar, L., Bach Knudsen, K. E., Munck. L., Axtell, J., J. Cereal Sci. USA 1983, 1, 127–137. [5] Kanzans, N., Fields, M. L., J. Food Sci. 1981, 46, 819–821. [6] Elkhalifa, A. O., El Tinay, A. H., Food Chem. 1994, 49, 265– 269. [7] Hassan, I. A. G., El Tinay, A. H., Food Chem. 1995, 53, 149– 151. [8] Elkhalifa, A. O., El Tinay, A. H., Univ. Khartoum J. Agric. Sci. 2002, 10, 136–142. [9] Wood, B. J. B., Food Res. Int. 1994, 27, 269–272. [10] El Tinay, A. H., El Mehdi, Z. M., El Soubki, A., J. Agric. Food Chem. 1985, 21, 679–687. [11] Zamora, A. F., Fields, M. L., J. Food Sci. 1979, 44, 234. [12] Dahlqvist, A., Anal. Biochem. 1964, 7, 18–25. [13] Bernfeld, P., in: Colowich, S. P., Kaplain, N. O. (Eds.), Methods in Enzymology, Academic Press, New York 1955, 1, pp. 149– 158. Nahrung/Food 48 (2004) No. 2, pp. 91 – 94

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Elkhalifa et al. [14] Champ, M., Noah, L., Loizeau, G., Kozlowski, F., in: Cho, S., Prosky, L., Dreher, M. (Eds.), Complex Carbohydrates in Foods: Definition, Functionality and Analysis, Marcel Dekker, New York 1994. [15] Juliano, B. O., Cereal Sci. Today 1971, 16, 334–339. [16] Zhang, G., Hamaker, B. R., Cereal Chem. 1998, 75, 710–713. [17] Snedecor, G. W., Cochran, W. G., Statistical Methods, The Iowa State University Press, Ames, IA 1987, pp. 221–222. [18] Duncan, B. D., Biometrics 1955, 11, 1–42. [19] Mohammed, S. I., Steenson, L. R., Kirleis, A. W., Appl. Env. Microbiol. 1991, 57, 2529–2533. [20] Yousif, N. E., El Tinay, A. H., Plant Foods Hum. Nutr. 2001, 56, 175–182. [21] El Hidai, M. M., MSc Thesis, Faculty of Agriculture, University of Khartoum, Sudan 1978. [22] Rooney, L. W., Pflugfelder, R. L., J. Anim. Sci. 1986, 63, 1607– 1623.

[23] Waniska, R. A., Kotarski, S., Thurn, K., in: Ejeta, G. (Ed.), Proceedings of the International Conference on Sorghum Nutritional Quality, Purdue University, West Lafayette, IN 1990, pp. 177– 190. [24] Chandrashekar, A., Kirleis, A. W., Cereal Chem. 1988, 65, 457– 462. [25] Bach Knudsen, K. E., Munck, L., Eggum, B. O., Br. J. Nutr. 1988, 59, 31–47. [26] Radley, J. A., The Microscopic Structure of Starch: Examination and Analysis of Starch and Starch Products, Applied Science Publishers, London 1976. [27] Numfor, F. A., Walter, W. M. Jr., Schwartz, S. J., Starch/Strke 1995, 47, 86–89. Received April 25, 2003 Accepted November 3, 2003

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