J Sci Food Agric 1995,69,367-371

Biochemical Changes in Cassava Tuber during Fermentation and its Effect on Extracted Starch and Residue Mathew George,* S N Moorthy and G Padmaja Division of Post-Harvest Technology, Central Tuber Crops Research Institute, Trivandrum-695 017, India

(Received 23 November 1994; revised version received 29 March 1995; accepted 7 June 1995)

Abstract: The biochemical changes brought about in cassava tubers during fer- mentation (72 h) with a mixed culture inoculum are delineated. The initial starch content of the four cultivars studied ranged from 216 to 331.3 g kg- * and only slight decrease in starch occurred after 72 h fermentation, the final values being 200-302.2 g kg-I. There was a considerable decrease in the sugar content with fermentation in all the cultivars, indicating a predominant utilisation of the easily assimilable sugars by the microorganisms. A sharp decrease in pH (4.3-4.7) was observed after 24 h fermentation itself, resulting from the production of organic acids. There was increased recovery of the starchy flour from the fermented cassava tubers with concomitant decrease in the residue yield. The starchy flour had higher fiber content than that obtained from the non-fermented tubers. Optical and scanning electron microscopy revealed clustering of starch granules in the starchy flour from fermented tubers.

Key words: cassava, mixed culture, starch, residue, SEM studies.

iNTRODUCTION

Cassava (Manihot esculenta Crantz) is widely cultivated along the tropical belt from its starchy tubers, which are used as food, feed or as an industrial raw material. Although most cultivars contain 200-350 g kg-' starch, the recovery of starch extracted through conven- tional rasping and sedimentation processes is not com- plete (Balagopalan er a1 1988). As a result, part of the starch goes unrecovered along with fibrous residue. With a view to enhancing the recovery of starch from cassava tubers, the efficacy of a mixed culture inoculum source was studied.

Fermentation has been practised traditionally in many African and Latin American countries as a means of improving the textural qualities of the flour and imparting flavour to the product (Oyenuga 1968; Oyewole and Odunfa 1989). It has also been reported as one of the best ways of eliminating cyanogens from cassava (Ayernor 1985; Mahungu et a1 1987; Bokanga er a1 1988). Studies conducted earlier using the mixed culture inoculum in this laboratory have shown that it

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could enhance the starch extractability from tubers (Mathew George et a1 1991). It was also reported that this property of the inoculum source was due to its ability to elaborate pectinolytic and cellulolytic enzymes which helped to solubilise the membranes and release the trapped starch granules (Mathew George et a1 1991). The properties of the starchy flour extracted from fermented tubers have been studied in detail. It was observed that the gelatinisation temperature was enhanced while the paste viscosity was reduced by the fermentation. The changes have been attributed to the presence of fibrous material rather than any major change in the granular structure of starch (Moorthy et a1 1993). In addition, fermentation also imparted higher elasticity to the granules, rendering it suitable for baked products (Mathew George et a1 1993). The mode of cyanogen detoxification brought about by the mixed culture inoculum source was also studied and reported earlier (Padmaja et a1 1993). A comparative nutritional evaluation of the fermented and plain sun-dried cassava flour indicated that fermentation led to decreases in the crude protein and ash content as well as decrease in all the amino acids (Padmaja et a1 1994). In this paper, we report the gross biochemical changes taking place in the

367 J Sci Food Agric 0022-5142/95/$09.00 0 1995 SCI. Printed in Great Britain

M George et a1

tubers as a consequence of fermentation and correlate the increased recovery of starchy flour with the increased content of fiber as well as indicate the micro- scopic alterations in the starch granules.

MATERIALS AND METHODS

Four cassava cultivars of uniform maturity having varying starch contents, were used in the study. Studies conducted earlier by Mathew George et al (1991) showed that the foul smell during fermentation could be eliminated by providing an inoculum to facilitate lactic fermentation. Using curd and coconut toddy as rich sources for lactic acid bacteria and yeasts, respectively, an initial inoculum was prepared. The inoculum was enriched by successive fermentation of cassava using steep liquor of an earlier batch as the inoculum. Thus, a mixed culture inoculum comprising Lactobacillus sp, Streptococcus sp, Corynebacterium sp and yeasts was formulated. The mixed culture inoculum is stable and self-sustaining. The pH of the steep liquor was found to be lowered within 24 h of fermentation, which helped to prevent extraneous microbial contamination. Peeled tubers were cut into prismoid pieces of 3 x 2 cm base and were dispensed in double the quantity of water (w/v). The beaker was covered with a muslin cloth. One millilitre of mother liquor containing the inoculum source (Mathew George et al 1991) was added per 100 g tuber and allowed to ferment for 72 h at ambient tem- perature (30 k l°C). Three replications were maintained for each cultivar. Samples were drawn at 24, 48 and 72 h of fermentation. Initial (0 h) as well as fermented tuber samples were analysed for starch, sugar and dry matter (DM) contents. DM was determined by the

oven-drying method ; starch and sugar were quantified by the procedure of Moorthy et al (submitted) pH of the steep liquor at each interval was determined by an Elico pH meter using a combined electrode. Starchy flour was extracted from the initial as well as the fermented samples by disintegration in a blender, sieving through an 80 mesh sieve and then settling it overnight. The settled starchy residue was recovered through centrifu- gation, dried to constant weight in an oven and weighed. Crude fibre content of the starchy flour was determined by the standard procedure (AOAC 1975). The starch granules were examined using an Olympus Compound Microscope at 400 x . For scanning electron microscopy (SEM), the starch granules were coated with gold and viewed in a Joel scanning electron microscope under 2000 x and 4800 x magnification.

RESULTS AND DISCUSSION

The starch, sugar and DM changes occurring with fer- mentation are presented in Table 1. It was found that there was only a slight decrease from the initial starch content in the fermented tubers in all the varieties. On the contrary, the decrease in sugar with fermentation was quite noticeable. Cassava roots contain high amounts (ranging from 30 to 100 g kg-' in various varieties) of water-soluble carbohydrates (Padmaja and Balagopalan 1991). These are suficient to provide an initial easily assimilable energy source for the micro- organisms for their proliferation. As a result, there is not much necessity to degrade the starch to sugars and use them. However, when a sufficient depletion in sugar content occurs, the organisms may begin to utilise the starch which may account for the decreases occurring towards the latter stages of fermentation (Table 1). The

TABLE 1 Biochemical changes in cassava tuber through fermentation

Variety of tuber Duration of Starch Sugar Dry weight PH of fermentation (h) ( g k g - FW) ( g k g - FW) (g kg- FW) steep liquor

M4 0 331.3 12.1 396.0 5.83 24 322.1 5.9 388.0 4.70 48 307.0 5.5 366.0 4.70 72 302.2 tr 364.0 4.30

H-1687 0 2 17.5 18.0 332.4 6.02 24 212.0 12.9 31 1.0 4.60 48 2 17.8 11.0 311.0 4.60 72 206.5 3.7 309.2 4.30

T-300 0 2 16.0 8.6 300.0 5.67 24 215.0 5.8 280.0 4.30 48 200.0 5.6 280.0 4.20 72 200.0 5.0 280.0 4.10

CI-468 0 216.1 24.0 309.0 6.05 24 21 1.4 16.7 300.0 4.55 48 21 1.5 9.6 300.0 4.80 12 204.5 4.0 300.0 4.10

Changes in cassava by fermentation and effects on starch and residue 369

decrease in starch observed was in accordance with the decrease in the DM content of tubers (Table 1).

The initial pH values of steep liquor for the various cultivars ranged from 5.67 to 6.05. After fermentation for 24 h, the values indicated a steep fall (4.3-4.7). pH ranged from 4.1 to 4.3 in steep liquor of the 72 h fer- mented tubers indicating an appreciable enrichment of organic acids in the tuber due to fermentation. We have reported earlier that there was a 26% increase in the ether extractive fraction in fermented flour which had resulted from the organic acids (mainly lactic acid) formed during fermentation (Padmaja et a1 1994). Akin- rele (1964) also found that organic acids were enhanced in fermented cassava. Organic acid production during cassava retting has been reported by Ampe et a1 (1994).

The recovery of starchy flour and residue from non- fermented and fermented tubers (at various sampling frequencies) was compared for the various cultivars (Table 2). The initial yield of starchy flour was 492- 554 g kg- DM for the four cultivars. Fermentation increased the yield of starchy flour from all the cultivars and there was a concomitant decrease in the total residue recovery (Table 2). This indicates that the enhanced recovery of starchy flour was certainly resulted from the particle breakdown of the fibrous residue which enables the fibre also to pass along with the starch through the 80 mesh sieve. This is further substantiated by the data on the absolute starch content in the starchy flour (0 h versus fermented) as well as the crude fibre content of the starchy flour (Table 3). The comparison to the starchy flour obtained from the non- fermented tubers (0 h), that recovered from the fer- mented tubers (24-72 h) evidently had low true starch content. This has resulted from the contamination of

TABLE 2 Starchy flour recovery on fermentation of cassava tuber

Variety Duration of Recovered starchy Residue fermentation flour (9 kg- ' DM)

(h) (9 kg- ' DM)

M4 0 492 29 1 24 606 88 48 655 46 72 669 46

H-1687 0 515 329 24 703 202 48 803 32 72 812 32

T-300 0 495 388 24 553 303 48 806 125 72 813 77

CI-468 0 544 343 24 654 23 1 48 710 88 72 705 85

TABLE 3 Starch and fibre content in recovered starchy flour

Variety Duration of Starch Fibre fermentation (g kg-' DM) (g kg-' DM)

(h) ~~~

M4 0 24 48 72

H-1687 0 24 48 72

T-300 0 24 48 72

CI-468 0 24 48 72

819 756 746 722 861 815 790 765 842 823 755 750 870 866 852 825

10 59 86 104 16 82 89 93 22 56 105 116 26 47 54 64

the starch which fibre due to its increased passage through sieve. It was also found that the mixed culture inoculum source elaborated pectinolytic and cellulolytic enzymes into the steep liquor which facilitate lysis of the cell membranes (Mathew George e t al 1991). The insol- uble fibre is thus disintegrated during this lytic process, and automatically passes along with the starch.

The starch and fibre contents in the extracted starchy flour and residue are presented in Tables 3 and 4. The increase in yield of starchy flour was invariably accom- panied by an increase in its fibre content. The crude fibre content in the starchy flour from non-fermented

TABLE 4 Starch and fibre content in residue

Variety Duration o/ Starch Fibre fermentation (g kg-' DM) (g kg-' D M )

(h)

M4 0 706 98 24 619 119 48 50 1 142 72 497 166

H-1687 0 66 1 102 24 582 124 48 416 229 72 35 1 249

T-300 0 684 108 24 625 117 48 610 234 72 577 296 \

C1-468 0 666 115 24 578 141 48 552 143 72 552 162

370 M George et a1

tubers was only 10-6-26 g kg- l . However, it increased tremendously in the starchy flour (64.8-1 16.4 g kg-') from fermented tubers. The lysis of cell membranes also facilitated release of the trapped starch granules and their consequent extraction during the process. Though the starch content in the residue even after 72 h of fer- mentation remained at 351-577 g kg- ' (Table 4) on a dry weight basis, the significant reduction in the quan- tity of residue from fermented samples led to much less starch loss compared to that from unfermented samples. The difficulty experienced in passing the slurry obtained by mashing the fermented tuber through fine mesh sieves indicated that non-starchy components accom- pany the starch during extraction. In addition, the starchy flour recovered from the carotene-rich variety H-1687, with a yellow flesh colour, retained the yellow colour, in conformity with the above observation. Hence, the starchy flour obtained from non-fermented and fermented tubers was examined microscopically. While the granules of starch from non-fermented tubers were uniformly dispersed individually in the micro- scopic field, half of the granules from fermented tubers were found to be clumped together as agglutinated granules of different shapes and sizes, often adhering to hyaline tubular materials. This result was confirmed in the SEM photographs which also showed that many of the granules were adhering to each other (Fig 1) prob- ably due to the electrostatic attraction between the granules which have undergone loss of superficial layers during the fermentation. Camargo et a! (1988) on study- ing the starch modification in cassava fermentation found that the granular structure was similar to that resulting from treatment wild mild acid for a short period and that the chemical composition was not sig- nificantly affected. Martinez and Quiroga (1988) report- ed that as the fermentation time elapsed, some of the starch granules became smaller due to loss of the super- ficial layers while others remained intact. Our results also indicate that fermentation brings about only minor changes in the starch morphology mainly on the surface.

CONCLUSIONS

The fermentation by a mixed culture brings about a rapid decrease in sugar content, the culture using up the sugars. The starch content and the starch structure are only slightly affected. The extracted starch is accompa- nied by fibrous material produced by cell-wall-degrad- ing enzymes.

ACKNOWLEDGEMENTS

The authors wish to express their gratitude to the Director, CTCRI for the facilities provided and also to Dr c Balagopalan, Head, Division of Post Harvest Technology, CTCRl for encouragement. The authors

~i~ 1. SEM ,,,icrographs of starch. (a) Raw tuber ( 2 0 0 0 ~ ) ; (b) fermented tuber (2oOOx); (c) fermented tuber

(4800 x ).

Changes in cassava by fermentation and effects on starch and residue 371

also thank Director, Regional Research Laboratory, Trivandrum for extending facilities for the SEM studies.

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