THE EFFECT OF FERMENTATION ON CASSAVA STARCH MICROSTRUCTURE

M.J.A. MARCON, M.A. VIEIRA, K. SANTOS, K.N. DE SIMAS, R. DIAS DE MELLO CASTANHO AMBONI and E.R. AMANTE1 Department of Food Science and Technology Santa Catarina Federal University Rodovia Admar Gonzaga, 1346 Itacorubi CEP 88034-001, Florianpolis, SC, Brazil
Accepted for Publication March 9, 2006

ABSTRACT The effect of glucose syrup addition on sour cassava starch fermentation was studied on cassava starch from three locations in Santa Catarina State (Brazil), following four treatments: traditional, 0.10, 0.25 and 0.50% of glucose syrup added to total cassava suspension volume. A glucose syrup concentration of 0.50% contributed to doubling fermentation yield. The objective of this work was to observe the effect of the cassava starch fermentation process on starch granule microstructure, as compared with industrial processing. Micrography was performed using a scanning electron microscope, at 2000 magnication. Fermentations with 0.50% glucose syrup presented the greatest effects on starch granule microstructure.

INTRODUCTION Cassava starch is produced from cassava (Manihot esculenta, Crant) root by extraction, washing, purication and drying (Cereda 1983c; Vilela and Ferreira 1987; Leonel and Cereda 2000; Silveira et al. 2000). Using a fermentative process, cassava starch is modied by amylolytic enzymes and organic acids produced by microorganisms from the natural environment; the resulting product is called sour cassava starch (Demiate et al. 1999; Pereira et al. 1999). It is a sun-dried product, used mainly as an essential raw material in biscuit and cheese bread production (Nakamura et al. 1976; Cereda 1983a,b,c, 1987; Cereda and Bonassi 1985; Amante 1986, Ascheri and Vilela 1995, Silveira et al. 2000, Maeda and Cereda 2001, Balagopalan 2002). However, the process
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Corresponding author. TEL: +55 48 33315370; FAX: +55 48 33319943; EMAIL: eamante@ cca.ufsc.br Journal of Food Process Engineering 29 (2006) 362372. All Rights Reserved. 2006, The Author(s) Journal compilation 2006, Blackwell Publishing

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needs improvement in terms of increased yield, where product standardization is a decisive factor for preference denition (Cereda and Lima 1981; Cereda 1983a; Cereda and Bonassi 1985; Ascheri and Vilela 1995; Leonel and Cereda 2000). In the cassava starch fermentation process, the microorganisms responsible are not introduced as starters, but the use of natural contaminant microorganisms executing specic functions in each stage of the process does occur (Ascheri and Vilela 1995; Cereda et al. 1995). The fermentation process can take from 30 to 60 days and it requires longer times in colder regions, such as the Santa Catarina state, especially in the southern part of the state (Nakamura et al. 1976; Cereda and Cataneo 1986; Cereda et al. 1995; Plata-Oviedo and Camargo 1995; Pereira et al. 1999; Silveira et al. 2000). Along with a number of other factors such as raw material varieties, kind of soil, starch granule characteristics and enzymatic susceptibility, these differences in fermentation time can contribute to product quality changes such as in biscuit baking performance, color, physic-chemical and microbiological characteristics, as well as to economic loss, because of the need for more extensive vat use per batch and a lower frequency of product offer on the market. Several authors state that sour cassava starch undergoes enzyme and acid modications. The acid attack is demonstrated by a large organic acid production, including lactic, acetic, butyric and propionic acids, among others. Enzymatic action is observed as small perforations and ssures in some starch granules with the use of electronic microscopy (Crdenas and Buckle 1980; Cereda et al. 1995; Plata-Oviedo 1995). Other studies have conrmed the presence of diversied bacteria, fungi and yeast, most notably the amylolytic lactic acid bacteria, as dominant in the natural microbiota and responsible for sour cassava starch fermentation by attacking its amylolytic enzymes on starch granules and transforming them into substrates (Cereda 1987; Cereda et al. 1995; Carvalho et al. 1996; Silveira et al. 2000; Ampe et al. 2001; Gyuot and Morlon-Guyot. 2001; Sanni et al. 2002). Considering the current importance of this fermentation process, this work evaluated the effect of glucose syrup addition on sour cassava starch production, with the aim of increasing the yield and observing the effect of the improved process on starch granule microstructure in comparison with industrial cassava starches obtained from three locations in the Santa Catarina state in Brazil. MATERIALS AND METHODS Sour cassava starch was produced by both the traditional method and with 0.10, 0.25 and 0.50% glucose syrup additions to total cassava starchwater

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suspension volume (Marcon 2004). Cassava starch samples were randomly selected from three regions of the Santa Catarina state: Santa Rosa, Rio do Sul and Tubaro. Cassava starch samples were weighed (approximately 2.5 kg) in triplicate for each treatment and distributed in 20-L plastic vats. The starch was then suspended in 10 L of water, more than sufcient to cover the starch. Glucose syrup was added to the starch milk according to specied concentration levels (0.10, 0.25 and 0.50%, w/v). The vats were numbered 136, and pH and acidity were measured daily. Acidity was determined by the adapted AOAC method (BRASIL 1995) and pH was measured directly from the fermentation water, using a potentiometer (Instrutherm pH 1600 digital pH meter). Analyses were conducted in triplicate for each fermentation vat, for a total of 36 vats. The fermentation process was interrupted when pH value achieved a constant value varying around 3.03.8 according to the starch, determined by a curve pH time, and acidity was around 2.0 mL NaOH N. After this period, the mass of sour cassava starch was sun-dried for 3 days following standard procedures (Demiate et al. 1999, 2000; Silveira et al. 2000; Ampe et al. 2001; Gyuot and Morlon-Gyuot 2001). Samples were stored in high-density polyethylene bags (5 L) as a primary package, and in rigid plastic vats as a secondary package, and then placed in a storage room (25C) until further testing. The microstructure of the sour cassava starch granules was evaluated by scanning electron microscopy with a Phillips model XL 30 microscope, with a tungsten lament, electron source and secondary electron detection. The samples were xed on an aluminum sample support and covered with a thin gold layer, using a methalizator model D2 Diode Sputtering System from International Scientic Instruments.

RESULTS AND DISCUSSION Observations of the sour cassava starch obtained from different fermentation processes traditional and with added glucose indicated extensive pH reduction, from 6.0 to 3.0, to 3.8 until stabilization, with time variations occurring according to the origin of the cassava starch. This trend was similar to that observed by Cereda and Lima (1981), Cereda (1983c), Cereda and Giaj-Levra (1987), Ascheri and Vilela (1995), Carvalho et al. (1996), Demiate et al. (1999) and Pereira et al. (1999) conrming that the pH reduction is a result of organic acid production. Santa Rosa starch was fermented in half the time as compared with the Rio do Sul and Tubaro starches, using both traditional and glucose addition method, indicating that the fermentation process is dependent on the starchs origin.

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50 45 Fermentation time (days) 40 35 30 25 20 15 10 5 0 Rio do Sul Tubaro Santa Rosa

Traditional Glucose syrup 0.10% Glucose syrup 0.25% Glycose syrup 0.50%

Cassava starch origin

FIG. 1. EFFECT OF DIFFERENT GLUCOSE SYRUP CONCENTRATIONS ON SOUR CASSAVA STARCH FERMENTATION YIELD

Starches from different regions performed differently under fermentation because of natural starch variability, thus our interest in studying starch fermentation from different sources (Cereda and Lima 1981; Cereda 1983c, 1987; Cereda and Bonassi 1985; Cereda and Giaj-Levra 1987; Ascheri and Vilela 1995; Demiate et al. 1999; Sriroth et al. 1999; Chatakanonda et al. 2003). The effect of glucose syrup addition on fermentation yield was evaluated considering the time each treatment took to achieve an acidity value of 20 mL NaOH N. The fermentation yield of the 0.50% glucose syrup treatment was veried as approximately twice as fast as the traditional processes in starches from the three locations studied (Fig. 1). Micrography shows the microstructure of the starch granules from the three locations studied (Figs. 24). Crdenas and Buckle (1980) and Plata-Oviedo and Camargo (1995) also observed small holes in sour cassava starch granules, which proves that enzymatic attack occurred, probably by microbial enzymes produced during the fermentation process. The structures they observed reveal characteristic microstructural aspects of cassava starch (Amante 1986), predominantly rounded, sacciform and cupuliform granules, in agreement with the ndings of this work. Figures 2B, 3B and 4B represent the industrial sour cassava starch microstructure of starches from Rio do Sul, Tubaro and Santa Rosa, respectively. Considering the amylolytic enzyme action on the granules, it is possible to observe details of industrial fermentation damage in all products. Starches of all treatments were fermented until the excess liquid in the fermentation vats

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FIG. 2. SCANNING ELECTRON MICROSCOPY (2000) OF (A) CASSAVA STARCH, (B) INDUSTRIAL CASSAVA STARCH, (C) CASSAVA STARCH SOUR FERMENTED BY TRADITIONAL PROCESS, (D) CASSAVA STARCH SOUR FERMENTED WITH GLUCOSE SYRUP SOLUTION 0.10%, (E) CASSAVA STARCH SOUR FERMENTED WITH GLUCOSE SYRUP SOLUTION 0.25% AND (F) CASSAVA STARCH SOUR FERMENTED WITH GLUCOSE SYRUP SOLUTION 0.50% FROM RIO DO SUL

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FIG. 3. SCANNING ELECTRON MICROSCOPY (2000) OF (A) CASSAVA STARCH, (B) INDUSTRIAL CASSAVA STARCH, (C) CASSAVA STARCH SOUR FERMENTED BY TRADITIONAL PROCESS, (D) CASSAVA STARCH SOUR FERMENTED WITH GLUCOSE SYRUP SOLUTION 0.10%, (E) CASSAVA STARCH SOUR FERMENTED WITH GLUCOSE SYRUP SOLUTION 0.25% AND (F) CASSAVA STARCH SOUR FERMENTED WITH GLUCOSE SYRUP SOLUTION 0.50% FROM TUBARO

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FIG. 4. SCANNING ELECTRON MICROSCOPY (2000) OF (A) CASSAVA STARCH, (B) INDUSTRIAL CASSAVA STARCH, (C) CASSAVA STARCH SOUR FERMENTED BY TRADITIONAL PROCESS, (D) CASSAVA STARCH SOUR FERMENTED WITH GLUCOSE SYRUP SOLUTION 0.10%, (E) CASSAVA STARCH SOUR FERMENTED WITH GLUCOSE SYRUP SOLUTION 0.25% AND (F) CASSAVA STARCH SOUR FERMENTED WITH GLUCOSE SYRUP SOLUTION 0.50% FROM SANTA ROSA

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reached a constant pH and acidity, which occurred in a maximum of 46 days a much shorter period than that currently practiced in industrial fermentation processes. Figures 2C, 3C and 4C represent the laboratory-produced sour cassava starch using the starches from the three regions studied, as a simulation of the industrial process, without glucose syrup. Detailed observation showed that the shorter fermentation time of the rapid method resulted in less damage to the cassava starch granules. Figures 2D, 3D and 4D represent that laboratory-produced sour cassava starch with 0.10% glucose syrup using the starches from the three regions studied. Increased porosity was observed in the Rio do Sul and Santa Rosa sour cassava starch granules; this was not observed with the Tubaro cassava starch samples. This result indicates that the effect of adding glucose syrup on sour cassava starch fermentation could be related to the nature of the granule, with the possibility of a variety of effects according to the density and dimensions of the cassava starch used. Tubaro cassava starch presented granules with smaller dimensions, which tend to be more resistant to the enzyme action. Figures 2E, 3E and 4E represent the sour cassava starch produced with 0.25% of glucose syrup from the starches of all three regions studied. Increased granule porosity was observed, with similar performances for Tubaro, Santa Rosa and Rio do Sul cassava starches. Figures 2F, 3F and 4F represent the effect of 0.50% glucose syrup on cassava starch fermentation, showing the most effective damage to starch granules, as well as the shortest fermentation time, increasing fermentation yield by around 100% compared with traditional fermentation. The rst stages of the sour cassava starch fermentation process are characterized by sugar generation from amylolytic microbial enzymes, mainly of fungal origin. Thus, the addition of glucose creates an opportunity to bypass certain initial stages in the process, supplying a higher concentration of substrates for lactic acid bacterial action, which are the most predominant in sour cassava starch production. Acids and enzymes produced in the different stages of sour cassava starch fermentation promote important starch granule damage, as observed in the present work. It was possible to observe that native starches used as raw material to obtain sour cassava starch can be damaged in a number of ways, all of which can inuence the quality of the sour cassava starch obtained.

CONCLUSIONS The process proposed to improve sour cassava starch fermentation reduced the fermentation time by around 50%. Fermentation time reduction

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can contribute to increased product quality and sour cassava starch fermentation can be, at least, doubled by glucose syrup addition. Starch origin and fermentation rate affect starch microstructure. Both the effect of added glucose and the performance of the starches differ according to the origin of the native starches used in the fermentative process. This simple method to increase fermentation productivity with added glucose syrup has been transferred to the industry and is currently being used, especially in the Santa Catarina state cold regions, to reduce fermentation time.

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