Issue journal of the science of food and agriculture( fermentation of cassava)

FERMENTATION OF CASSAVA By I. A. AKINRELE Cassava (Manzhot utzlzsszma Pohl) is usually fermented during preparation when it This fermentation was found to be self-sterilising, exothermic and anaerobic, and to Lactic and formic acids becomes detoxified and develops a characteristic flavour. proceed in two stages at an optimum temperature of about 35". are produced with a trace of gallic acid. The process could be made continuous. Introduction The fermentation of cassava (Manihot utilissima) is a very important step during the preparation of ' gari ', a popular food among the low income group in the southern regions of Nigeria which is also eaten extensively along the coast of West Africa. Its significance has already been reported1 that it brings about the detoxification of cassava by the liberation of hydrocyanic acid at low pH, through the combined activities of Corynebacterium manilaot and Geotricum candida, and the development of the characteristic flavour of gari. This fermentation which normally takes 3-4 days during village processing can be shortened to 24 h. by seeding a fresh pulp with cassava juice 4 days old. In the work described here, a further attempt has been made to elucidate the nature of biochemical reactions taking place during fermentation, and to establish the optimum conditions which could be utilised in a modern industry based on this process. Experimental (I) Effects of temperature on fermentation Weighed quantities of grated cassava pulp were put into labelled vacuum flasks and the progress of fermentation followed by the measurement of the temperature with mercury thermometers. Also, samples of about 5 lb. of the grated pulp were placed in beakers, half of which were ' seeded ' by treatment with I pint of 3-days-old cassava juice added to I cwt. of mash. The beakers in pairs, each consisting of ' seeded ' and untreated mash, were covered and incubated at room temperature (about 26"), 35", 40°, 45" and 50" respectively in thermostats and the progress of fermentation followed by measuring the pH of the expressed juice with a Cambridge direct reading pH meter. (2) Comparison of batch and continuous fermentation and the effects of sunlight, aeration and Two 3-ft. columns were used-one of glass and the other of porcelain. The columns were marked externally into three portions and filled with grated pulp. Internally, the portions were marked by rings of dyed cotton wool which slid along with the mash. After each 24 h., the mash at the bottom third portion of the column was pushed out and fresh pulp added at the top. The pH of the juice expressed from each portion of pulp was measured as before. At the same time, control experiments were set up with fresh pulp in open shallow plastic buckets. Their pH were similarly measured after each 24 h. (these experiments were done with unseeded pulp). Three other samples of grated cassava pulp were put into labelled beakers and treated as follows : Sample S was inoculated and frequently mixed, Sample A was also inoculated but not mixed, while Sample C was neither seeded nor mixed and served as a control experiment. The progress of fermentation in these samples was again followed by measuring the pH of their juices. (3) Identijcation by paper chromatography of the organic acids produced during fermentation Preparation of sample Cassava juice was expressed from a fermenting mash and kept in a beaker labelled A. A sample of gari was also treated with cold water and the extract put in another beaker labelled B. Both samples were treated separately with ethanol to precipitate starch and the mixtures frequent mixing of the mash J. Sci. Fd Agric., 1964, Vol. 15, September 00 590 AKINRELE-FERMENTATION OF CASSAVA were filtered. Each filtrate was then passed through Amberlite resin IRA-400 in the carbonate form in order to absorb the organic acids,2 and subsequently eluted with O*IN-ammOniUm carbonate. The excess of ammonium carbonate was decomposed by heat during concentration. Alphatic acids3 Solutions from these samples were spotted with micro-pipettes on to Whatman No. I papers with other spots of the following reference acids : orthophosphoric, lactic, succinic, oxalic and tartaric. Duplicate papers were prepared, one paper was developed with mesityl oxide-85% formic acid-water (75 25 : I) and the other with phenol-water-formic acid (75 : 25 : I) using descending chromatography. The chromatograms were dried, sprayed with bromocresol green indicator (0.04 g. in 15 ml. of ethanol + 5 ml. of water, pH adjusted to 5.5) and the R, values measured. Aromatic acids Sample solutions prepared as above were spotted on two Whatman No. I papers and developed with butanol-acetic acid-water (4 : I : 5) by descending chromatography. Reference standards were chosen from the following phenolic compounds, viz., vanillin, pyrocatechol, pyrogallol, P-hydroxybenzoic acid, gallic acid, hydroquinone, salicylic acid and phloroglucinol. One chromatogram was sprayed with ferric chloride and the other with ammoniacal silver nitrate and the RF values measured. Volatile acids4 Another portion of the juice expressed from a fermenting cassava mash, which should contain all the volatile acids, was treated with alcohol to precipitate starch and then filtered. The filtrate was neutralised with o.og~-bariurn hydroxide (phenolphthalein indicator) and centri- fuged. The supernatant solution was decanted and evaporated almost to dryness. Excess of saturated ammonium oxalate solution (about 5 ml.) was added and the whole mixed and filtered. The filtrate which contained the ammonium salts of the volatile acids was used for the chromatographic examination. This solution was next spotted on two Whatman No. I papers and developed with butanol-~.g~-aq. ammonia (50 : 50). Reference spots were made with formic, acetic, propionic, butyric and lactic acids which had been made alkaline with ammonia. The chromatograms were dried and one was sprayed with Bromocresol green and the other with ammoniacal silver nitrate to distinguish between acetic and formic acids. The Rp values of the developed spots were then measured. Results The increases in temperature accompanying fermentation in the vacuum flasks are typified by curves shown in Fig. I for two series of results. The pH readings taken to compare batch and continuous fermentation and also the effects of sunlight and exposure to air are recorded in Table I. The effect of the frequent mixing of the mash on the progress of fermentation, measured by the fall of pH with time, is shown 0 20 40 60 80 TIME, h. 0 FIG. I Temperature rise during fermentation of cassava pulp (two sets of results) J. Sci. Fd Agric., 1964, Vol. 15, September AKINRELE-FERMENTATION OF CASSAVA 591 Table I pH readtngs durzng batch and contznuous fermentatzon of cassava mash Batch fermentation in open buckets Continuous fermentation in tubes Control (exposed to light) Glass Porcelain Sample 24 h. 48 h. 72 h. 96 h. 24 h. 48 h. 72 h. 96 h. 24 h. 48 h. 72 h. 96h. -_ 4.20 3.85 3.85 - 4.1 4.05 4.0 4.0 4.0j 4.1 - A H 4.25 4.3 4.3 4.2 4.0 4.1 c 4'35 4'3 4'3 4.2 4.1 4.05 4.2 4.1 4.1 - E 4.j 4.3 4.1 - 4.r.j 4.1 4.0 - 4.1 4.2 4.15 - - 4'25 3'9 3'9 - D 4.2 4.2 4.25 4-25 4.2 4.1 - 4'2 4.15 4'15 - 1; 4'3 4.15 - 4.15 4.2 4.0 - 4'1 4.2 4.0 - 4'0 G 4.25 - 4.1 3.8 4.15 - 4'0 3'7 4'1 4'1 3'7 4'2 3-9 3'9 Average 4.3~ 4.26 4.12 3.97 4.19 4.11 3.98 3.86 4-13 4.11 4-08 3.9 in Table I1 ; whilst Fig. z shows the relationship between the rate of fall of pH and time at 45". The effect of incubating the mash at various temperatures is shown in Fig. 3. The RB. values obtained during the chromatographic separation of the organic acids of cassava mash and gari are recorded in Tables 111-V. Passage of the gas evolved during fer- mentation through lime water produced a turbidity, but the remaining gas was not absorbed by 40% sodium hydroxide solution. It is inferred that the formic acid present is decomposed to carbon dioxide and probably hydrogen. - 4.15 3.8 3.8 - 4'3 3'9 4'05 - H Table I1 PH of fermenting cassava mash at various times Incubated at 4j0 __ Time, Incubated at 35' h. S A C S A c 0 2 4 7 I1 12 I4 16 I8 20 22 2 5 28 30 5'90 5.60 5'51 5'05 4'25 4.05 3'94 3'83 3'70 3.67 3'67 3.62 3'45 3'44 5'90 5'58 5.50 5.06 4.46 4'24 4.02 3.88 345 3'72 3.69 3.66 3'51 3'49 5.90 5'74 5.50 5.38 4'63 4'30 4'22 4.12 4'0 4'0 4'0 3'94 3.78 3.78 5.93 5'75 5'50 5'35 4% 4'55 4'25 4'2 4'0 3.85 3'7 3'7 3'65 - 5'93 5'93 5'70 5'75 5'46 5-65 5'05 5'55 4'95 4'95 445 4'95 4.65 4'75 4'65 4'6 4'37 4'45 4-25 4'40 4.05 4.2 3'97 4'1 4.20 4'25 - - S = inoculated acid mixed frequently ,, but not mixed C = control - - 0.1251 I I TIME, h FIG. 2 Rate of fall of PH during fermentation of cassava pul9 at 45" x inoculated, unmixed 0 inoculated, mixed 0 uninoculated, unmixed (control) J. Sci. Fd Agric., 1964, Vol. 15, September 592 AKINRELE-FERMENTATION OF CASSAVA w 3 A > I a a -0 10 20 30 TIME, h. FIG. 3 pH values after diflerent times for fermentation of cassawa pulp at different temperatwe: (uninoculated) 0 50' 0 45' A 40' A 35' X 26" (inoculated) 0 - - - 0 Table 111 RF Values of acids in fevmentang cassava mash Developing solvent Mesityl oxide-formic Phenol-water-formE ~ acid-water Orthophosphoric acid 0.24 Lactic acid 0.67* Succinic acid 0.71 Gari acid 0.62 Pulp (fermented) acid 0.61 Tartaric acid - Oxalic acid 0'52 acid 0.17 0.13 0.72 0.71 0.70 0.69 0.41 0.38 0.71 0.71 0.72 0.70 0.29 0.25 * Three spots were obtained at RF 0.67, 081 and 0.88 Table IV RF Values of aromatic acids in fermenting cassawa mash (developing solvent butanol-acetic acid-water 4 : I : 5) Phenols and aromatic acids Vanillin Pyrocatecliol Pyrogallol p-Hydroxybenzoic acid Gallic acid Salicylic acid Hydroquinone Phloroglucinol Gari acids Pulp (fermented) acids 0.87, 0.83 0.80, 0.82 0.70, 0.71 0.84, 0.85 0.60, 0.64 0.81, 0.82 0.09, 0.66 ; 0.11, 0.66 0.09, 0.65 ; 0.13, 0.67 __ -_ Standard values 0'92 0.85 0'77 0.90 0.69 0'95 0.88 0.76 - - Discussion ,4 very striking feature of the fermentation of cassava is the good reproducible results obtained in various experiments in which crude culture inoculants were used. It would appear that the medium is self-sterilising against adventitious infestation by undesirable micro- organisms and that biochemical studies could be carried out satisfactorily on it. The acceptability of gari is to a large extent influenced by its sourness and this in turn is directly related to the degree of fermentation. In Table VI is seen the direct relationship of the pH of fermenting cassava mash to the flavour quality of gari prepared therefrom. The J. Sci. Fd Agric., 1964, Vol. 15, September AKINRELE-FERMENTATION OF CASSAVA 593 least acceptable flavour is found with material of pH 3.95 which is the average value obtained in fermentation for 4 days under normal conditions (see Table I). This value can sometimes be attained more quickly, particularly in locations where cassava processing is a regular practice. Table V Rp Values of volatile acids in feiwenting cassaua mash (developing solvent butanol-I.5~-ammonia 50 : 50) Acids Bromocresol Ammoniacal silver green spray nitrate spray Formic 0'10 + Dark brown Acetic 0'12 - - Propionic 0'2 I Butyric 0.31 - Lactic 0.08, 0.15 + Yellow Pulp (fermented) 0.08 + Dark brown Table VI Effect of PH and acidity on $avow quality of gari pH of cassava mash 3'70 3.60 3.80 3.87 3.90 3'93 Total acidity (yo as lactic) of gari made therefrom 0.92 I '04 0.66 0.58 0'43 0.40 Taste and flavour Good Satisfactory Good Satisfactory Just Rather Swelling index 4'75 4'40 4.20 4.60 5.10 4'30 satisfactory poor That this fermentation is accompanied by the evolution of heat is well established in Fig. I. The two peaks shown in each fermentation appear to be characteristic and coincide with the periods of dominant growth of the micro-organisms isolated and identified in the cassava mash. This would, therefore, seem to confirm the two-stage fermentation hypothesis put forward by Collard & Levi.l The results in Table I indicate that continuous fermentation is possible and that it is more effective than the batch type. The process could therefore be streamlined, probably by the use of a tunnel without a great risk of serious contamination. The effect of sunlight (comparing the results for porcelain and glass tubes in Table I) also becomes observable only after 48 h. of normal fermentation, when a differential lowering of the pH of the mashes is indicated, It is significant that this period should again coincide with the onset of the second stage of fermentation during which the fungus, Geotricmn calzdida, is re- ported to proliferate and to produce a variety of aldehydes and esters. This could mean that light may have a stimulating effect on the growth of the fungus whose optimum pH for growth is known5 to be about 3. Cassava pulp when exposed to air soon acquires a brownish discoloration from the oxidation of its leuco-anthocyanins, viz., delphinidin and cyanidin. The deeper layers, however, which are rendered anaerobic by the evolution of carbon dioxide and hydrocyanic acid during fer- mentation, do not undergo this change. When cassava was fermented in open shallow vessels with a wire mesh bottom so as to increase the surface area exposed to air, a significant slowing down of the rate of acidification was noticed. This therefore raises a strong presumption that aeration during fermentation could be inhibitory. From Fig. 3 it could be inferred, in general, that 35" is the optimum temperature for the fermentation. This is confirmed by the extremely rapid fall of pH in the seeded mash incubated at this temperature. While it may be said that fermentation will take place at 45" quite satis- factorily, a marked retardation is noticeable at 50" which results in the suppression of the second stage. From the results in Table 11, it would appear that it is advantagous to mix the mash during fermentation, but it can be observed from Fig. 2 that this advantage is manifested only after 7 h. of uninterrupted incubation. Two organic acids have been identified, as products of the fermentation of cassava (Tables 111-V), viz., lactic and formic acids, but only lactic acid was found in gari. During the J. Sci. Fd Agric., 1964, Vol. 15, September 594 MONEY-ANALYTICAL DATA OF COMMON FRUITS chromatographic separation of the polyphenols and aromatic acids, two spots with equal RF values derived from the fermented pulp and gari respectively were located close to the value obtained for gallic acid, but they were much paler in colour than the standard, probably due to their low concentrations. It is quite likely that the gallic acid may have been produced from the tannins of the inner cortex of the cassava root by an enzyme ' tannase ' which is known5 to occur in some fungi. Conclusions It is confirmed that the fermentation of cassava proceeds in two stages during which the mash is gradually sterilised against adventitious microbial growth. During the first phase, cassava bacteria ' Corynebacterium manihot ' attack the starch with the production of lactic and formic acids, a reaction accompanied by the evolution of heat. When the pH of the medium has fallen to about 4-25, a mould Geokicum candida, begins to proliferate rapidly bringing about further acidification and the production of the characteristic aroma of gari. Hydrogen cyanide is liberated during fermentation through the spontaneous hydrolysis of the cyanogenic glucoside of cassava at low pH. It is also believed that some of the formic acid breaks down by a hydrogenase system to form carbon dioxide and probably hydrogen. All these gases tend to render the medium anaerobic. Fermentation seems to proceed best at a temperature of about 35', and with pulp inoculated with fermented cassava juice a satisfactory product has been produced under 15 h. Frequent mixing of the mash and exposure to light appear to accelerate fermentation particularly during the second stage. A continuous system is possible and, in fact, gives a better fermentation than does the batch process. Exposure to air or oxygen and contact with iron should be reduced to a minimum to avoid discoloration. Federal Institute of Industrial Research Private Mail Bag 1023 Ikeja Airport Nigeria Received TO December, 1963 ; amended manuscript 28 January, 1964 References Gawler, J. H., J. Sci. Fd Agric., 1962, 13, 57 a Bryant, F., & Overell, B. T., Nature, Lond., 1951, Cochrane, V. W., ' Physiology of Fungi' (New Akinrele, I. A., Cook, A. S., & Holgate, R. A., Fed. Inst. industr. Res. Rep., 1962, No. 12 (Lagos) Collard, P., &Levi, S., Nature, Lond., 1959,183,620 Lederer, E., & Lederer, M., ' Chromatography' 167, 361 York: Wiley) (London : Elsevier) ANALYTICAL DATA OF SOME COMMON FRUITS POTASSIUM AND PHOSPHORUS CONTENTS By R. W. MONEY Analytical data of some common fruits were published in 1950' and 1958 ;2 these have been extended by determinations of the potassium and phosphorus contents of further samples taken over a period of about 3 years. Some consideration has been given to the use of these values to calculate the fruit content of products such as jam, but as will be shown below, care must be exercised when such calculations are applied to samples of unknown origin. The potassium content was determined with an E.E.L. flame photometer and without ashing ; the results are given as mg. of K/IOO g. The phosphorus was determined after dry ashing J. Sci. Fd Agric., 1964, Vol. 15, September . in thermostats and the progress of fermentation followed by measuring the pH of the expressed juice with a Cambridge direct reading pH meter. (2) Comparison of batch and continuous fermentation. fermentation and the effects of sunlight, aeration and Two 3-ft. columns were used-one of glass and the other of porcelain. The columns were marked externally into three portions and filled. two series of results. The pH readings taken to compare batch and continuous fermentation and also the effects of sunlight and exposure to air are recorded in Table I. The effect of the frequent