AKINRELE Cassava Manzhot utzlzsszma Pohl is usually fermented during preparation when it This fermentation was found to be self-sterilising, exothermic and anaerobic, and t o Lactic an
Trang 1By 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 t o
Lactic and formic acids
becomes detoxified and develops a characteristic flavour
proceed in two stages a t 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 a t 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 a t 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 m a s h
J Sci Fd Agric., 1964, Vol 15, September
Trang 2were 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
R p 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
TIME, h
0
FIG I.-Temperature rise during fermentation of cassava pulp
(two sets of results)
J Sci Fd Agric., 1964, Vol 15, September
Trang 3Table I
p H readtngs durzng batch and contznuous fermentatzon of cassava mash
Batch fermentation in open buckets Continuous fermentation in tubes
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 a t 45" The effect of incubating the mash a t 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
P H of fermenting cassava mash at various times
Incubated at 4 j 0
Time, Incubated - a t 35'
0
2
4
7 I1
1 2 I4
16 I8
2 0
2 2
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
4 4 5 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
-
-
TIME, h
FIG 2.-Rate of f a l l 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
Trang 4w
3
A
>
I
a
a
TIME, h
FIG 3.-pH values after diflerent times f o r 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
Pulp (fermented) acid 0.61
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, 0 8 1 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
Trang 5least acceptable flavour is found with material of p H 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
R p Values of volatile acids in f e i w e n t i n g cassaua m a s h
(developing solvent butanol-I.5~-ammonia 50 : 50) Acids Bromocresol Ammoniacal silver
green spray nitrate spray Formic 0'10 + Dark brown
- Propionic 0'2 I
Lactic 0.08, 0.15 + Yellow
Table VI
Effect of PH and acidity on $ a v o w 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 Swelling index Good 4'75 Satisfactory 4'40 Good 4.20 Satisfactory 4.60 Just 5.10 Rather 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 a t 45" quite satis- factorily, a marked retardation is noticeable a t 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
Trang 6chromatographic 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
t o 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 a t 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 T O December, 1963 ; amended manuscript 28 January, 1964
References
Gawler, J H., J Sci F d 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 1 2 (Lagos)
Collard, P., &Levi, S., Nature, Lond., 1959,183,620
Lederer, E., & Lederer, M., ' Chromatography'
(London : Elsevier)
ANALYTICAL DATA OF SOME COMMON FRUITS
POTASSIUM AND PHOSPHORUS CONTENTS
B y 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