Fermentation Kinetic Studies

imSEARCH PAPERS

3, 205-209

J. Food Scí. TechnoL., 1999, Vol. 36, No.

Fermentation Kinetic Studies

of Nigerian Palm

Raphia hookeri by Bottling

Wines-Elaeis guineensis and for Preservation

R.C. AGU*i,MtP. OKENCHP, C.M. UDE1, A.I. ONY1, A.H. ONWMELU2 AND V.I.E. AJIW2 Natural Sciences, if;" ¡Faculty of Applied

Enllg,tiState University of Science andTechnology, P.M.B. 01660, Enugu, Nigeria.

2Faculty of Sciences, Nnamdi Azikiwe University, P.M.B. 5025, Awka, Nigeria.

Fermentation kinetic studies of two Nigerian palm wines-Elaeisguineensis and Raphia hookeri showed that.gas the fermentation experiment due to heavy

evolution, used as the fermentation rate index, was continuous throughout

microbial growt in the fermenting broths. Both Elaeis and Raphia Wie fermentationsfollowed a similar first-order

rate constant three times that of Raphia

kinetics. Elaeis wine fermentation was more vigorous~ having a fermentation

wine. Elaeis wines from different sources showed slight varations in their fermentation rates. The unsterilized sapfermented

faster and produced higher levels of alcohol than sterilized Elaeis sap inoculated with yeast, Saccharomyces cerevisia.

after botting of fermented wine. The

Sterilization of the sap followed by yeast inoculation did not prevent exlosion

of the sap, followed by pasteurization of lermented wine was more effective in controlling exlosion durig the. bottling of palm wine. However, the sterilization process changed the appetizing whitish colour of palm wine to sterilization

a dull brownsh colour.

Keywords: Kinetics, Fermentation, Palm wine, Sterilization, Bottling, Preservation.

palm wine in natural state by bottling has not been achieved because any attempt to cap the

Palm wine, a generic name for :; group of

of

alcoholic beverages obtaied by fermentatlon from

tapped wine without chemical treatment or use of

the saps of palm' trees, is usually a whitish effervescent liquid:

preservatives usually results iri a violent explosion, no suitable Presently, especially with Elaeis wine.

This propert is derived from

numerous microorganisms that are metabolically active. (Okafor 1978, 1987; Mmegwa 1987). Rokosu

preservation method for palm

wine, has been

evolved except refrigeration at near freezing point.

and Nwisienyi (1980) have reported thatfresh palm

wine is a sweet drink, which easily becomes sour, when left for a few' hours before consumption. The alcohol content ranges from 0.5 to 7.0% (v/v)

This preservation method

depending onthè lqcality, where the sap is obtained

populace. During the

is expensive because of

high energy requirement and it is not readily affordable by over 98% of the low income group

..

peak production period of palm wine, most of the wine is wasted or distilled

and the éxtent of fermentation (Bassir 1962;

for the production of "ilicit gin" (known locally as

Faparst and Bassir 1972 a, b ; Okafor 1990). Palm

kai-kai orekpeteshi ), which is deleterious. to had earlier suggested the

wine is rich in non-pathogenic microorganisms of industral importance. High ethanol-resistant yeàsts

health. Okafor (l990)

have been isolated from palm wine_ (Agu et al.

production

of synthetic palm wine by inoculating

"palm wine yeast" or "Bussa' yeast" into maltose for industrial production. of palm wine. Palm wine is relished by milions of people in the Eastern part

1993). Other yeasts of importance in brewing,

distilling and wine production are S. ellipsoides, S. cerevisiae, Schizosaccharomyces pombe,' as well as

of Nigeria. The ar of tapping the palm tree for wine phased out

Saccharomyces ludwigii, which are useful in the

production of low alcohol beer because of their

production is tedious and is being

(Okafor 1990). Hence synthatic palm wine is gaining importance. However, the synethatic palm

inability to ferment maltose (Okafor 1975 a, 1990). Tapped and naturally fermented palm wine,

is likely to be different from that of the naturally inoculated and fermented wine.

or "Bussa yeast" is inoculated into maltose (Okafor 1990), has

wine

where "palm wine yeast"

. ~

Although extensive studies have been done on

nutrtional benefis and supplies approximately 300

palm wine, most of

kcal of energy (Bassir 1968). It is important

these studies were centred on

their microbiology (Van Pee and Swing 1971;

therefore, that palm wine should be preserved in

Okafor 1972, 1975 b; 1990; Faparsi 1973). These

its natural state as an alcoholic beverage by

microorganisms were reported to be inoculated advertently from previously used collecting vessels,

bottling and consumed, when desired. Preservation * Corresponding Author

205

206

tapping knives, bamboo funnels, air and palm inflorescence (Okafor 1971). Studies on fermentation

3 '

allowed to stand for 15 min, after which 0.5 g of

NaHCO was added and the volume made up. An

study was, therefore, undertaken to investigate the

transferred to a 250 ml volumetric flask. After addition of 5 ml of 80%

fermentation kinetics in the conversion of fresh sugar saps of Elaeis guineensís and Raphia hookeri

was made up, shaken vigorously and allowed to

to wine for possible preservation in its natural state

stand for 10 min. The absorbance was read at 520

by bottling. Carbon dioxide evolution rather than ethanol production was used as fermentation rate

blank was distiled water, while the amount of

scanty. The present

,kinetics of palm wine are very

aliquot of 10 mL, was

phenol and 5 ml concentrated HßO 4' the volume

nm using a spectrophotometer (Spectronic-20). The

from the metabolic activities of the various

sugar in the sample was determined from a glucose curve. standard Determination of protein, ash ànd spectfc gravity of the saps: Micro':Kjeldahl method' was employed

microorganisms during the conversion of sugars in

for the determination of the nitrogen materials

index for two reasons. Firstly, the explosion

experienced when the wine is capped, is a result of excessive pressure build-up from the gas generated

the sap to ethanol and gas. 'Secondly, the ethanol

present in the sap. The protein content of the sap

carbon source for some of the

was obtained by multyplying by the factor 6.25.

mixed microbial flora present in the fermentation

About 10 ml of the sap saturated with concentrated H2SO4 contained in a' parcelain boat was dried and ignited at 550°C in a muffle furnace to determine

produced may be a

broth, thus giving a false monitoring of this product in the kietic study.

Materials and Methods

Sample collectin : The samples used in this study (from three different sources-Okpuno in

Awka, Umuabi in Udi and Enugu-EziÌte in Nsukka areas, of Eastern par of Nigeria) were obtained from local tappers specially commissioned to collect the

fresh saps. For the purpose of this study, the tapper's collecting vessels were used to collect the

sap for the initial' advertent inoculation of the sap with the mixed flora present in the collectig vesseL.

The saps were quickly transferred to sterile containers to reduce furter entr of microorganisms

from other sources. Although fermentation was

already in progress, the alcohol value (-0.7%) was

regarded as the starng alcohol value. The sugar sap was then used for the laboratory studies. The palm saps in sterile containers were divided into

the ash content. The specific gravity was determined using the SG botte method. Alcohol was measured

using the specific gravity method (Instiute of Brewing Recommended Methods 1989).

Fermentation and rate studies : The "fresh" sugar samples (150 ml) of the saps from Elaeis or Raphia palms were measured into their respective 250 ml fermentation flasks set up in duplicates and allowed to ferment for 24 h at 32°C. The volume

of carbon dioxide evolved, obtained by, downwrud displacement of water, was measured using a 2000

cm3 calibrated cylinder on an hourly basis. The mean value of carbon dioxide from two replicate measurements was used in the calculations. Calculation procedure: The moles of carbon dioxide gas may be obtained from the following equations.

two portons. The first porton was fermented as

CsHi20s ...............;, 2C2HsOH + 2C02 (1)

such, while the second portion was sterilized and

Hence, the rate of disappearance of sugar is

after cooling, yeast, Saccharomyces cerevisiae was inoculated. Fermentation of sterilized sap inoculated

-r CSHi20S .............;, d (CsHI20s) ............... (2)

with yeast and unsterilized saps were carried out as described below.

Determination of sugars in the fresh samples

dt and the rate of appearance of carbon dioxide is r CO2 .............;, d (C02) ............................ (3)

dt

ofE. guineensis : A modifcation of the 'colorimetric

method described by Dubois et al (1956) for the determination of sugars was employed. To the fresh

fermenting palm sap, 2 g, contained in a flask, 30

while the rate of appearance of ethanol is r C2HsOH .............;, d (C2HsOH) ............... (4)

ml of 80% ethanol was added and extracted (3 times) for 1 h with constant strirring, followed by

decantation after each extraction. To 20 ml of the extract contained in ioo ml volumetric flask, 5 ml of 10% (w Iv) lead acetate solution was added and

Using the production

dt of carbon dioxide in our

model, then, -d CsHi20s

dt

.............;, k d (C02) dt

............... (5)

207

where k, is the fermentation rate constant for

carbon dioxide evolved. Assuming an ideal gas dioxide, then,

behaviour for carbon

PV = nRf

.. . . . . .. . .. . .. . .. .. . . . .. .. .. . .. . .. . . .. .. . .. . . (6)

where P = gas pressure, - 0.1 atm in this experiment; V= gas volume per hour; n = number

of moles of CO2 gas collected; R = gas constant,

TABLE 2. COMPOSITION OF Elaeis SAPS FROM DIFFERENT SOURCES

Unsterilized

Parameters

Uu*

Protein, % Ash, % Specific

Sterilised

Ou*

Us**

Sap Os**

164.00 7.80 4.80 1.06

158.00 8.20 4.70 1.05

188.00 8.60 \ 4.40 1.06

sucrose, mg/rn

Sap

152.00 7.20 5.00 1.04

0.08206 litre atm mol-IKI; T = temperature of

gravity (20°C/20°C)

fermentation = 305 K Since all the values except for n, in equation

brown brown Uu* = unsterilized palm sap from Umuabi; Ou*= Unsterilized

(6) are known, the number of moles of carbon

palm sap from Okpuno; Us**=Sterilized palm sap from Umuabi

Light Light

White

White

Colour

dioxide gas may be obtained from the relation:

Rf RT

n = Pv; P = 3.6774 x 10-3 ............ (7)

(Fig 1) with Elaeis broth fermenting faster than

Raphia broth. The slopes of the graphs for the

The values of the volumes of carbon dioxide released durig the fermentation of Elaeis and Raphia saps to wine and their corresponding

number of moles are shown in Table 1. Results and Discussion

fermentation rate constant, k, of 2.1 x 10-4 mol-I sec-I obtained for Elaeis guineensis was three times (7.4 x 10-5 mol-I sec-I) that obtained

for Raphia hookerL Although the rèason for this both large difference in the fermentation vigour of

wies is not clear, it is not known if the

palm

The sap composition is shown in Table 2. Palm sap contains both sugars and protein required for metabolic activitits of the different microorganisisms en responsible for the wine fermentation. It has he

growt environment of these palm trees would play

reported that other sugars such as sucrose, fructose

palms grow best in -swampy areas, Elaeis palms

(Chiarasa

and raffiose are also present in palm sap

1968; Okafor 1990). Various growt factors such

a role in determining the sap composition. Elaeis

sap contains relatively higher levels of sugars than Raphia sap (results not shown). While Raphia

grow best in dry sandy/loamy soils. It is' also not clear how these growt environments will affect the

as vitamins C and BI2 have also been reported to

quality of the wine derived from these palm trees

be present in palm sap (Okafor 1990).

because wine from Elaeis is usually preferred to

A preliminar study of the fermentation kinetics

that from Raphia for unknown reasons. Again,

of Elaeis guineensis and Raphia hookeri (another

when the wines from Elaeis obtained from different

type of palm tapped for wine production) wines

geographical locations are ranked higher than others, suggesting unknown differences in palm wine production. However, regarding the fermenta-

showed that although, both fermentations followed a similar pattern, the vigour of fermehtations in Elaeis and Raphia broth 'differed

tremendously

TABLE 1. CARBON DIOXIDE PRODUCED DURING

sources are considered, the wines from some

tion kinetics of Elaeis and Raphi.wines reported above, a more iprbba1:HetJtplanation fotthe

FROM

FERMENTATION OF Elaeis WINE

DIFFERENT SOURCES

Unsterilized sap Volumes of Moles of

CO2 (cm3) CO2 Time

4

Steriized sap Volumes of Moles of

3

CO2 (em3) CO2

U ° U 0 U 0 U 0

(xlO3)

sec 2.28 2.26 620 620 2.28 2.28 620 615 3.6 670 665 2.46 2.45 2.61 2.43 710 660 7.2 3.46 3.02 860 815 3.16 3.00 940 820 10.8 4.65 4.23 1200 1080 4.41 3.97 1265 1150 14.4 5.81 5.24 1505 1355 5.53 4.98 1580 1425 18.0 7.35 6.95 1940 1640 7.13 6.03 21.6 2000 1890 U= Umuabi; 0 = Okpuno; Moles of CO2=3.67741 x 10-3 x volume

of CO2; SD for values of CO2 = :! 15 to 38 cm3

Elaeis wine

3,5

o 2.5

u

'õ 2 .. ..

'Raphia wine

~ 1.5

0,5 o o

5

10

15

20

Time (sec) x 10"

Fig. 1.

Kietics of ElaisardRaphia wie fermentation

208

differences in the fermentation vigour observed in this study for Elaeis and Raphia wines is that a

TABLE 3. FERMENTATION PROPERlIES OF Elaeis WINES FROM

local preservative, Saccoglott gabonensis, is usually added to the collecting vessels when tapping Raphia

Parameters

wine. This is not the usual practice when tapping

Rate constant

Elaeis wine. It is however, not clear if this local preservative will inhibit the entry of microorganisms or the activities of th~se microorganisms during the

wine fermentation. Since wine from Elaeis palm is preferred to the

wine from Raphia palms, further studies were carred out on the fermentation kinetics of .Elaeis wine from different sources. It is obvious from the

results shown in Table 1 that the unsterilized saps had faster fermentation rate and released more

carbon dioxide irrespective of the source of the sap.

Fig. 2 and 3 indicate tha.t growt location might have afected the fermentation rates of both sterilied and unsterilized saps. This is probably because of the sap composition or varation in

a difference in

8'

UJ

7

a.

DIFFERENT SOURCES

Unsterilised Sap

Sterilised

Sap

U

0

U

0

1.8 xW-4

1.8 xIO-4

1.8 xIO-4

1.8 xlO"

(mole'l sec-I)

Ethanol production (v/v %)

4.7 5.8 5.8 6.5 7.0 7.2 Fermentation rate constant was calculated from the slope of the graphs U= Umuabi; O=Okpuno

After 3 h After 6 h

6.2 8.6

the microflora of the sap obtained from the different

growth areas. This observation supports earlier reports in this regard (Faparusi and Bassir

1972 a, b; Okafor 1990). The microflora of the unsterilized sap (Okafor 1975a, 1990) might have also inuenced the faster ferementation rate observed in the broth containing unsterilized sap. The

unsterilized sap also had higher fermentation rate constant and produced higher ethanol (Table 3). In contrast, the lower fermentation rate constant

obtained for the sterilized sap inoculated with yeast

6

(Table 3) was partly due to the destruction of some of the microflora present in the sap and partly due to the lower levels of sugars found in the sterilzed

85 'õ 4 el

~ 3

sap. During the sterilization process, it is possible

E 2

that Mailard reaction may have taken place, resulting not only in the depletion of sugar and

1

o o

10

5

15

25

20

Fermentio time (sec) x 1()

UJ = lJtailizO sa fi UT; a. = lJtailizoo sa fi Oq Fig. 2, Fermentation kinetics of unsterilized Elaeis palm wine

(first 3h) was more of a mixed order than first order

8 7 6

U;

kinetics (Fig 2 and 3). As the fermentation

0;

progressed, a more defined first order reaction

05 0

kinetics occured (Fig 2 and 3). Therefore, as the

fermentation progressed and the ethanol yield

'õ 4

increased (Table 3), the resistant-yeast that survved

II

.! 3

ethanol toxicity would, continue the fermentation

0

wine contains ethanol-resistant-yeasts (Agu et aL. 1993).

:l 2

process. (Day et aL. 1975; White 1978). Palm

1

o

the mixed microbial flora, especially in ,the unsterilized sap, fermentation at the early stages

from diferent sources

..

protein levels in the sap, but also resuIted in the colour change (Table 1). The pattern of alcohol production as shown in er Fig 2, and 3 indicated' a first order kinetics (And and Sonnessa 1965). However, in the presence of

o 5 ID ff 20 ~

Similar observations in the fermentation kinetics of sterilized sap inoculated with yeast and unsterilized

Fenn ti (se x 10'

saps (Fig 2 and 3) suggest that the sterilization process was not effective in destroying the microflora

U; = stailiz sa fi Un; 0; = stEJliz 5a fi Oq

of palm sap prior to yeast inoculation. This

Fig. 3. Fermentation kinetics of sterilized Elaeis palm wine from

different sources

observation is furter supported by the fact that

when the sterilized sap inoculated with yeast was

209

fermented and the wine was bottled, similar explosions occurred with the unsterilized fermented wine. However, explosion also occurred when the

unsterilzed fermented wine was capped and pasteurized (pasteurization conditions; 75°C/lh). Iw contrast, when the sterilized/yeast-inoculated

fermented wine was capped. followed by

pasteurization, no explosion occurred. The sterilization process resulted in a colour change

from the known appetizing whitish colour to a dull brownish colour.

Though the results of this study are final solution to the botting of palm wine in its natural state could be found. The fermentation kinetic study reported here is limited to wine obtained from three sources. ,Wide varations in the

microflora of saps from different sources have been

li R,¡"

.

i a ,

ï',~.~

ù ~ ifl fl

tl t¡

F

-,.

i

t

t!i l;

Research Report. Federal Minstr of Industr. No. 8. Lagos.

Nigeria

Day A. Anderson E, Marins PA (1975) Ethanol tolerance of brewing yeasts. In:Proceedings of 15th Congress European' Brew Conference, Nice, pp 371-39 I Dubois M. GiIles KA. Hamlton JK. Rebers PA, Smith F (1956)

substances. Anal Chem 28:350-356

Faparusi SI. (1973) Origin of initial microflora of palm trees. J Appl,Bacteriol 36:559-565

Faparusi SI, Bassir 0 (1972a) Factors affecting the quality of African J palm wine: Period of tapping a palm tree. West

BioI Appl Chem 15:17-23 Faparusi SI, Bassir 0 (1972b) Factors afecting the quality of palm wine: Period of storage. West African J BioI Appl Chem 15:24-28

Presently, "botted" palm wine is found in NigeriaI

Mmegwa SVA (1987) Aspects of the preparations of formulated

markets, but it is not dear if these products are

palm wine. Ph. D. Thesis. University of Nigeria. Nsukka.

naturally fermented or synthetic palm wines, which

Nigeria

into sugars

Okafor N (1971) The sources of microbes in palm wine: In:

(Okafor1990). The effervescence, which characteries

Proceedings Symposium Nigerian Society of Microbiology. Ibadan. Nigeria. ppl02~106

the naturally fermented palm wine is lacking in the "bottled" palm wine. The fermentation kinetics of palm wine revealed that gas production was the major problem because of heavy contamination of

Okafor N (1972) Palm wine yeasts from pars of Nigeria. J Food

Sci Agric Chem 23: 1399-1407 Okafor N (1975a) Microbiology

of Nigerian palm wine with

paricular reference to bacteria. J Appl Bacteriol 38:81-88

the palm sap. Although the sterilized sap inoculated slower fermentation, sterilization

Okafor N (1975b) Preliminar microbiological studies on the

followed by inoculation with pure yeast culture alone, did not achieve the desired objective. The

Okafor N (1978) Microbiology and biochemistr of oil palm wine.

with yeast had

study showed that, fermentation of the yeastinoculated sterilized sap, followed by pasteurization

seemed to control the microbial activity and resulted in successful botting of the

palm wine. However,

the change in colour of the sterilized and fermented

product may not be appetizing to consumers of palm wine. In, this regard, sensory evaluation of the product is essential. References

,

Bassir 0 (1968) Some Nigerian wines. West African J BioI Appl Chem 10:41-45 ChinarasaE (1968) The preservation and bottlng of palm wine.

reported (Faparusi 1973; Okafor 1975 a, 1990).

are obtained by inoculating yeast

I

West African J BioI Appl Chem 6:20-25

Colorimetrc method for determination of sugars and related

encouraging, further studies are required before a

i

Bassir 0 (1962) Observations on the fermentation of palm wine.

Agu RC, Anyanwu TU, Onwumelu AB (1993) Use of highethanol-resistant yeast isolates from Nigerian palm wine in lager beer brewing. World J Microbiol Biotechnol 9:660-661 ,

Ande( P, Sonnessa A (1965) The rates of chemical reactionchemical kinetics: In : Principles of Chemistr, An Introduction

preservation of palm wine. J Appl Bacteriol 38: 1-7

Adv Appl Microbiol 24:237-256 Okafor N (1987) Industral Microbiology. 1st edn, University of Ife Press, Ile-Ife, Nigeria

OkaforN (1990) Traditional alcoholic beverages of tropical Africa: Strategies for scale-up. Process Biochem 27:213-230 Rokosu AA, Nwisienyi JJ (1980) Varation in component of palm'

wine during fermentation. J Enze Microbiol Technol 2:63-

65 The Institute of Brewing (1989)Recommended Methods of Analysis Van Pee M. Swing JG (1971) Chemical and microbiological

studies on congolese palm wine (Elaeis. guineesis) J East African Agric Foresty 36:311-314

White FH (1978) Ethanol tolerance of brewing yeasts. In: Proceedings of 15th Congress Soc Inst Brew, Australia and New Zealand Section. pp 133-146

to Theoretical Concepts. The MacMilan Co, New York. Collin-

McMilan Publishers, London. pp 656-585

i§;

l-!. Î~

I;

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Received 11 July 1997; revised 8 March 1999; accepted 16 April 1999