EFFECT OF PRESERVATION WITH SACCOGLOTTZS GABONENSZS ON THE BIOCHEMISTRY AND SENSORY

ATTRIBUTES OF FERMENTING PALM WINE

PHILIPPA C. OJIMELUKWE

Micheal Okpara (Federal) University of Agricullure Umudike P.M.B 7267 Umuahia. Abia state

Nigeria

Received for Publication September 21, 2000 Accepted for Publication December 27, 2000

ABSTRACT

Fresh palm-wine, from Raphia hookeri was treated with different amounts of Saccoglottis gabonensis. The alcohol content, refractive index, assimilation of sugars and assimilation of nitrates were monitored for 4 days) in treated samples and untreated controls (containing no S . gabonensis). Fermentation rates were monitored by determining CO, production as well as pH and titratable acidity changes within the same period. Sensory attributes of fresh and treated palm wine were compared. Sensory evaluation studies were carried out on ( I ) The fresh palm wine, (2) Palm-wine stored for 4 days at ambient temperature (27C-29C) and (3) Palm-wine stored for 30 days at ambient temperature.

Treatment with S . gabonensis led to increased alcohol content and CO, production with lower sugar content (sucrose, glucose and maltose) and pH, than in control samples. There were no significant variations in the refractive indices of treated and untreated samples. CO, production decreased with each successive fermentation day in both treated and untreated samples.

Panelists preferred the sensory properties of fresh palm-wine to that of palm-wine treated with S. gabonensis. However, by the second day the sensory characteristics of treated palm-wine was preferred. Until the 4th day, the color and taste of treated palm-wine was acceptable while the untreated samples became unacceptable. By the 30th day both treated and untreated palm-wine samples became unacceptable.

Journal of Food Biochemistry 25 (2001) 411-424. All Rights Reserved. Vopyright 2001 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 411

412 P.C. OJIMELUKWE

INTRODUCTION

Palm-wine is an alcoholic beverage obtained by fermenting the sap of palm tree (Family, Palmae) (Anon 1966). Okafor (1972) defined palm-wine as a collective name for an assortment of alcoholic beverages prepared from fermented sap derived from various palms (Family Palmae). It is sometimes referred to as “toddy” (Corner 1966) and is drunk throughout the tropics (Van Pee and Swing 1971). The sap is a sweet clear syrup containing 10-12% sugar which is fermented by yeasts to produce alcohol. Ordinarily, palm wine is not considered acceptable after about 48 h from the time it was brought down from the tree. An incision is made towards the top of the palm tree to penetrate the phloem tissue and a receiving vessel is attached to collect the phloem exudates. Every 24 h or less, the exudates found in the receiving vessels are collected. In the traditional setting palm wine tapping instruments, especially receiving vessels are used repeatedly without cleaning the inner surfaces to remove the microbial deposits (Okafor 1978; Loumbe et al. 1985). Consequently there are slight variations in the flavor of palm-wine from different locations and the end products of the subsequent fermentation process (Rokosu and Nwisienyi 1980; Faparusi and Bassir 1982).

Rapid deterioration in organoleptic characteristics is observed shortly after the fresh exudate is obtained. This is the result of fermentation of endogenous sugars by the natural flora of the fermenting sap. High alcohol and acid contents render the palm-wine unacceptable within 24 h.

To prolong the shelf-life of fresh palm-sap, in some traditional settings the pulverized dust of the bitter bark tree (Saccoglottis gabonensis) is added to fresh palm-wine. In the present research the scientific merit of this practice was evaluated by comparing the biochemical changes in untreated and treated palm-wine.

MATERIALS AND METHODS

Preparation of Materials

Fresh palm-wine from Raphia hooken‘ was collected in previously unused sterile plastic containers. Arrangements were made with a local tapper who collected the samples at 6:OO am on each day of the experiment. The samples were used within 3 h of collection. Dried bark of S. gabonensis was obtained from the commercial stock in Aba Nigeria. It was first pounded in a clean mortar and finally ground to fine powder using a Wiley Mill (to pass 0.5 mm aperture).

EFFECT OF PRESERVATION WITH S. GABONENSIS 413

Determination of Alcohol Content

Two liters of fresh palm-wine were measured into fermentation vessels with slits in their covers to allow elimination of C 0 2 gas. Different amounts of S. gubonensis (0-lg/L) were added into each fermentation vessel, covered and mixed thoroughly. For the determination of alcohol content, 100 mL of palm wine was withdrawn from each sample, centrifuged at 3000 rpm for 10 min to remove particles of S. gubonensis and the supernate was added to 50 mL distilled water in a 500 mL distillation flask. About 100 mL of the sample was distilled off. Distilled water was used to dilute the distillate and a refractometer was used to measure its refractive index (Eil 7060) as described by Ranjahan and Krishan (1980). Triplicate determinations were made. Refractive indices of 1-10% ethanol, prepared in distilled water were measured and used to plot a standard graph from which the percentage alcohol contents (v/v) of palm-wine samples (which contained different amounts of S. gubonensis) were estimated.

Test for Assimilation of Glucose, Maltose and Sucrose

Sugar assimilation tests were carried out using the supernatant after centrifugation of palm-wine (at 3000 rpm for 10 min). Sugar assimilation tests were carried out on a daily basis for 5 days.

Palm-wine (0.01 mg) was applied to a 20 x 20 cm silica gel TLC plate (250 pm thick) (along side standard glucose, sucrose and maltose) using a sample applicator. The plates were developed in a solvent system of ethylace- tate/methanol/water (37:40:23, v/v/v), for 48 h. Developed plates were dipped in glucoamylase solution (prepared from 125 mg of glucoamylase) in 200 mL of cold acetone for 1 min and air-dried. The plates were incubated at 40C for 30 min, dried at lOOC for 5 min and soaked successively in

(a) 1 mL of saturated AgNO, in 200 mL acetone (b) 4 g NaOH in 10 mL of water and 190 mL of ethanol (c) 240 g of sodium thiosulfate, 25 g Na,SO, and 10 g sodium bisulfite

dissolved in 1 L of distilled water.

Plates were dried after each dipping and were finally immersed in water to remove the reagents before drying (Marero ef ul. 1990). The areas containing the sugars were removed and diluted with 5 mL distilled water. The amount of the various sugars present were determined spectrophotometrically according to the method of Mmegwa ef al. (1988) which is based on the reaction of 0.2% anthrone in concentrated H2S0, to give a blue green color. Standard graphs were calibrated using different concentrations of maltose, glucose and sucrose. Amounts of these sugars in the sample were then extrapolated from the standard graphs.

414 P.C. OJIMELUKW

Test for Assimilation of Nitrates

Test for the assimilation of nitrates was carried out on the supernatants from centrifuged samples using the Nitron method (Vogel 1971). Five mL, of each supernatant was mixed with 0.2 mL of 2N H2S0, and heated to nearly its boiling point. The sample was cooled for 2 h and 1.5 mL of Nitron reagent (5 g nitron in 50 mL of 5% acetic acid) was added and the mixture was filtered and washed. The precipitate on the filter paper was dried at 60C and weighed to obtain the value of assimilated nitrates.

Determination of Fermentation Rates

Rate of fermentation was monitored using the following methods:

Rate of C02 Production. Weight of C02 evolved was determined as the difference between the initial and final weights of the fermenting unit on a daily basis. This change in weight was monitored for fermenting palm-wine samples containing different concentrations of S. gabonensis.

Titratable Acidity. Five mL of each sample was titrated with 0.1 NaOH using phenolphthalein as end point indicator

%TTA = 0.1 x 100/5 mL where TTA = Total Titratable acidity. 1 mL of 0.1 NaOH is equivalent to Xmg TTA.

pH of Fermenting Samples. The pH of fermenting samples was deter- mined on daily basis using the pH meter (Electronic Instruments, model 7060).

Sensory Attributes of Fresh and Preserved Palm-Wine

Palm-wine were pitched in clean fermentation vessels with different concentrations of S. gabonensis. Sensory analysis was carried out on fresh and preserved palm wine on day 0, day 1, day 4, and day 30. The same samples that were used for other experiments were also used for sensory studies. The parameters evaluated were taste, aroma, color, and overall acceptability. They were ranked using a 7-point intensity scale where 0 = dislike extremely and 7 = like extremely. Fifteen experienced judges (people who had been drinking palm wine for at least 10 years) were used as panelists. Samples were presented at ambient temperature (27-29C) in a random manner in a properly ventilated and illuminated room. Samples were presented in glass tumblers. Distilled water (at room temperature) was provided to the panelists for the removal of residual flavors, while tasting the samples.

EFFECT OF PRESERVATION WITH S. GABONENSIS 415

Statistical Analysis

Analysis of variance was employed on the data, to determine the variations among the treatments. Duncan’s multiple range test was used to separate the means at 5% level (Steel and Torrie 1980).

RESULTS AND DISCUSSION

Figure 1 shows the change in alcoholic content of treated palm wine and controls within a 4-day storage period. There was an increase in the alcohol content of preserved and unpreserved palm-wine up to the fourth day after which the alcohol content decreased, possibly due to the utilization of alcohol for the production of vinegar. The alcohol content of samples containing > 490-510 S. gabonensis were generally higher than the alcohol content of unpreserved samples. There was no significant difference (p 50.05) in the refractive indices of both fresh and treated palm-wine samples. There was also no significant difference (p ~ 0 . 0 5 ) in the alcohol content of palm-wine samples preserved with 0.125 - 0.5 g/L S. gabonensis. This might imply that yeast fermentation to produce alcohol was affected in the same manner by these levels of additive (S. gabonensis). Yeast fermentations (evaluated in terms of alcohol production) with pure sugar substrates are known to take a different pattern from such fermenta- tions in a heterogenous system (Delegenes et al.1990; Du Preez and Prior 1985).

Sugar Assimilation Test

Table 1 shows data from sugar assimilation tests. Sugar quantities decreased more in samples containing S. gabonensis than in unpreserved samples. Amounts of sucrose, maltose and glucose were lowest in palm-wine samples preserved with 0.625-1 .OO S. gabonensis. Sucrose assimilation was the fastest, followed by glucose and maltose utilization.

Nitrites

Table 2 presents data on the assimilation of nitrites during fermentation. Untreated samples retained higher amounts of nitrites than samples containing > 0.5% S. gabonensis. Nitrate assimilation was therefore higher in treated samples than in controls. Levels of nitrate decreased as the storage period increased. Nitrates are required by fermenting microorganisms particularly the yeasts. Higher assimilation of nitrates may imply a higher metabolic rate and higher activity by fermenting yeasts.

416

6

5

4

- 0 c 0 2 3 a s

2

1

0

P.C. OJIMELUKW

.I*-*-* /

.-.-.-.-.-.-.-.-.

0 5 10

% Sgabonensis powder (x lo-’) FZq

-0- Day3 --+::+--- Day4 I//

FIG. 1 . ALCOHOL CONTENT OF PALM WINE PITCHED WITH VARYING AMOUNTS OF S. GABONENSIS

EFFECT OF PRESERVATION WITH S. GABONENSIS 417

TABLE 1 . EFFECT OF PRESERVATION OF PALM-WINE W I M SASCCOGLO7TIS GABONENSIS ON ASSIMLLATION

OF SUGARS

Concentration of Suecoglottis gabonensis (g/100ml palm-wine)

0 0.125 0.250 0.375 0.500 0.675 0.750 0.850 1.00

Day 0 (Fresh sample)

Day 1

Day 2

Day 3

Day 4

% Sucrose 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 %Maltose 0.50 0.50 0.60 0.50 0.50 0.50 0.50 0.50 0.50 %Glucose 1.92 1.92 1.92 1.92 1.92 1.92 1.92 1.92 1.92

%Sucrose 2.13 1.93 1.73 1.60 1.40 1.13 1.13 1.47 1.40 %Maltose 0.35 0.35 0.35 0.35 0.35 0.30 0.35 0.35 0.35 %Glucose 1.50 1.50 1.33 1.25 1.25 1.08 1.25 1.42 1.50

%Sucrose 1 13 087 067 040 033 027 040 047 0 6 0 %Maltose 035 035 035 030 020 020 020 036 035 %Glucose 075 068 055 055 055 038 055 068 075

%Sucrose 0.60 0.40 0.33 0.27 0.13 0.70 0.20 2.70 0.40 %Maltose 0.25 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 YO Glucose 0.55 0.55 0.55 0.55 0.30 0.30 0.30 0.47 0.55

YO Sucrose 0.40 0.27 0.27 0.20 0.00 0.00 0.00 0.70 0.20 %Maltose 0.20 0.20 0.20 0.20 0.00 0.00 0.00 0.00 0.00 %Glucose 0.55 0.47 0.30 0.30 0.30 0.00 0.00 0.30 0.30

Effect of Treatment with S. gabonensis on the Fermentation Rate

Evolution of CO,. Figure 2 shows the % CO, evolved from treated and untreated palm wine samples. Within a fermentation period of 24 h, 1.8-2.9% CO, was evolved from the samples. More CO, was evolved from palm wine samples containing S. gabonensis than from untreated palm wine samples. By the second to the fourth day of fermentation, the reverse was the case. There was more CO, evolution from the control than from the treated samples.

418 P.C. OJIMELUKWE

Generally, evolution of COz reduced with increased fermentation period as expected. Within 3 days, oxidative yeasts and species of Acetobacter oxidize the ethanol produced by natural palm wine fermentation into vinegar (Uzochukwu ef al. 1991).

TABLE 2. ASSIM~ATION OF NITRATES IN PALM-WINE SAMPLES PI'TCIED WITH DIFFERENT

CONCENTRATIONS OF SACCOGLOlTIS GABONENSIS DURING STORAGE

Concentration of Saccoglortis gabonensis (g/100ml palm-wine)

0 0.125 0.250 0.375 0.500 0.625 0.750 0.875 1.00

Day 0 (fresh 1.69 1.69 1.69 1.69 2.69 1.69 1.69 1.69 1.69 Sample) %Nitrate 39.02 +0.02 M.02 M.02 M.02 f0.02 M.02 M.02 M.02

Day 1

Day 2

Day 3

Day 4

1.03 0.99 0.95 0.89 0.80 0.73 0.74 0.76 0.79 %Nitrate M.01 M.00 M.00 M.01 M.O1 10.01 M.01 i-O.01 fl.011

0.85 0.81 0.90 0.85 0.75 0.65 0.69 0.73 0.76 %Nitrate M.01 M.01 M.01 M.01 M.01 M.01 M.01 10.01 M.01

0.79 0.75 0.69 0.62 0.57 0.40 0.44 0.47 0.41 %Nitrate M.01 M.01 M.01 fO.OO M.01 M.00 M.00 M.01 M.O1

0.61 0.52 0.50 0.43 0.41 0.35 0.37 0.39 0.43 %Nitrate M.01 10.02 M.01 M.01 39.01 M.01 M.O1 M.01 B.01

Means and standard deviation values are shown in the Table.

Titratable Acidity. Figure 3 shows the changes in titratable acidity of treated and untreated samples as storage/ferrnentation period increased. There was an increase in the titratable acidity of the control as fermentation period increased (0.9-2.5 within 4 days). The titratable acidity of treated samples only showed a slight increase within this period (Fig. 3). Observed results indicate that S. gabonensis retards this process.

EFFECT OF PRESERVATION WITH S. GABONENSIS

1.00

0,90

0,80

0.70

-u 1 0.60 P w 0) 'D-

j 5 030

f '0

0.40 s

0.30

0.20

0.10

0,oo 0 0.125 0.25

1 1 1 0375 0 5 0625

% S. gabonensis

ODay 1 Day 2

0 Day 3

0.75 1

0.875 1

419

1

FIG. 2. EVOLUTION OF CARBON DIOXIDE FROM PALM WINE PITCHED WITH VARYING AMOUNTS OF S. GABONENSIS

At each concentration of S. gabonensis, bars from left to right are for 1 , 2, 3 and 4 days. The values for 1 day were all greater than 1 %.

420 P.C. OJIMELUKWE

\> \

\\+ .-.-.-. 6 ) I) t* 0 t* -.-. -.-.--.-.-.-. \ \

~

0 0-5 I 1 3

Amount of S.gabonensis (g/l)

. - - I --.---Day2

-0- Day3

FIG. 3. TITRATABLE ACIDITY OF PALM WINE TREATED WITH DIFFERENT AMOUNTS OF S. GABONENSIS

EFFECT OF PRESERVATION WITH S. GABONENSIS 42 1

8 -

7 -

6 -

5 -

5 4 -

pH. Figure 4 shows the pH changes for the treated and untreated palm wine samples during a 4-day period of storage. Decrease in pH was higher for palm wine samples, which were not treated with S. gabonensis. As the quantity of S. gabonensis used to preserve palm wine increased, the change in pH became less. There were no significant differences (p 2 0.05) in the pH of palm wine samples treated with 2 0.06% S. gabonensis. The drop in pH observed in the present study was not as sharp as was observed by Rokosu and Nwisienyi

3 -

2 -

1 -

04 1

0 2 4 6 a 10

K S.gabonensis

. . ... . - Day4 -a- Day2 -0- Day3 -::- Day4

FIG. 4. pH CHANGES IN PALM WINE STORED WITH VARYING AMOUNTS OF S. GABONENSIS

Sensory Properties of Palm Wine

Table 3 shows the sensory characteristics of (1) fresh palm wine, (2) palm wine stored for 4 days, and (3) palm wine stored for 1 month at ambient temperature (27-29C). The taste and color of fresh palm wine was preferred to

TAB

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EFFECT OF PRESERVATION WITH S. GABONENSIS 423

that treated with S. gabonensis. The aroma of treated and untreated palm wine samples was the same. After 24 h the taste and aroma of palm wine treated with 2 0.06% S . gabonensis was preferred by panelists. Treatment with 0.5-0.6% S. gabonensis gave the best organoleptic properties. By the fourth day, untreated palm wine was completely unacceptable in terms of the sensory parameters that were evaluated. However, palm wine treated with > 0.06% still retained 2 60% of the initial aroma, 2 80% of the original taste and 2 60% of the general acceptability (in terms of ratings by panelists). There was no notable color change. After 30 days of storage, both treated and untreated palm wine became unacceptable.

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CORNER, E.J.A. 1966. The Natural History of Palm, (Wiedenfeld and Nicholson, eds.) University of California Press, California.

DELEGENES, J.P., MOLETT, R. and NAVAMO, J.M. 1990. Acid hydrolysis of wheat straw and process consideration for ethanol fermentation by Pichia stipites YT124. Proc. Biochem. 25, 132-135.

DU PREEZ, J.C. and PRIOR, B.A. 1985. A quantitative screening of some xylose fermenting yeast isolates. Biotechnol. Lett. 7, 241-246.

FAPARUSI, S.I. and BASSIR, 0. 1982. Factors affecting the quality of Palm-wine (ii); period of storage. West African J. Biological Appl. Chem.

LOUMBE, D.S., KABAWILA, C., MALOAGA, M., BAYEADA, J.P. and MANDA, V.G. 1985. Etude preliminaire dela microbiologie et de biochemie du van de palm-elaeos guineensis. A paper presented at a Conference organized by the Intern. Found. for Sci. and the United Nations University Japan in Douala, Cameroon.

MARERO, L., PAGAMO, E.M., AGUINALDO, A.R. and HOMMA, S . 1990. Maltooligosaccharide composition of flours, weaning foods and gruels prepared from germinated rice corn mungbean and cowpea. J. Nutr. Sci. Vitaminol. 36, 55-64.

MMEGWA, S.V.A., BALOGH, E. and NGODDY, P.O. 1998. Fermented palm wine. Comparative studies. Nigerian Food J. 6 , 36-43.

OKAFOR, N. 1972. Palm-wine yeast from Nigeria. J. Sci. Food Agric. 23,

OKAFOR, N. 1978. Microbiology and chemistry of oil palm-wine. Adv. Appl.

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1399-1407.

Microbiol. 24, 273-256.

424 P.C. OJIMELUKW

RANJAHAN, S.K. and KRISHNAN, C. 1980. Laboratory manual for Nutrition Research, pp. 11 1-1 12, Vika Publisher Intern., New Delhi.

ROKOSU, A.A. and NWISIENYI, J.H. 1980. Variations in the components of Palm-wine during fermentation. Enzyme Microbial Technol. 2, 63-65.

STEEL, R.G.D. and TORRIE, J.H. 1980. The Principle and Procedures of Statistics. McGraw Hill, New York.

UZOCHUKWU, S.V.A., BALOGH, E. and NGODDY, P.O. 1991. Standard pure culture innoculum for natural and fermented palm sap fermentation. Nigerian Food J. 9, 67-77.

VAN PEE, W. and SWING, J.G. 1971. Chemical and Microbiological studies on Congolese palm wine (elueis guineensis). East African Agric. Forestry J. 36, 311.

VOGEL, A.L. 1971. Principles of Ejcperimental Chemistry, pp. 1088, Heinmon Publisher , London.