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MICROBIOTA EVALUATION OF FOUR-MONTH AGED PALM WINE (Elaeis guneensis) FROM OGBA UMUOSIGIDE, UDENU L.G.A. ENUGU STATE, NIGERIA

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Abstract

Aged palm wine is usually discarded by most people as it is considered to be spoilt because of its unpalatable acidic taste. Thus, the study was aimed at microbiota evaluating of four month aged palm wine (Elaeis guneensis). The sample of fresh palm wine was collected from traditional palm wine tapper from Ogba Umuosigide Obollo Orie and stored for 4 months. The physicochemical parameters were first assessed by the method of Association of Official Analytical Chemist (AOAC), while all microbiological analyses were carried out using Standard Microbiology methods. Only the predominant organisms that were identified by phenotypic and genotypic characterization were subjected to further studies. The results revealed a lower pH (4.12 ± 0.02) and sugar content (0.74mg/ml ± 0.01) with higher alcoholic content (35.83% ± 0.10) in aged palm wine than the fresh palm wine. A higher lactic acid bacterial and yeasts count (4.26 x 10 6 and 3.39 x 10 6 cfu/ml) were recovered from aged palm wine than fresh palm wine (5.1 x 10 5 and 2.56 x 10 6 cfu/ml). Coliforms, E. coli were present in fresh palm wine while they are absent in the aged palm wine. The identified organisms were Lactobacillus rhamnosus strain HUMBO7379 and Lactobacillus buchneri strain SS90 for bacteria and Kodamaea ohmeri strain PMN09-931L and Candida parapsilosis strain URM7235 for fungi using 16S rRNA and 1TS rRNA sequencing analysis.
International Journal of Advanced Academic Research | ISSN: 2488-9849
Vol. 7, Issue 6 (June, 2021) | www.ijaar.org
Journal DOI: 10.46654/ij.24889849
Article DOI: 10.46654/ij.24889849.e7631
1
MICROBIOTA EVALUATION OF FOUR-MONTH AGED
PALM WINE (Elaeis guneensis) FROM OGBA UMUOSIGIDE,
UDENU L.G.A. ENUGU STATE, NIGERIA
Odo, I. A.1 and George-Okafor, U. O.1
1Department of Applied Microbiology, Faculty of Applied Natural Sciences, Enugu State
University of Science and Technology, P. M. B. 01660, Enugu, Nigeria.
ABSTRACT
Aged palm wine is usually discarded by most people as it is considered to be spoilt because
of its unpalatable acidic taste. Thus, the study was aimed at microbiota evaluating of four
month aged palm wine (Elaeis guneensis). The sample of fresh palm wine was collected
from traditional palm wine tapper from Ogba Umuosigide Obollo Orie and stored for 4
months. The physicochemical parameters were first assessed by the method of Association of
Official Analytical Chemist (AOAC), while all microbiological analyses were carried out
using Standard Microbiology methods. Only the predominant organisms that were identified
by phenotypic and genotypic characterization were subjected to further studies. The results
revealed a lower pH (4.12 ± 0.02) and sugar content (0.74mg/ml ± 0.01) with higher
alcoholic content (35.83% ± 0.10) in aged palm wine than the fresh palm wine. A higher
lactic acid bacterial and yeasts count (4.26 x 106 and 3.39 x 106cfu/ml) were recovered from
aged palm wine than fresh palm wine (5.1 x 105 and 2.56 x 106cfu/ml). Coliforms, E. coli
were present in fresh palm wine while they are absent in the aged palm wine. The identified
organisms were Lactobacillus rhamnosus strain HUMBO7379 and Lactobacillus buchneri
strain SS90 for bacteria and Kodamaea ohmeri strain PMN09- 931L and Candida
parapsilosis strain URM7235 for fungi using 16S rRNA and 1TS rRNA sequencing analysis.
Key words: Elaeis guneensis, Aged palm wine, Bacteria, Yeasts.
International Journal of Advanced Academic Research | ISSN: 2488-9849
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Journal DOI: 10.46654/ij.24889849
Article DOI: 10.46654/ij.24889849.e7631
2
INTRODUCTION
Palm wine “Nkwu ocha” in Igbo, “Emu” in Yoruba and “Bammi” in Hausa is a cloudy,
whitish alcoholic beverage resulting from spontaneous fermentation of the sap of the Raphia
palm (Raphia hookeri) or oil palm (Elaeis guineesis). The fermentation has been attributed to
yeasts and bacteria (Onwuka, 2011; Opara et al., 2013). It is collected by tapping at the top of
the trunk after felling the palm tree and boring a hole into the trunk. It has a short shelf life of
only 24h (Ogbulie et al., 2007). The palm wine is consumed in a variety of flavours from
sweet unfermented to sour fermented and vinegary alcoholic drink (Chandrasekhar et al.,
2012). It is traditionally the major brand of wine in some parts of the southern part of Nigeria.
According to Oyeku et al. (2009), it consists mainly of water, sugar, vitamins, aroma and
flavor components in small amounts. In traditional African societies, the palm wine plays a
significant role in customary practices. For example, the distilled product of the palm wine is
a potent gin called by various names in West Africa (Amoa-Awua et al., 2006). Over ten
million people consume palm wine in West Africa and it is consumed by both men and
women including pregnant women (Onwuka, 2011). Pregnant women consume it fresh for
the sweetness and nutrition, while nursing mothers drink it warm to enhance breast milk
production (Eluwa et al., 2009). According to Onwuka (2011), palm wine also enhances
men‟s potency due to yeast cell concentration. Microorganisms such as yeast (Candida sp.,
Saccharomyces sp.) and bacteria (LAB, Micrococcus sp., Streptococcus sp., Pediococcus sp. and
Leuconostoc sp.) have been isolated from palm wine (Amoa- Awua et al., 2006; Santiago-Urbina
and Ruiz-Teran, 2014; Detto et al., 2016). Yeasts, LAB and AAB have been identified as the major
microorganism in the aged palm wine (Rita et al., 2017). The yeast contained in palm wine has
been reportedly used for industrial purposes such as baking and production of alcoholic
drinks or potable ethanol (Chandrasekhar et al., 2012; Nwachukwu et al., 2006; Chilaka et
al., 2010; Mbuagbaw and Noorduyn, 2012). Despite all these good qualities of palm wine, it
is a highly perishable sap due to fermentation which starts soon after the sap is collected and
within an hour or two becomes reasonably high in alcohol (up to 4%) (Detto et al., 2016). If
palm wine is allowed to continue to ferment for more than 24hrs, it starts to turn into vinegar.
This makes the wine unacceptable to consumers and serves as sources of economic losses to
palm wine tapper and sellers. It also creates losses to the food service industries.
Thus, there is need to explore its possible utilization for an economic values. Hence this study
is aimed at evaluating the microbiota of four- month aged palm wine (Elaeis guneensis).
MATERIALS AND METHODS
Source of Sample
The test palm wine from Oil palm (Elaeis guneensis) was collected from traditional palm
wine tapper from Ogba Umuosigide Obollo Orie in Udenu L. G. A. Enugu, Nigeria and
stored for four months.
Physicochemical Analysis
The pH, alcohol content and sugar content of the aged and fresh palm wine were determined
before the palm wine was subjected to microbiological analysis.
International Journal of Advanced Academic Research | ISSN: 2488-9849
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Journal DOI: 10.46654/ij.24889849
Article DOI: 10.46654/ij.24889849.e7631
3
Determination of pH of palm wine
The pH of aged and fresh palm wine was determined using a pH meter (Hanna, Mode Hi,
1950) after calibration using the standard buffer such as 6.86 and 9.86 (Amoa Awua et al.,
2007).
Determination of Sugar Contents
Method of Miller (1959) was adopted. Three milliliters of 3, 5-Dinitrosalicyclic acid (DNS)
reagent were added to 3ml of glucose standards and 3ml of sample separately in a lightly
capped test tube. Thereafter, the mixture was heated at 90oC for 15 minutes in a water bath to
develop the red-brown color. Then 1ml of 40% potassium sodium tartrate (Rochelle salt)
solution was added to stabilize the color and was allowed to cool at room temperature in a
cool water bath. The absorbance was recorded using Uv-vis spectrophotometer (725N,
Lemfield Medical England) with wavelength 575nm. From the reading obtained, a glucose
graph was used to estimate the values of the sugar content of the palm wine.
Determination of Alcohol Content
The alcohol content of fresh palm wine was determined using alcohol meter (model an iso
9001: 2000, JK hydrometer India as described by Washington (2000). The alcohol content of
aged palm wine was determined by subjecting the palm wine to several distillation processes.
After cooling, the distillate was dispensed into a 50cm3 measuring cylinder to obtain the
volume of the alcohol distilled.
Percentage of alcohol (%) = volume of distillate
volume of aged palm wine ×100
1
Microbiological Analysis
Isolation of Bacteria and Yeasts
The palm wine was homogenously mixed and 1ml of it was taken and tenfold serial dilution
up to 10-5 was made (Amoa-Awua et al., 2007). A 0.5ml of each dilution (up to 10-5) of the
palm wine was transferred aseptically onto sterile Petri dish. Lactic acid bacteria were
enumerated by pour plate on De Man Rogosa and Sharpe (MRS) agar containing 0.01g of
nystatin to suppress yeast growth and incubated anaerobically at 370C for 48h. Acetic acid
bacteria was enumerated through pour plate on Glucose Yeast Extract and Calcium
Carbonate (GYC) agar containing 0.00125g of penicillin and 0.01g of nystatin to suppress
bacteria and fungi growth and incubated at 370C for 48h.Yeasts were enumerated by pour
plate on Potato Dextrose agar (PDA) containing 0.1g of chloramphenicol to suppress bacteria
growth and incubated at 300C for 24h. The presence of coliforms was enumerated through
pour plate on MacConkey agar and Eosin Methylene Blue agar incubated at 370C for 48h.
Developed discrete colonies were counted using Colony counter prior to sub-culturing into
different fresh sterile media plates to obtain pure cultures. The grown cultures were later
subjected to identification.
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Journal DOI: 10.46654/ij.24889849
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Identification of the Isolates
Phenotypical Identification of Isolates
Isolates were identified using standard morphological characteristics and identification keys.
The tests used in the identification of bacteria include morphology, gram reaction,
biochemical test and sugar fermentation. The test used in the identification of yeast includes,
morphology, methylene blue staining, catalase test and sugar fermentation.
Molecular Characterization of Isolates
This was done using 16S rRNA and 1TS rRNA sequence method to identify the predominant
isolates.
Statistical Analysis
The parameters were evaluated using Analysis of Variance (ANOVA) to identify means that
are significantly different.
RESULTS
Table 1: Major Physicochemic and Parameters of the Palm Wine Samples
Type of Palm Wine Sample
Parameters Fresh Aged
(2hour) (4months)
pH 3.53 ± 0.01 2.94 ± 0.02
Sugar (mg/ml) 0.93 ± 0.10 0.22 ± 0.01
Alcohol (%) 0.00 ± 0.00 15.00 ± 0.10
The table shows physicochemical parameters of the palm wine samples. It shows that the pH
and sugar content were reduced while alcoholic content increased in aged palm wine than the
fresh palm wine and the difference were significant ( p ≤ 0.05).
International Journal of Advanced Academic Research | ISSN: 2488-9849
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Journal DOI: 10.46654/ij.24889849
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Table 2: Mean Total Microbial Loads Recovered from Fresh and Aged Palm Wine
Microbial Counts (cfu/ml)
Recovered
Microorganisms Media Used Fresh Palm Wine Aged Palm Wine
Yeast PDA 2.56×106 3.39×106
LAB MRS 5.1×105 4.26×10 6
AAB GYC - _
Coliform MacConkey 1.6×105 _
E. coli EMB 5.0×104 _
Key: LAB = Lactic Acid Bacterial, PDA = Potato Dextrose Agar, MRS = De Man Rogosa
and Sharpe agar, AAB = Acetic Acid Bacteria, EMB = Eosine Methylene Blue agar, GYC =
Glucose Yeast Extract and Calcium Carbonate, E. coli = Escherichia coli and - = Absent.
The table shows microbial loads recovered from fresh and aged palm wine. It shows that the
total lactic acid bacteria and yeasts counts were more in the aged palm wine than the fresh
palm wine. The differences of colony forming unit in aged palm wine as compared to fresh
palm wine were significant (p< 0.05). The coliforms recovered from fresh palm wine were
absent in aged palm wine. The acetic acid bacterial was not recovered from both of the palm
wine.
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Journal DOI: 10.46654/ij.24889849
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Table 3: Phenotypic Properties of Lactic Acid Bacterial Isolates from Aged Palm Wine
Colonial Appearance
On Sugar Fermentation
Isolates
MRS
Gram Reaction
Oxidase
Glucose
Galactose
Fructrose
Sucrose
Lactose
Maltose
D-Xylose
Suspected
Organisms
LS1
Milky, round, small, raised,
buttery, opaque, smooth
colonies
+ short rod in
chains/single
-
+
+
+
+
+
+
+
Lactobacillus sp1
LS2
Milky, round, raised,
medium, buttery, opaque,
smooth colonies
+ cocci in
clusters and pairs
-
+
+
+
+
_
_
+
Micrococcus sp
LS3
milky, round, raised,
medium, buttery, opaque,
smooth colonies
+ short rod in
chains
-
+
+
+
+
+
+
+
Lactobacillus sp2
LS4
Cream, round, small, raised,
buttery, opaque, smooth
colonies
+ cocci in chains
_
+
_
+
_
_
_
_
Streptococcus sp
LS5
Milky, round, small, raised,
buttery, opaque, smooth
colonies
+ cocci some in
pairs and some in
chains
_
+
+
+
+
-
+
+
Leuconostoc sp
Key: MRS= De Man Rogosa and Sharpe, += Positive, -= Negative, LS= Lactic acid bacterial isolates 1-5 and sp = species.
The table shows phenotypic properties of lactic acid bacteria isolates from aged palm wine. The organisms isolated were mostly
Lactobacillus species and their genotypic characterization indicated the species to be L .buchneri and L. rhamnsous.
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Table 4: Phenotypic Properties of Yeast Isolates from Aged Palm Wine
Colonial Appearance
On Sugar Fermentation
Isolates
PDA
Microscopic features
Catalase
Glucose
Galactose
Fructose
Sucrose
Lactose
Maltose
D- xylose
Suspected Organism
Y1
Cream, round, raised, medium,
opaque, butter, smooth colonies
Single oval cell
+
+
+
+
+
-
-
+
Saccharomyces sp1
Y2
Cream, round, raised, small,
opaque, butter, smooth colonies
Single elongated cell
+
+
+
+
+
-
-
-
Saccaromyces sp2
Y3
Cream, round, raised, medium,
opaque, butter, smooth colonies
Single oval cell
+
+
-
+
+
-
-
+
Candida sp
Y4
White, round, raised, Small,
opaque, dry, rough- colonies
Oval cell in chains
+
+
+
+
+
-
-
+
Schizsaccharomyce sp
Y5
Cream, round, raised, big,
opaque, butter, smooth colonies
Oval cell some in
single and some in
pairs with budded cell
+
+
+
+
+
-
-
+
Saccharomyces sp3
Y6
Cream, round, raised, medium,
opaque, butter, smooth colonies
Budded cell in single
+
+
+
+
+
-
-
+
Saccharomyces sp4
Key: PDA= Potato Dextrose agar, Y= Yeast isolate 1-6, + = Positive, - = Negative, sp= species
The table shows phenotypic properties of yeasts isolates from aged palm wine. The organisms isolated were mostly Saccharomyces
and Candida species and their genotypic characterization indicated their species to be Kodamaea ohmeri and Candida parapsolosis.
International Journal of Advanced Academic Research | ISSN: 2488-9849
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Journal DOI: 10.46654/ij.24889849
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DISCUSSION
The pH of fresh palm (5.80 ± 0.01) shows significant (p< 0.05) increase when compare with
aged palm wine (3.62 ± 0.02). The reduction in pH of aged palm wine indicates the increase in
acid concentration of the palm wine which could be due to the fermentation activities of lactic
acid bacteria in the aged palm wine. This result agreed with the findings of Schlundt, (2002)
which indicated that fermentation lowers the pH and makes the medium favorable for lactic acid
bacteria. High alcohol content (15%) of aged palm wine obtained after distillation is in line with
the reports from Detto et al. (2016), which stated that fermentation process of palm wine starts
soon after the sap is collected and within an hour or two hours becomes reasonably high in
alcohol (up to 4%). The sugar level of aged palm wine showed significant (p≥ 0.05) decrease
when compared with the fresh palm wine. This result agrees with the findings of Amoa Awua
et al. (2007), Ouoba et al. (2012) and Santiago Urbina et al, (2013) that revealed that sap
undergoes spontaneous fermentation which promotes the proliferation of yeasts and bacteria for
the conversion of the sweet substrate into several metabolites mainly ethanol, lactic acid and
acetic acid.
The microbial counts (cfu /ml) of yeasts and lactic acid bacteria in aged palm wine showed
significant (p< 0.05) increase when compared with the fresh palm wine (table 2). This is contrary
to the findings of Faparusi and Bassir, (1971) which showed that yeast and bacteria counts
decreased with increase in aging of palm wine but in line with the findings of Chilaka et al.
(2010), which indicated 3.7 to 4.8 log10 cfu/ml of viable yeasts that increased steadily with
increase in storage time to a maximum population range of 7.6 to 8.9 log10 cfu/ml after 24h. The
rate of multiplication of microbial cells in a medium is dependent on several factors which
include pH, temperature, water activity, nutrient, redox potential and antimicrobial agents. The
combination of one or more of these factors results into series of successions and subsequently,
predominance by yeast species in the palm wine samples. Lactic acid bacteria count also
increased significantly (p< 0.05) in aged palm wine and this may attributed to the spontaneous
sugar fermentation which is also a rich carbon source for the proliferation. Thus, storage
increased the lactic acid bacteria count after 24h.The presence of the coliforms and E. coli in the
fresh palm wine sample could be attributed to the poor hygienic conditions during the extraction
of the sap by producers. The result is similar to the report from Olawale et al. (2010) that
implicated water used in the extraction, dilution of palm wine and washing of the container as
possible source of contamination. Other implication sources of coliform contamination were the
environment and vectors such as insects that feed on sweet sap (Tapsoba et al., 2014). However,
the absence of coliforms and E. coli could be linked to the high alcoholic content and acidity of
the wine sample that were toxic to them. This can be confirmed with the previous findings of Obi
et al., 2015, Akinrotoye, 2014, Tiepma et al., 2013 and Santiago Urbina et al 2013 that
revealed that pathogens in the fresh sap of palm trees disappeared during the fermentation due to
the alcohol content and acidity (pH between 3 and 4) of the medium. It could also be due to
antimicrobial substances (bacteriocins, H2O2) produced by lactic acid bacteria (Tapsoba et al.,
2011 and Akinrotoye, 2014). The non- recovery of acetic acid bacteria could be due to ethanol
concentration, acetic acid concentration, oxygen, temperature and nutrient available in the palm
wine. This result agreed with the findings of Awad et al. (2011), who stated that bacteria cells
were affected by the concentration of alcohol.
International Journal of Advanced Academic Research | ISSN: 2488-9849
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Journal DOI: 10.46654/ij.24889849
Article DOI: 10.46654/ij.24889849.e7631
9
The microbiological analysis identified predominant microorganisms among 11 isolates as
Lactobacillus rhamnosus and Lactobacillus buchneri for bacteria and Kodamaea ohmeri and
Candida parapsilosis for yeast. This result agreed with the findings of Ouoba et al. (2012) which
indicated that Lactobacillus genus is responsible for the acidification of palm wine, which not
gives its sour taste but also increases its stability. These species could enable the protection of
palm wine against undesirable species. Aidoo et al. (2006) and Amoa-Awua et al. (2007)
reported that Saccharomyces cerevisiae are the most predominant yeasts but other yeasts such as
Kodamaea ohmeri, Schizosaccharomyces pombe, Candida tropiccalis and Pichia ohmeri are also
commonly detected in palm sap and wine which promote the fermentation of sugar to alcohol
which is in line with the work.
CONCLUSION
Palm wine and its distillate are important solvent in herbal medicinal administration. Pregnant
women consume it fresh for the sweetness and nutrition while nursing mothers drink it warm to
enhance breast milk production. In addition, majority of people drink palm wine during social
activities and other ceremonies. The presence of these organisms in the samples as identified in
the present study is of public health importance, if not properly handled. Therefore, there is need
to promote the quality of these products to enhance their nutritional and health benefits as widely
consumed in this parts of country.
The micro-organisms isolated from the aged palm wine can be useful industrially such as in
bread making, dairy production e.t.c.
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Journal DOI: 10.46654/ij.24889849
Article DOI: 10.46654/ij.24889849.e7631
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International Journal of Advanced Academic Research | ISSN: 2488-9849
Vol. 7, Issue 6 (June, 2021) | www.ijaar.org
Journal DOI: 10.46654/ij.24889849
Article DOI: 10.46654/ij.24889849.e7631
12
Washington, D. C. (2000). Official Methods of Analysis of the Association of Official Analytical
Chemists 15th edition volume 2: 739- 750.
... A vast microbiota is responsible for its attributes such as taste, ethanol content and aroma 3,4 . Various microorganisms of commercial importance have been isolated from the microbial pool present in palm wine 2,5 . The predominant microbes commonly isolated from palm wine microbiota community includes bacteria and fungi. ...
... The predominant microbes commonly isolated from palm wine microbiota community includes bacteria and fungi. The most common bacteria isolated include strains of the following family: Acetobacteriaceae, Lactobacillaceae and Leuconostocaceae, while the most frequently isolated yeast belong to the genera of Saccharomyces cerevisiae and Candida parapsilosis 2,5 . Strains of Saccharomyces cerevisiae are one of the most versatile cell factories applied for biotechnological applications including commercial enzyme production. ...
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