Evaluation of fermentation period on the proximate composition and tannin

Journal of Microbiology and Biotechnology Research
Scholars Research Library
J. Microbiol. Biotech. Res., 2014, 4 (1):21-27
(http://scholarsresearchlibrary.com/archive.html)
ISSN : 2231 –3168
CODEN (USA) : JMBRB4
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Available online at www.scholarsresearchlibrary.com
Evaluation of fermentation period on the proximate composition and tannin
concentration of sheanut (Vitellaria paradoxa) meal
Nelson Winston Agbo*
and Collins Doudu Prah
Department of Fisheries and Watershed Management, Kwame Nkrumah University of Science
and Technology, Kumasi, Ghana
_____________________________________________________________________________________________
ABSTRACT
This study evaluated the effect of fermentation period on the proximate composition and tannin concentration of
sheanut (Vitellaria paradoxa) meal. Sheanut meal (SNM) was taken through a fermentation process involving
oyster mushroom (Pleurotus ostreatus) to serve as a biocatalyst for 8 weeks. Proximate composition of SNM
followed standard protocol and tannin concentration was determined using quantitatitive test (stiasny number)
before and after fermentation. The results showed that the proximate composition of SNM improved during
fermentation. Crude protein and ash significantly (p<0.05) had a total increment of 50.71% and 197.46%
respectively whiles moisture content, crude fibre and crude lipid showed significant decrease with total reductions
of 46.88%, 25.62% and 18.36% respectively after 8 weeks of fermentation. Tannin content in the SNM decreased
significantly (p<0.05) with a total reduction of 72.99%. The increase in crude protein and ash in SNM and
reduction in crude fibre and tannin contents is indicative of improvement in the nutritional status of SNM. In this
study although the fermentation period was 8 weeks some components such as CP did not increase significantly
after the fifth week and CF and tannin content also did not decrease significantly after the second week. Therefore,
a minimum of 5 weeks of P. ostreatus fermentation could be considered as an optimum fermentation period to
significantly improve the proximate composition and reduce the tannin level of SNM.
Key words: Sheanut meal, fermentation, Pleurotus ostreatus, tannin, proximate composition.
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INTRODUCTION
The major reason for poor performance of animals in developing countries is the seasonal inadequacy of feed, both
in quantity and quality [1]. Inadequate nutrition is one of the major constraints limiting animal production in African
countries. Many feed resources that could have a major impact on livestock production continue to be unused,
undeveloped or poorly utilised. An important class of non-conventional feeds is by-product feedstuffs which are
obtained during harvesting or processing of a commodity in which human food or fibre is derived. According to
Agbo [2], feeds are mostly based on agricultural by-products available in an area and may be of modest quality but
of a reliable quantity.
Shea nut meal (SNM) is a by-product of shea butter and traditionally regarded as waste with no economic value and
its increasing output of late has become an environmental issue [3]. Some researchers have fed SNM based diets to
certain farm animals and have recorded low growth performance, digestibility and general reduction in feed intake
[4]. These observations were due to poor nutrient composition and possible nutrient utilization limiting factors in
SNM like saponins and most particularly tannins in the diets [5]. According to Miles and Chapman [6] the presence
of these components is thought to be contributing obstacles to efficient utilization of proteins in many economical

Nelson Winston Agbo and Collins Doudu Prah J. Microbiol. Biotech. Res., 2014, 4 (1):21-27
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22
plant-based feedstuffs. Wood and Fermor [7] reported that higher fungi or mushrooms have the ability to bio-
transform fibrous agro-residues into value-added products through their enzyme activities. These could also
counteract the anti-nutritive factors such as tannins and saponins in agro-residues to improve nutrient composition as
demonstrated through the use of Trichoderma viride [8], Rhizopus stolonifer [9], Pleurotus ostreatus [10] and
Volvariella volvacea [11]. Reddy and Pierson [12] reported that fermentation process can create conditions for the
growth of microbes that break down tannins. This study was conducted to investigate the effect of fermentation
period on the proximate composition and tannin concentration of sheanut meal in the quest of improving it for
animal feed.
MATERIALS AND METHODS
Test sample
Shea nut meal (SNM) (solvent oil-extraction method) the test sample was obtained from Ghana Nuts Ltd., an animal
feed and edible oil manufacturing company located in Techiman in the Brong-Ahafo Region, Ghana. It was then
solar-dried and milled into a fine powder using corn mill and stored in a polythene sack until needed for use.
Process of fermentation
The fermentation process followed the procedure described by Alemawor et al. [10] and Ecklu [13] by using oyster
mushroom (Pleurotus ostreatus). Oyster mushroom grain spawn used in this study was procured from a local
mushroom producer at Kenyase, a suburb of Kumasi, Ghana. The fermentation process started by taking 7kg of
dried sheanut meal (SNM) and mixing it thoroughly with clean water to a moisture content of about 60-70% in a
plastic container. The mixture was then covered with polythene sheet and then put in a dark room between 23-25ºC
for 3 days. The mixture was sprinkled with water each day and turned several times to maintain the moisture
content, and then autoclaved at a temperature of 121ºC for 15 minutes at a pressure of 242KPa, cooled to room
temperature before inoculating with oyster mushroom grain spawns. Six hundred and sixty grams (660g) of
mushroom grain spawns were inoculated into 7000g of autoclaved SNM and mixed thoroughly with clean water to
about 60% moisture content under aseptic conditions. Thirty transparent polythene bags were filled with 500g of the
inoculated SNM and put through the fermentation process (i.e incubation in a dark room between 23-25o
C to allow
for spawn running for eight weeks). The relative humidity of the room was kept at approximately 70% by wetting
the floor and walls of the room with water.
Prior to the fermentation process, the raw SNM was analysed for proximate composition and tannin concentration
and this was repeated during and after the fermentation process, which took eight weeks. Three samples were taken
randomly each week for proximate composition and tannin analyses. The randomly selected samples were dried in
an oven at a temperature of 600
C for 24 hours, ground using mortar and pestle into powder and sieved through 0.5
mesh before analyses.
Chemical analyses
Sheanut meal samples were analysed in triplicates for proximate composition using standard methods [14]. Moisture
content (MC) was determined by oven-drying at 110o
C for 24 h, crude protein (CP) by the Kjeldahl method, crude
lipid (CL) by the ether-extraction, crude fibre (CF) by boiling a defatted sample in 1.25% H2SO4 and 1.25% NaOH
for 12h and ashing by incinerating a weighed amount of sample at 5500
C for 4 hours. Nitrogen free extract was
calculated by subtracting the sum of MC, CP, CL, CF and ash from 100.
Tannin concentration of SNM was determined using quantitatitive test (stiasny number) as described by Darkwa and
Jetuah [15]. First of all, tannin was extracted from the SNM sample by weighing 2.00g of the dried sample into a
thimble and adding 150ml of 95% alcohol and placed into a Soxhlet extractor for 4 hours. After extraction the
solvent was transferred into a pre-weighed empty conical flask and the solvent distilled to near dryness and the flask
dried in an oven for 1 hour at 1050
C, cooled in a desiccator and weighed. The tannin extract then underwent a
qualitative test to determine the presence of tannin in the SNM extract by adding three drops of Iron (III) chloride
solution to about 15ml of the extract and the colour change observed. Concentration of tannin (stiasny number) was
determined by the addition of concentrated HCl and formaldehyde (1/2v/v) to the dried extracts to form a
precipitate. The precipitate formed was washed with hot distilled water, dried and weighed. The weight of the
precipitate over the original weight of extract expressed as percentage gives the Stiasny number (percent tannin).
Statistical procedure

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23
Statistical analyses in this study were conducted using GraphPad Prism 5 statistical software. Data were subject to
one-way ANOVA and Tukey’s Multiple Comparison Test applied to evaluate differences between means (P<0.05).
All data in this study are presented as means ± SD.
RESULTS AND DISCUSSION
Proximate composition of sheanut meal
When SNM was taken through fermentation over a period of eight weeks its proximate composition changed with
time. Crude protein revealed a significant increase (p<0.05) with fermentation period and ranged between 13.59-
20.03% which represent a 50.71% total increment after eight weeks of fermentation (Table 1, Figures 1).
Table 1 Proximate composition and tannin content (%) of sheanut meal after eight weeks of fermentation
Parameter Unfermented Fermented* % Increment % Reduction
MC 10.09 ± 0.05a
5.36 ± 0.17b
- 46.88
CP 13.59 ± 0.05a
20.03 ± 0.07b
50.71 -
Ash 1.18 ± 0.05a
3.52 ± 0.02b
197.46 -
CF 5.23 ± 0.02a
3.89 ± 0.04b
- 25.62
CL 10.13 ± 0.03a
8.27 ± 0.26b
- 18.36
NFE 59.79 ± 0.06a
58.94 ± 0.26a
- 1.41
Tannin 47.37 ± 0.01a
12.80 ± 0.14b
- 72.98
MC: Moisture Content, CP: Crude Protein, CF: Crude Fibre, CL: Crude Lipid, NFE: Nitrogen Free Extract, * Eight weeks of fermentation,
values are means ±SD (n=3). Values within the same row with different letters are significantly different (p<0.05).
Figure 1. Crude protein content of sheanut meal over eight weeks of fermentation
Crude protein content of SNM increased significantly (p<0.05) and correlated positively (r = 0.9537) with
fermentation period (Figure 1). One week of fermentation saw a major increase (30.2%) in protein content after
which there were small and gradual increases. However, after the fifth week there was no significant change till the
end of the eighth week. Studies conducted by Balagopalan [16], Leifa et al. [17] and Alemawor et al. [10] on fungal
fermentations of agro-residues i.e. coffee husk, cassava by-products and cocoa pod husk also reported similar
increase in protein content. The increase in protein in this study could be as a result of the bioconversion ability of
Pleurotus ostreatus (Oyster mushroom) of some of the soluble carbohydrates in the colonised substrate into mycelia
protein or single cell protein by the growing fungus [18] and could also be due to the release of polysaccharide
bound protein which makes the substrate nutritionally better [19].

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24
Ash content of SNM also revealed a significant increase (p<0.05) with fermentation period and ranged between
1.18-3.52% which represent a 197.46% total increment after eight weeks of fermentation (Table 1, Figures 2). A
more drastic increase was observed for the ash content of SNM (increased significantly (p<0.05) and correlated
positively (r = 0.984)) with fermentation period with an increase of 103.4% by the fourth week and a peak of
197.5% at the end of the eighth week.
The significant high increase in ash content may be due to contribution from the mycelia of the mushroom and
undeveloped mushroom grain spawn. Alemawor et al. [10] recorded ash content for fermented cocoa pod husk to
range between 91.0–134.0 g/kg after eight weeks of fermentation. They attributed the increase in ash to the decline
in some organic fractions during the bioconversion process [20]. The increase in ash content in this study is positive
since it is indicative of the fact that the fermented SNM has reasonable quantity of mineral elements.
Figure 2. Ash content of sheanut meal over eight weeks of fermentation
Crude fibre content of the SNM under fermentation in this study decreased significantly (p<0.05; r = 0.678) from
5.23% to 3.89% (Figure 3) by week 8 representing 25.62% total reduction (Table 1). An average reduction of
14.91% was obtained by the end of the second week with no significant changes after that. A general decrease in
CF content of palm kernel cake, saw dust, wheat bran, rice bran and wheat straw have been reported [11, 21]. In a
similar study by Lateef et al. [9] and Alemawor et al. [10] they reported 7.2% and 17.08% reduction of CF in cocoa
pod husk respectively. The reduction in CF may be an indication of P. ostreatus having enzymatic system for
degradation of polymeric lignocelluloses of SNM [10].
A sharp reduction of 17.97% in CL was observed within the first week of fermentation after which there was no
significant reduction for the rest of the period (Figure 4). The total reduction of CL at the end of fermentation was
18.36% (Table 1). The reduction may be attributed to fat use by the fungus (P. ostreatus) as an energy source.
Oliveira et al. [22] observed that crude lipid content of fungal-fermented whole rice bran decreased significantly and
may be as a result of lipid use by fungus, possibly in the synthesis of phospholipids constituents of the cell
membrane of fungal tissue.

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25
Figure 3. Crude fibre content of sheanut meal over eight weeks of fermentation
Figure 4. Crude lipid content of sheanut meal over eight weeks of fermentation
There was no significant (p<0.05) change in the NFE content of the sheanut meal during the fermentation period
(Table 1, Figure 5). This observation may be due to the oyster mushroom’s ability to utilize carbohydrate in the
production of cellulase and amylase [11].

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26
Figure 5. Nitrogen free extract (NFE) content of sheanut meal after eight weeks of fermentation
Figure 6. Tannin content of sheanut meal over eight weeks of fermentation
Tannin concentration in the shea nut meal
The tannin concentration of SNM during fermentation showed a reduction from 47.37% to 12.80% depicting a
significant (p<0.05) reduction of 72.98% (Table 1, Figure 5). However, an average reduction of 61.77% was
obtained by the end of the second week with no significant changes thereafter. In a research conducted by Alemawor
et al. [10] and Leifa et al. [17] to determine the tannin content of cocoa pod husk and coffee husk after Pleurotus
fermentation respectively, their result revealed that the tannin content of cocoa pod husk was reduced significantly

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27
(P < 0.05) by 93.28% and 79.19% in coffee husk. Dei et al. [3] also recorded lower tannin contents in fermented
SNM than in unfermented sheanut meal. These results are similar to what was achieved in this study which recorded
a significant 72.98% reduction in tannin between the unfermented and fermented SNM after 8 weeks of Pleurotus
fermentation.
CONCLUSION
Results from this study indicate that the composition of SNM improved through P. ostreatus fermentation. The
increase in crude protein and ash after fermentation as well as reduction in crude fibre contents and tannin could be
an indication of improved nutritional value of SNM. Reduction in crude fibre contents in particular could lead to
better digestibility and that of tannin could lead to improved palatability and therefore better feed intake and
utilization by animals. In this study although the fermentation period was 8 weeks some components such as CP did
not increase significantly after the fifth week and CF and tannin content also did not decrease significantly after the
second week. Therefore, a minimum of 5 weeks of P. ostreatus fermentation could be considered as an optimum
fermentation period to produce positive and significant changes in SNM composition, i.e. 38.6% and 138.14%
increment in CP and ash respectively; 19.69% reduction in CF and 70.4% reduction in tannin.
Acknowledgement
The authors are grateful to the entire staff of the Department of Fisheries and Watershed Management of the Kwame
Nkrumah University of Science and Technology for their immense contribution in terms of equipment.
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