Analysis of the Catalytic Behavior of Osyris Alba Bark and Indigofera Amabelacensis Leaves Extract in Ethanoic and Lactic Fermentation
Gitonga Maxwell, Osano Aloys and Bakari Chaka*
1Department of Mathematics and Physical Sciences, Maasai mara university, Kenya
2Department of Mathematics and Physical Sciences, Maasai mara university, Kenya
3Department of Mathematics and Physical Sciences, Maasai mara university, Kenya
Received Date: 14/11/2020; Published Date: 11/12/2020
*Corresponding author: Bakari Chaka, Department of Mathematics and Physical Sciences, Maasai mara university, Kenya. P.O Box 861-20500 Narok, Kenya
Cite this article: Gitonga Maxwell, Osano Aloys and Bakari Chaka*. Analysis of the Catalytic Behavior of Osyris Alba Bark and Indigofera Amabelacensis Leaves Extract in Ethanoic and Lactic Fermentation. Op Acc J Bio Sci & Res 6(2)-2020.
Fermentation process naturally occur spontaneously though the process is quite slow. To optimize on fermentation process, high temperature systems and enzymes are used. However, these processes are quite expensive and lead to increased production costs. This is notwithstanding the importance attached to fermentation process in food, diary, baking, brewery, pharmaceutical, chemicals and biofuels synthesis. Osyris alba bark and Indigofera amabelacensis leave extracts were traditionally used to hasten saccharification and fermentation of biomass during preparation of communal alcohol. This study aimed at exploiting the potentials of these two extracts in catalyzing ethanoic and lactic fermentation at ambient temperature conditions. The extracts were fused into maize and sorghum flour (ethanoic) and milk (lactic fermentation) in 1.5-liter batch reactors. The reactors were monitored for 30 days while closely assessing the amount of CO2 gas produced. The products were qualitatively screened by Iodoform and Benedicts methods as well as infra-red spectroscopy. The products were quantitatively analyzed by ultraviolet-visible (UV-VIS) spectroscopy and gas chromatography (GC) techniques. The results indicated that the catalyzed samples generated 3-to-5-folds more CO2 gas compared to the control samples. These findings were supported by the qualitative tests done which showed the catalyzed samples began producing ethanol and lactic acid before the control samples. The catalyzed lactic fermentation setup produced more lactic acid (2.79μg/L) compared to the control one (1.48μg/L). Catalyzed ethanoic fermentation setups yielded 11.55% (sorghum) and 2.16% total ethanol while the control samples had negligible ethanol concentrations at similar conditions.
Keywords: Fermentation; Bio-catalysts; Osyris alba; Indigofera amabelacensis; Ethanol; Lactic acid
Fermentation is process by which carbohydrate (glucose, starch and sugar) is used up in absence of oxygen by an organism. There are many products which results from the fermentation process and include; ethanol, lactic acid, butyric acid, acetone, carbon dioxide and hydrogen . Fermentation is a vital domestic and industrial process responsible for food preparation as well as preparation of drugs and pharmaceuticals, enzymes, biofuels and food products. Fermentation process is exclusively bio-chemical thus primarily controlled by enzymes and other factors affecting enzymes . The process has been widely studied with regards to generating chemical and industrial products as well as environmental clean-up.
There are two types of fermentation; ethanol (alcoholic) and lactic acid fermentation. Ethanoic fermentation primarily occur in a single route to give ethanol and carbon dioxide as the major products. Lactic acid fermentation takes place in two ways, homolactic fermentation (which produces lactic acid exclusively) and heterolactic fermentation (which involves production of lactic acid, other acids and alcohols . In homolactic fermentation the pyruvate formed during glycolysis undergoes simple redox reaction and it forms lactic acid . This happens inside the muscles of animals when the affinity for oxygen is high and the blood supply might not meet the requirement.
During fermentation process, glycolysis of glucose and other biomass take place in an anoxia environment to yield pyruvate . The process occurs in the cytosol and is powered by adenosine diphosphate (ADP) molecules. The pyruvate molecule formed is decarboxylated to acetaldehyde before conversion to ethanol in ethanolic fermentation . In the process, nicotinamide adenine dinucleotide hydrogenase, NADH is reconverted back to nicotinamide adenine dinucleotide ion, NAD+ molecules. The homolactic (lactic fermentation) process, pyruvate formed is directly converted to lactate ions (deprotonated lactic acid).
Fermentation chemistry generally involves recycling NADH to NAD+. NAD+ is molecule that is used in one of the glycolysis steps to produce energy . NAD+ is used for the conversion of glyceraldehyde-3-phosphate to 1, 3-biphosphoglycerate. This happens in the cytoplasm of the cell. NAD+ is recycled because it is used and one of the ways used to recycle is the electron chain transport. In aerobic organisms the terminal electron acceptor is oxygen but in anaerobic organisms the terminal electron acceptor can vary from species to species and include some of the metal species like Fe, Mn, Co, CO2, nitrates and sulfur . Methanogenic bacteria use CO2 as the electron acceptors reducing the precursor molecule (acetaldehyde) to methane gas . This leads to formation of NAD+ to NADH through a reduction and then it’s ready for the redox reaction in the sixth step of glycolysis .
Fermentation process can be optimized by providing appropriate conditions and use of catalysts (enzymes). High temperature is one of the vital conditions required in fermentation and increase the energy costs of the process. Enzymes are thus seen as a cheaper means to optimize the process. There are several types of enzymes used in fermentation at various stages. Hydrolysis and liquefaction of biomass substrate to short dextrin molecules require alpha amylases . Saccharification of the resultant starch molecules and dextrins to fermentable sugar require glucoamylase enzymes . Protease enzymes are added to provide nutrients for growth of yeast while beta glucoase and pentosanase enzymes reduce the viscosity of the broth for faster fermentation .
Several plant extracts have been characterized to comprise of single or multiple of fermentation enzymes. Osyris alba barks and Indigofera amabelacensis leaves are such extracts common amongst the Aembu community of central Kenya. The extracts were used to hasten saccharification and fermentation of biomass during communal preparation of alcohol and porridge (Indigofera amabelacensis leaves) and sour milk (Osyris alba bark). This study aimed at analyzing the catalytic potential of these extracts in ethanoic fermentation of maize and sorghum and lactic fermentation of milk.
Materials and Methods
1.1. Experimental Design
Osyris alba barks and Indigofera amabelacensis leave extracts were obtained by maceration of the crudes. The extracts were added onto three substrates comprising of milk (for lactic fermentation) and sorghum and maize flour (for ethanolic fermentation). Batch experiments were conducted in 1.5-liter reactors for a 28-day retention period with the amount of gas formed being monitored. A total of 6 experimental set-ups for each of the three substrates plus their controls were used. The products of the fermentation processes were thereafter assessed qualitatively by fourier transform infra-red spectroscopy (FT-IR), UV-VIS, iodoform and Benedicts test and quantitatively by UV-VIS (lactic acid) and gas chromatography with a flame ionization detector, GC-FID (ethanol).
All chemicals and reagents used were analytical grade and were sourced from Sigma-Aldrich company. The FT-IR model was Shimadzu while the GC-FID model used was also Shimadzu. Jasco 1800 UV-VIS spectrometer was used for characterization of the absorption bands of the products of fermentation process.
1.3. Preparation of Bio-Catalytic Extracts
Osyris alba barks were ground to fine powder using a grinding machine. Indigofera amabelacensis leaves were macerated to yield crude sap extracts. The catalysts were then stored at 4 °C to inactivate any enzymes present before dosing into the fermentation substrate.
1.4. Monitoring of the Fermentation Process
All the six bio-digesters were fed with the respective substrate. The digesters were filled up to the 1-liter mark leaving the other part (one third) free for gas collection. The substrates had 80% water content. The digesters were maintained at room temperature ranging between 20-25°C in an unstirred environment. The volume of gas produced was closely monitored and the products of the fermentation process were analyzed after the retention period.
1.5. Qualitative Analysis of the Fermentation Products
The fermentation substrate was tested for presence of acetaldehydes and ethanol by iodoform test after every 7 days. The lactic fermentation substrate was also subjected to Benedicts test in a similar range of time. The products were also characterized by FT-IR and UV-VIS spectroscopy.
1.6. Quantitative Analysis of Lactic Acid by Uv-Vis Spectrometry
Lactic acid content in the substrate was determined by checking the absorbance at 340nm. The samples were prepared by dissolving 10.g of the substrate solutions into 60.0ml distilled water followed by addition of 2.0ml of potassium hexacyanoferrate (ii) (3.6g/100ml distilled water). After stirring, 2.0ml of zinc sulphate (7.2g/100ml distilled water) and 4.0 ml sodium hydroxide (100mM) were added. The mixture was again re-agitated before adjusting the volume to 100.0ml solution using distilled water. A clear solution was obtained by filtering this mixture using Whatman number 41 filter papers. A 1.0 ml aliquot sample was used for analysis. Distilled water was used as the blank while l-lactic acid solution (0.15mg/ml) was used as the standard solution. The formula below was used to calculate the concentrations of lactic acid in the samples;
C(lactic acid) = 0.3204 x ΔAL-lactic acid (1)
1.7. Quantitative Analysis of Ethanol by Gc-Fid
The substrates undergoing ethanolic fermentation were analyzed for ethanol content after every seven retention days. The substrates were subjected to fractional distillation prior to analysis to remove traces of water vapor present. The following chromatographic conditions were maintained; Headspace Wait Sample temperature: 60°C, Loop fill time: 0.15 min Cycle time: 12 min, GC Inlet: 60°C, split less mode. Helium carrier gas flow rate: 3 mL/min, constant flow mode, Oven temperature program: 35 °C for 2 min, then 25°C/min to 90°C with a final hold time of 4.3 min. FID temperature 250°C, FID Hydrogen flow rate: 40 mL/min Air flow rate: 450 mL/min.
1.8. Data Analysis
Data obtained from analyses of the experiments was evaluated at 8 degrees of freedom, at 95% confidence level. Ms Excel and Originlab statistical softwares were used.
Results and Discussions
Quantitative Analysis of Gas Produced during Fermentation Process
The samples containing Osyris alba and Indigofera amabelacensis additives were found to produce more carbon dioxide gas compared to their control setups. The gas was found to precipitate lime water; confirming its identity as CO2 gas. CO2 levels in the catalytic samples differed by about 5-folds in ethanoic fermentation using sorghum substrate as illustrated in (Figure 1).
From (Figure 1), CO2 generation increased exponentially in the samples with Indigofera amabelacensis over the retention period. Generation of CO2 gas is a direct indicator of progressive fermentation process . This is affirmed by the reduced volume of CO2 gas generated by the fermentation setups of maize compared to that of sorghum substrate. (Figure 2) illustrates the production of CO2 gas during ethanoic fermentation of maize substrate. From the figure, the net CO2 generated by the end of the retention period was 3.0ml for the control setup and 10.0ml for the sample with Indigofera amabelacensis additive. This was about one-third the amount of gas generated by fermentation of sorghum. The disparity in CO2 formation lies in the composition of the two substrates. Diether and Willing  explains that fermentation bacteria and enzymes require proteins to grow and develop; which is scarce in maize. Sorghum has a larger net protein composition compared to maize , which has an effect on the general kinetics of ethanoic fermentation. Use of the additives can therefore be concluded to have a positive effect on ethanoic fermentation kinetics. Inclusion of the bio-catalytic additives into the substrates provide a matrix of enzymes, trace metals and nutritional composition that boost growth of fermentation micro-organisms .
The end product of glycolysis is pyruvate molecule which is converted to acetaldehyde in the process yielding CO2 gas as a by-product . More CO2 gas is generated in conversion of the acetaldehyde to ethanol. The production of CO2 gas is therefore proportional to the rate of fermentation process taking place. Lactic fermentation process also yield CO2 gas as illustrated by (Figure 3).
From (Figure 3), use of Osyris alba additives in the fermentation of lactose accelerated the formation of products by three-folds. This is attributable to addition of possible enzymes, trace metals, conjugated organic compounds in the extracts which aid in hydrolysis of the biomass, its saccharification as well as fermentation. Pervez et al.,  found out the roots of Osyris lanceolata (genetically related to O. alba) to have abundant amylase enzymes responsible for saccharification of biomass. The average rate of CO2 generation for the lactic fermentation process was by far much higher than that of ethanolic fermentation (P > 0.05, n = 8).
Figure 1: Variation in production of CO2 gas in ethanoic fermentation using sorghum substrate.
Figure 2: Variation in CO2 generation between a control and sample with additive in ethanoic fermentation using maize substrate.
Figure 3: Variation in CO2 production between the substrate with Osyris alba and a control sample in lactic fermentation process.
Qualitative Test for Ethanol and Lactic Acid
Presence of ethanol and lactic acid in the samples was confirmed by Iodoform and Benedict’s test as shown in (Table 1). All the samples with additives showed the potential to exhibit presence of the test fermentation products at a quicker rate compared to their control counterparts. The maize setups were however slower compared to the other samples and could only exhibit presence of ethanol after 14 retention days. This sample produced extensive amounts of ethanol only after 28 retention days. This is quite slow compared to Graham  who describes that the primary fermentation process should take about five days at the right temperature and in presence of yeast. Ethanolic fermentation of sorghum yielded products quickly with the first fermentation phase potentially ending after 28 retention days in the sample with a catalyst. From the table, primary ethanolic fermentation occurred between the 7th and 21st retention day in the additive sample. The control setup for the sorghum substrate took a longer period (i.e up to the 28th day) for this phase to conclude. The products of ethanolic and lactic fermentation for the samples with additives reduced on the 28th retention day. This is because primary fermentation accounts for about 70% of the fermentation products while secondary fermentation yield less products. Lactic fermentation instantly produced lactic acid with the sample containing O. alba additives exhibiting extensive amounts of the product after only 7 retention days. Such concentrations were only possible after 21 retention days in the sample without O. alba additive. Ethanol and lactic acid from the experimental setups were confirmed by FT-IR spectroscopy as illustrated in (Figure 4).
From the above spectra, there was presence of carboxylic and alcoholic OH groups as well as C=O and C-O-H peaks in both samples undergoing lactic fermentation. These groups confirm presence of organic acids in the sample. However, the spectra of the sample with Osyris alba additive had steeper peaks compared to the control sample. Doublet alcoholic OH groups were also visible resulting from the germinal (α)-OH position and β-OH position of lactic acid structure. There was concise resemblance in the FT-IR spectra of the samples containing ethanol. All samples exhibited a shallow peak at around 3800cm-1, probably due to water in the samples. The samples also had a sharp doublet peak (except the control setup of sorghum substrate) at around 3000cm-1, resulting from alcoholic OH groups. The samples from sorghum substrate had peaks between 2200-2600cm-1 due to C ≡N- groups resulting from amino groups found in sorghum.
Table 1: Qualitative test for ethanol and lactic acid in the fermentation products.
Figure 4: FT-IR spectra of the products of ethanolic and lactic acid fermentation processes.
Quantitative Test for Lactic Acid
Indigofera amabelacensis extracts were found to increase the concentrations of lactic acid in milk substrate fermented. Lactic acid concentrations linearly increased from 0.32μg/L at the beginning to 2.79μg/L at the end of the fermentation process. This is a larger margin compared to that of the control sample which increased from 0.42μg/L to 1.48μg/L within the same duration. The increment of the concentrations was almost perfectly linearly correlated with the retention period. The change in concentration of lactic acid in the homolactic fermentation setup is illustrated in (Figure 5).
The slope of the trendline with a catalyst (0.088) was twice that of its control (0.0406). This implies that fermentation process with Osyris alba additives occur twice that without the additives. The concentration of lactic acid in the sample with a catalyst (i.e 2.79μg/L) is one of the highest possible concentrations of lactic acid in a milk sample . According to Kuhl and Lindner, , the concentration of lactic acid in milk and other dairy products range between 0.3-3.0μg/L. This positions I. amabelacensis extracts as a prime candidate for hastening homolactic fermentation process. Continuous generation of CO2 gas in the setup (Figure 3) gradually lower the pH of the digester further accelerating the fermentation process. Tshikantwa et al.,  confirmed that reduced pH provides a better environment for several fermentation bacteria and fungi to thrive.
Figure 5: Change in concentration of lactic acid with increasing retention days.
Quantitative Analysis of Ethanol
The ethanol standard used (98%) had the main peak appearing at a retention time of 1.632 minutes and correlation area of 427,111 sq. units. A minor peak (suspected to be that of methanol) appeared earlier at a retention time of about 1.561 minutes. Other minor peaks with the retention time of 3.0 to about 4.0 seconds were found (suspected to be propanol and acetone respectively). Nevertheless, the ethanol peak was large enough to deem the other peaks negligible. The chromatogram of the ethanol standard is illustrated in (Figure 6a). The samples containing Indigofera amabelacensis extracts were proven to yield more ethanol content compared to those without as illustrated in (Figures 6b, 6c and 7).
The sample containing I. amabelacensis extracts had an identical chromatogram to that of the ethanol standard. This was illustrated by the number, position and intensity of peaks in the two chromatograms. The ethanol peak in the standard appeared after 1.632 minuts while that of the catalyzed sample appeared after 1.638 minutes. The correlation area of the catalysed samples chromatogram ethanol peak was 50,353 sq. units. This yields a purity of 11.55% total ethanol concentration. This highlights the intensity of ethanol concentration in the catalysed sample. The methanol peaks in the standard and catalyzed samples were also close together at 1.561 minutes and 1.562 minutes respectively. The correlation area of the standard sample was 7091 sq. units while that of the catalyzed sample was 1566 sq. units (22.08% total methanol concentration). The chromatogram of the sample without I. amabelacensis additive greatly differed with the rest with only a short peak at the ethanol retention time. The chromatogram also had other multiple peaks at varying positions illustrating presence of impurities. The same pattern was repeated in the fermentation of maize substrate as illustrated in (Figure 7).
The catalyzed maize sample was identical to that of the ethanol standard in terms of peak positions and intensity. The major ethanol peak of this sample appeared at 1.644 minutes while that of the standard was at 1.632 minutes. However, the intensity of the catalyzed maize sample ethanol peak was quite low (correlation area of 9406 sq. units). This yielded only 2.16% total ethanol sample in the catalyzed maize sample (five times less than the catalyzed sorghum sample). The standard industrial rate of ethanol conversion from biomass using yeast and standard conditions is 12-15% . The catalyzed maize sample also had propanol and acetone peaks at retention times of 1.753 and 1.825 minutes, similar to the standard. However, the chromatogram of the control sample of the maize sample greatly differed with that of the standard. Like the control of the sorghum substrate, this chromatogram had multiple minute peaks. Many impurities were thus formed by the end of the retention period used.
Figure 6: The chromatograms of ethanol standard (a), ethanol from sorghum with I. amabelacensis (b) and ethanol from the sorghum control sample (c).
Figure 7: The chromatogram for ethanol production from maize with I. amabelacensis additives (a) and that of maize without any additives (b).
Indigofera amabelacensis and Osyris alba extracts gave positive results in catalyzing ethanolic and lactic fermentation. The catalyzed samples of ethanolic fermentation using sorghum produced CO2 gas 5-folds higher than the control sample. Ethanolic fermentation using maize and lactic fermentation using milk substrates produced 3-folds more CO2 gas with reference to the control setups. Iodoform and Benedicts tests conducted confirmed that the catalysts actually hastened fermentation process. There was concise resemblance in the FT-IR spectra of the ethanolic fermentation products, all having an alcohol OH peak. The lactic acid products also resembled each other with slight variations in the intensities of the peaks of the catalyzed samples. The lactic acid from the sample with O. alba catalysts was way higher (2.79μg/L) compared to that of the control sample (1.48μg/L). The chromatograms of the catalyzed ethanolic fermentation products were quite identical with the ethanol standard used. Ethanol from the catalyzed sorghum substrate was 11.55% pure while that of the catalyzed maize substrate was 2.16% pure. The chromatograms of the control samples had multiple short peaks, illustrating many impurities present.
The authors wish to sincerely thank Mr. Rutto Kenneth, Mr. Patrick Lumumba and Md. Linda Mesoppirr for their hearted support in technical analysis during the research. The authors are also grateful to Maasai mara university mathematics and physical sciences (mps) department as well as the centre for innovation, new and renewable energy (cinre) directorate for their continuos support during the research.
Conceptualization, M.G. and A.O.; Methodology, M.G.; Software, B.C.; Validation, M.G., A.O. and B.C.; Formal Analysis, B.C.; Investigation, A.O. and B.C.; Resources, A.O.; Data Curation, B.C.; Writing – Original Draft Preparation, M.G.; Writing – Review & Editing, A.O and B.C.; Visualization, B.C.; Supervision, A.O.; Project Administration, A.O.; Funding Acquisition,-
Conflicts of Interests
The authors declare to have no conflicts of interest whatsoever.
Sources of Funding
No funding was received for this research.
Data Availability Statement
All data used in this work is enclosed within the manuscript and any supplementary sheets herein attached.
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