Evaluation of starter culture fermented sweet potato flour using FTIR spectra and GCMS Chromatogram

1 Department of Biotechnology, Federal Institute of Industrial Research, Oshodi, PMB 21023, Ikeja, Lagos State, Nigeria. 2 Department of Food Technology, Federal Institute of Industrial Research, Oshodi, PMB 21023, Ikeja, Lagos State, Nigeria. 3 Department of Production, Analytical Services and Laboratory Management (PALM), Federal Institute of Industrial Research, Oshodi, PMB 21023, Ikeja, Lagos State, Nigeria. 4 Department of Product Design and Development, Federal Institute of Industrial Research, Oshodi, PMB 21023, Ikeja, Lagos State, Nigeria.

includes sugars, starch and cellulose.These compounds are classified as monosaccharides (such as, glucose, fructose), disaccharides (such as, sucrose, lactose) or polysaccharides (such as, starch, cellulose) (Kim et al., 2007).All carbohydrate compounds have been used as a source of biomass and a large number of microorganisms use them as energy source as a result, carbohydrates are essential for maintaining life (Kim et al., 2007).Glucose is the key compound as most life systems are built around it.
Starch is a carbohydrate consisting of glucose compounds joined to form a polysaccharide (Dias et al., 2008).It is a plant natural energy source that is most abundant and valuable which needs to be converted to simple sugars before it can be utilized as a carbon source (Yoo and Jane, 2002;Mosier et al., 2005;Yang et al., 2006;Gray et al., 2006).
The starch stored in the seeds and tubers of various agricultural crops including maize, wheat, rice, barley, potato and cassava provide the main sources of energy in the human diet (Evers et al., 1999;Perez et al., 2009;Perez andBretoft 2010, Schwartz andWhistler, 2009).Starch is the major component of cereal grains, and changes in its biophysical and biochemical properties are related to the amount and ratio of amylose and amylopectin, which influences and affect properties such as viscosity, gelatinization, that will determine its end use properties (such as, bread, malt, beer, polymers) (Evers et al.,1999;Schwartz and Whistler, 2009;Willett, 2009).
Starch functions mainly as a carbohydrate source for the growing plant (such as, for germinating seeds and leaf tissue development) and is consequently the primary source of stored energy in the plant.Depending on the plant, starch can be found in a variety of tissues, including leaves, tubers, fruits, and seeds.It is the primary source of stored energy in cereal grains.Although the amount of starch contained in grains varies, it is generally between 60 and 75% of the weight of the grain and provides 70 to 80% of the calories consumed by humans worldwide.
Root vegetables are plant roots used as vegetable, they are generally storage organs enlarged to store energy in form of carbohydrates, starch root vegetable are important staple food particularly in tropical regions overshadowing cereals throughout much of west Africa, Central Africa, they are used directly or mashed to make fufu or poi.Storage roots can be categorised in bulb, rhizome and tubers.Examples of tuberous root include desert yam (Ipeoma costata), sweet potato (Ipeoma batatas), cassava (Manihot esculenta), etc. Sweet potato carbohydrate has been reported to contain pectin substances, lignin, cellulose and hemicellulose, which are all converted to simple sugars when fermented (Yokoi et al., 2001).
Sweet potato has been processed into prickles and consumed as lacto-juices by processing it with lactic acid bacteria as the fermenting organism and the juice produced has been reported to be very rich in minerals and vitamins (Smita et al., 2007;Panda and Ray, 2007).Sweet potato has also been processed into chips in much the same way as Irish potato (Brigato et al., 2010;Hagenimana and Owori, 1998).It can also be eaten boiled, fried and in roasted form.In addition, it can be sliced, dried in the sun and ground to give flour that remains in good condition for a long time (Wheatly, 2009).Sweet potato can be fermented, dried and milled into flour.Fermentation is the conversion of carbohydrate into alcohols and short chain fatty acids by enzymes of microorganisms (Silva et al., 2008;Yuan et al., 2008).It is a basis of many biological products which involves a process of chemical reactions with the use of microbes such as bacteria, yeast and filamentous fungi (Huang and Tang, 2007;Fortman et al., 2008).The primary benefit of fermentation is the conversion of sugars and other carbohydrates to usable products.As stated earlier, not all bacteria can readily use starch as their energy and carbon source (Nigam and Singh, 1995).This means that some starches need to be broken down to simple fermentable sugars so they can be utilized by bacteria (Nigam and Singh, 1995).On the other hand, sweet potato is rich in βamylase, which converts long chained starch into readily used maltose units making it a good energy and carbon source for bacteria (Yoshida et al., 1992;Brena et al., 1993;Cudney and McPherson, 1993;Nigam and Singh, 1995).Starter cultures are living microorganisms of defined combination used for fermentation purposes.They help to elicit specific changes in the chemical composition, nutritional value and sensorial properties of the substrate (Opere et al., 2012) and they are generally recognised as safe (Aguirre and Collins, 1993).Moreover, their properties are as follows: they are harmless, initiate and control the fermentation process, typical for product, help in rapid acid formation, and help protect against spoilage organisms.Starter cultures are cheaply reproducible in large amount; they also help provide desirable sensory properties and assist in reducing fermentation period.Ajayi et al. (2016) have done work on the fermentation of sweet potato into flour using starter culture.
Fourier transforms infrared (FTIR) spectroscopy is one of the most important and emerging tool used for analysing functional groups present in test samples.This technique is rapid and sensitive with a great variety of sampling techniques.FTIR is a rapid, non-destructive, time saving method that can detect a range of functional groups and is sensitive to changes in molecular structure.FTIR provides information on the basis of chemical composition and physical state of the whole sample (Cocchi et al., 2004).In addition the sensitivity and accuracy of FTIR detectors along with wide variety of software algorithms have dramatically increased the practical use of infrared for quantitative analysis (Dowell et al., 2006).FTIR works because of functional groups and provide information in the form of peaks.
GCMS combines the features of gas chromatography and mass spectrometry to identify different substances within a test sample.Gas chromatography portion separates the chemical mixture into pulses of pure chemicals and the mass spectrometer identifies and quantifies the chemicals.It reveals the compounds eluted at different retention times with mass spectra corresponding to compounds present (Siong et al., 2014).
This study aimed at investigating the functional groups of the Starter culture fermented sweet potato flour using Fourier transform infrared (FTIR) spectroscopy as well as reporting the effect of the functional group on the products, the chemical compounds present in the starter culture fermented sweet potato flour will be detected using the GCMS.

Sourcing of raw materials
Yellow-fleshed sweet potatoes were obtained from Oshodi market (Oshodi), Lagos, Nigeria.The samples were transported to the biotechnology department of the Federal Institute of Industrial Research for immediate use.

Starter cultures
The starter cultures used were obtained from the biotechnology department of the Federal Institute of Industrial Research Oshodi.
Potato dextrose agar and De man Rogosa Sharpe (MRS) agar were prepared using manufacturers specification and sterilized using the autoclave at 121°C for 15 min.L. brevis and D. polymorphous stored in MRS and PDA slants were subcultured into freshly prepared MRS and PDA agar plates.

Preparation of innoculum
This was carried out using the method of Asmahan et al. (2009).Lactobacillus brevis were cultivated by streaking on MRS agar plates (Oxoid) and incubated anaerobically at 37°C for 24 h.A colony was picked from each pure culture plate, grown successively in MRS broth before centrifugation at 5000 rpm for 15 min.The pellet was washed in sterile distilled water centrifuged again and redistributed in distilled water.This procedure achieved a culture preparation containing about 10 9 colony forming units cfu/ml, checked as viable count on MRS agar.Pure cultures of D. polymorphous were cultivated by streaking on Potato dextrose agar (Oxoid), incubated at 37°C for 24 h and the picked colony was inoculated into yeast extract peptone dextrose broth (YEPD) and incubated at 28°C for 24 h.These cultures were centrifuged and washed as described above.This procedure achieved a culture preparation containing 10 7 cfu/ml, as viable count on potato dextrose agar.

Preparation of starter culture fermented sweet potato flour
The sweet potatoes were washed to remove adhering soil particles and peeled.The peeled tubers were chipped into slices (4-5 mm).Starter cultures were prepared and inoculated into the sweet potato, then left to ferment for a period of two days (48 h).
After this period has elapsed, the fermented chips were drained and dried in a cabinet drier (Mitchel, Model SM220H) at 55°C for 9 h and milled into flour (≤ 250 μm) using the method of Ajayi et al. (2016) and the starter culture fermented sweet potato flour was produced.

Preparation of fermented sweet potato flour (control)
The sweet potatoes were washed to remove adhering soil particles and peeled.The peeled tubers were chipped into slices (4-5 mm) and soaked in potable water for a period of two days (48 h).
After this period has elapsed, the fermented chips were drained and dried in a cabinet drier (Mitchel, Model SM220H) at 55°C for 9 h and milled into flour (≤ 250 μm) (Oluwole et al., 2012).

Fourier Transform Infrared (FTIR)
FTIR spectra illustrate absorption bands with characteristic frequency attributed to different functional groups and all spectra were obtained, using a Bruker FTIR CLASS 1 ALPHA.The spectra were collected at a resolution of 4 cm −1 in the range of 500-4000cm −1 .Each spectrum was rationed against a fresh background spectrum recorded from the bare crystal.Prior to collection of each background spectrum, the crystal was cleaned with absolute ethanol to remove any residual.Each sample was scanned in triplicate.

Gas chromatography mass spectrometry (GCMS)
Ten grams of samples were dissolved in 15ml of ethanol.The sample was analysed on a Shimadzu GC-MS system model QP2010, with a medium polarity capillary column SLB-5ms supelco column (length 30.0m x thickness 0.20 mm × Diameter 0.20 mm), with helium as the carrier gas.Column oven temperature was at 40°C, injection temperature was at 250°C, injection mode split (10:1), temperature program was 40°C (Hold 3 min) 9°C/min to 290°C (Hold 6mins), MS ion source temperature at 200 o C interface temperature at 250°C.Detector voltage = Relative to the tuning result solvent cut time 4min, Acqusition mode-scan, scanning range 40-550 mz.One microlitre of the sample was injected using splitless injection with injector temperature 300°C according to the following scheme: 50°C for 2min with 10°C/min up to 300°C.The final temperature was held for 10 min.The total runtime for each sample was 37min.For MS detection, electron ionization with 70 eV was applied and mass fragments were detected between 40 and 500 m/z.The ion source temperature and transfer line temperature were 200°C and 300°C, respectively.The detector was activated after 5min.

RESULTS
The FTIR Spectra were recorded in regions below 800 cm −1 , 500 cm −1 (the fingerprint region), the region between 2,800 and 3,000 cm −1 (C-H stretch region), and finally the region between 3,000 and 3,600 cm −1 (O-H stretch region) (Table 1).
The O-H stretching for the raw sweet potato occurred at 3322.15 cm -1 .The peaks at 2928.10 cm -1 was observed as a result of C-H bond stretching.The peaks at 1097 cm  Various functional groups such as hydroxyl group, carboxyl group were also observed in Figure 2. The peak at 3298.87 cm -1 is attributed to O-H stretching .The O-H stretching was also observed and after 24h fermentation using starter cultures it reduced to 3298.87cm -1 .The 2930.33 cm -1 peak observed is attributed to C-H bond stretching.The peaks at 1412.94 cm -1 is attributed to the bending modes of O-H.The GC/MS analysis of the starter culture fermented sweet potato for 24 h of wheat extract showed the presence of 27 compounds corresponding with retention time from 5.043 to 33517 s.The most intensive peak was the lactic acid with an area percentage of 30% and a retention time of 7.118 s.The chromatogram had the following organic acids identified: octadecanoic acid at 25.123 s with an area percentage of 3.53%.n-Hexadecanoic acid at 23.025 s with an area percentage of 8.86% and oxirane had an area percentage of 27.40% and retention time of 14.241 s.The presence of lactic acid in the starter culture fermented sweet potato flour will help serve as a biopreservative, extend shelf life, and give a tart and tangy flavour (Schnurer et al., 2005;Cizeikiene et al., 2013).
The absorbance at 3292.59 cm -1 is attributed to O-H stretching ; while 2929.48cm -1 is attributed to C-H bond stretching .The peaks at 1412.83 cm -1 is attributed to the bending modes of O-H.The various functional groups detected are similar to those obtained in the 24 h fermented sweet potato flour.
Table 3 shows the chromatogram of starter culture fermented sweet potato flour after 48 h with 24 peaks.Lactic acid was detected and it also had the most intensive peak after 48 h fermentation using starter cultures (Figure 3), it had an area percentage of 55.53% and a retention time of 7.378 s, oxirane was detected at 14.117 s with an area percentage of 11.70%.Other compounds such as 9,12-octadecanoic acid (Z,Z) are polysaturated fatty acid which are antiinflammatory, hypocholestrolemic, and cancer preventive according to Adeoye-Issijola et al. ( 2018) were detected with an area percentage of 10.265% and a retention time of 24.850 s.At 23.026 s, n-Hexadecanoic acid was detected with an area percentage of 11.87%.
The absorbance spectra obtained from Figure 4 shows O-H stretching ,C-H bond stretching and O-H bending.(Table 4).The peaks at 1414.11 cm -1 is attributed to the bending modes of O-H.
The chromatogram for 72 h starter culture fermented sweet potato flour is showed in Table 5 and Figure   s was detected.9,12octadecanoic acid (Z,Z) had a retention time of 24.850 s and an area percentage of 10.26% was detected.The chromatogram had 26 peaks and the most intensive peak was lactic acid with an area percentage of 58.645% and a retention time of 7.295 s. n-Hexadeacanoic acid (palmitic acid) is a fatty acid which is are antioxidant, antibacterial, antiinflammatory, cancer preventive amongst other (Adeoye-Isijola et al., 2018).
In Figure 5 3274.59cm -1 could be attributed to O-H stretching .2927.29 cm -1 are attributed to C-H bond stretching.The peaks at 1410.41 cm -1 was attributed to the bending modes of O-H.
The chromatogram of the control (spontaneously fermented sweet potato flour) was observed in Figure 5 with 22 peaks.It also had lactic acid has the most intensive peak with an area percentage of 28.71% at 6.92 s retention time, octadecanoic acid (Z,Z) was also detected at 24.843 s with an area percentage of 10.64%.n-Hexadecanoic acid was also detected with an area percentage of 11.8755% at a retention time of 23.026 s (Table 6).

DISCUSSION
The peaks obtained were at 3322.15, 3311.92 , 2928, 1638.48 cm -1 for the raw sweet potato, 3298.87, 2930.33,1636.54, 1412.94 and 995.52 cm -1 for the starter culture fermented sweet potato flour for 24 h.Then, 3292.59, 2929Then, 3292.59, .48,1637Then, 3292.59, .64, 1412.83 and 994.83 and 994.41 cm -1 for starter culture fermented sweet potato flour for 48 h 3279.59, 2929.31, 1636.20, 1414.11, 994.75 cm -1 the starter culture fermented sweet potato flour for 72 h.Also, 3274.59,2927.29,1635.65,1414.41 and 995.42  cm  -1 for the spontaneously fermented sweet potato flour for 72 h.This is in line with earlier works carried out on starch contents spectra of starch were recorded using FT-IR, key bands by Belton et al. (1991).Spectra were recorded in regions below 800 cm −1 , 500 cm −1 (thefingerprint region), the region between 2,800 and 3,000 cm −1 (C-H stretch region), and finally the region between 3,000 and 3,600 cm −1 (O-H stretch region).Fourier transform infrared (FTIR) spectroscopy is a tool used to differentiate between patterns of amylose in different granule.Peaks near 3500, 3000, 1600, 1400, 1000, 800, described the IR spectrum of starch samples and Manley et al. (2002) to determine the presence of moisture used 500 cm -1 as seen in the study by Zeng et al. (2011) and it.Moisture contents were also on mid infrared range.Peaks for water were observed on 1,640 and 3,300 cm −1 .The absorption is done on the base of functional groups H and OH.The absorption spectra of starter culture fermented sweet potato flour and the control shows strong peaks in the same region indicating the presence of moisture in the    flour.FTIR is one of the most elusive methods for the analysis of moisture.Water absorbs strongly in the infrared region of the spectrum due to its O-H stretching and H bending vibrations however its quantization is frequently complicated by spectral interferences from other OH containing constituents such as alcohols, phenols and hydroperoxides and confounded further by hydrogen bonding effects (Dong et al., 2000).
The O-H stretching for the raw sweet potato occurred at 3322.15 cm -1 and after 24h fermentation using starter cultures, it reduced to 3298.87cm -1 ; at 48 h it was 3279.59 cm -1 and at 72h it was 3279.59 cm -1.The decrease in the wavelength with increase in time depicts dilution of the crystalline (amylose) region, leading to the breakage of α-1,4-glycosidic linkage.Consequently, the hydrophilic O-H group will contribute to the increasing amorphous fraction.
The peaks at 2928.10, 2930.33,2929.48,2929.31  and 2927.29 cm  -1 are attributed to C-H bond stretching.The C-H stretching for raw potato, starter culture fermented sweet potato flour at 24 h, 48 h, 72 h were 2928.10,2930.33,2929.48 and 2929.31cm  -1 respectively.These values fall within the same range.This indicates that there was a change in the functional group and this could be the oxidation of an aldehyde group (CHO) during the fermentation.Supriya et al. (2015) observed that Crude fat of flour samples had peaks at 1,600 cm −1 to 1,700 cm −1 and 1,550 to 1,570 cm −1 .The absorption peaks are determined on the basis of C-H bonds.The absorption spectra of starter culture fermented sweet potato flour also show strong peaks in the same region, which indicates the presence of fat in flour.It has been reported that FTIR spectroscopy could be utilized as a quality control method for fat and moisture determination in butter and high-fat products ( Van de Voort et al., 1992).Che-Man and Setiowaty (1999) and Rai et al. (2013) also reported similar results.Supriya et al. (2015) observed absorption bands that are two primary features of the protein, amide I and amide II bands at approximately 1,660 cm −1 and 1,550 cm −1 , respectively.Amide I arises from the stretch of C=O of the peptide group in the protein.Peaks were also observed around this region for the starter culture fermented sweet potato flour as well as the control.
A cursory look at the spectra shows a slight decrease in the wavelength of the carboxyl (C=O stretching ) of the α 1,4-and α1,6glycosidic linkeage (1638.48cm -1 for raw sweet potato,1636.54cm -1 after 24 h,1637.20 cm -1 after 48 h and 1636.64 cm -1 after 72 h.The slight decrease in wavenumber indicates arial oxidation to carboxylic acid such as, acetic acid /ascorbic acid which can serve as preservative to the flour.
The stretching vibration for α 1,4glycosidic linage at the amylose region decreased from 1003.96 cm -1 for raw sweet potato to 994.75 cm -1 after 72 h, this will The peaks at 1097 and 1019 cm -1 were assigned as the C-O bond stretching.The bands at 1047 and 1022 cm -1 were associated with the ordered and amorphous structures of starch respectively.
The starter culture fermented sweet potato flour had more peaks than the control after 72 h fermentation.It also had higher concentration of lactic acid, which indicates a faster rate of fermentation with the use of starter cultures.The presence of high concentration of lactic acid will also help inhibit the presence of spoilage organisms, the elimination of spoilage organisms and it will serve as preservatives.
Some of the compounds detected by the GCMS include 9,12-octadecadienoic acid; hexadecanoic acid which are antioxidant; ethanol which is used as food preservative and propanoic acid which is widely used as an antifungal agent amongst others Supriya et al. (2015).
Generally, the chemical components were identified in each sample.The purpose of the study was identify the chemical components and compare presence of components in the various sample.Similar chromatograms were also obtained for all the starter culture fermented sweet potato samples.This study has shown that it is possible to compare the chemical compounds of samples with GCMS chromatography.

Conclusion
The study reveals that sweet potato contain starch (amylose and amylopectin) and sucrose as sugar.
Starter cultures L. brevis and D. polymorphous fermented the sweet potato thereby breaking down the carbohydrate (starch) to produce alcohol, organic acid and CO 2 hence lactic acid fermentation occurred.
Functional groups such as hydroxyl, aldehydes, alcohol and carboxyl are present in the fermented samples.Lactic acid fermentation occurred and it caused a shift in some of the functional groups.
The chemical shift indicates that the starter culture fermented sweet potato flour containing compounds such as carboxylic acids, alcohols, aldehydes, hydroxyl and alkenes.
Starter culture fermented sweet potato flour had a higher concentration of carboxylic acids, alcohols, aldehydes etc. Alcohol and carboxylic acid was produced in situ from the fermentation process, which will help inhibit spoilage organisms moreover, it will also serve as preservatives thereby increasing shelf life of the product.
The FTIR showed a similar spectrum for all the fermented sweet potato flour and it was possible to verify the main organic functions associated, the results corroborate the wide variety of volatile organic compounds identified by the CGMS.The presence of OH group detected by FTIR which was further emphasized by the presence of butanol detected by the GCMS, and the presence of the C=O group detected by FTIR which was also emphasized.
5.Compounds such as n-Hexadecanoic acid with area percentage of 11.875% with a retention time of 23.026

Figure 4 .
Figure 4. FTIR absorbance spectra for starter culture fermented sweet potato flour for 72 h (J).

Figure 5 .
Figure 5. FTIR absorbance spectra for starter culture fermented sweet potato flour for 72 h control (K).
corresponding to compounds present.Figure1shows the GCMS chromatogram of raw sweet potato sample from 5.72 to 34.066 s.It showed 45 peaks, compounds such as pyrimidine-2,4(1H,3H) dione was detected with a retention time of 5.72 s and an area percentage of 0.27%, propanoic acid had the retention time of 66.556

Table 2 .
GCMS Peak report for starter culture fermented sweet potato flour 24 h (H).

Table 3 .
GCMS Peak report for starter culture fermented sweet potato flour 48 h (H).

Table 4 .
GCMS Peak Report for starter culture fermented sweet potato flour 72 h (J).

Table 5 .
GCMS Peak Report for starter culture fermented sweet potato flour 72 h (J).

Table 6 .
Summary of some compounds obtained from GC-MS analysis.