Volatile organic compound modification by lactic acid bacteria in fermented chilli mash using GC-MS headspace extraction

This study was conducted to assess the volatile compound generated in fermented chili using the static headspace gas chromatography (GC-MS). Three types of lactic acid bacteria (LAB) inoculant were used; Lactobacillus plantarum Alo1, Lactobaciluss pentosus Alo2 and Lactobacillus platarum Au2 for the lactic acid fermented chili mash. Raw chili and natural fermented chili mash were served as negative and positive control. The volatile compound was grouped into 7 main compounds consist of ester, alcohol, alkane, acid, hydrocarbon, ether and nitrogen-containing group. Study showed that LAB inoculated chili mash has lower amount of ethanol as compared to naturally fermented chili mash. Besides, volatile compound generated among each inoculant was different. A compound known as n-Hexadecanoic acid was the primary compound detected in all LAB-inoculated chili mash. Result proved that LAB can be used as the potential starter culture in modifying the aroma of fermented chili mash.


Introduction
Flavor compounds that imparts aroma to chillies is influenced by volatile organic compunds. Chili flavours were characterised as a mixture of volatile organic compound (VOC) build from pyrazines such as 3-isopropyl-2-methoxypyrazine, 3-butyl-2-methoxypyrazine and 3-isobutyl-2-methoxypyrazine [1]. The pungency is contributed by capsaicinoids, synthesised from vanillyl amides of various acids. The presence of volatile organic compound (VOC) interfered directly to the sensory quality of the fresh chilli fruit as well as the processed product. Detection of aromatic component in food is generally conducted by GC-MS [2]. The simplest way of extracting the volatile compound from a sample is using the headspace extraction technique. This technique has been used to extract VOC from a variety of natural products and it is now considered a matured extraction technique. It is able to quantitatively quantify wide numbers of volatile compounds in passion fruit juice, rice, chilli cultivars, orange and blackcurrant [3, 4 and 5]. The beauty of applying this technique is aroma detected directly from the portion of the air in contact with the odor source, without any other sample treatment step. Due to this, volatile flavour detected by static headspace GC-MS provide volatile profiles close to the aroma profiles perceived by humans.
Applying the fermentation process can alter the VOC of a wide range of food products. Adding starter culture such as lactic acid bacteria (LAB) has proven to produce a more complex aroma as the bacteria can convert the complex structure of carbohydrate, fats and protein into simpler compounds. Research conducted by Abolhassani et al. (2009) [6]  LAB has modified the flavour of the Merlot wines by the production of different VOC such as ester and di-acetyl in winemaking [7]. LAB has excellent potential to be applied as starter culture for development of fermented chili mash as it can improve the microbial quality as well as odour profile perceived by hedonic test [8,9]. Inoculated LAB can survive well in the fermented chili mash instead of controlling the mold and yeast growth ( Figure 1) [8]. However, the knowledge of volatile compound generated by the LAB inoculant is remain unknown. Understanding the volatile components generated in the fermented chili mash will differentiate the capability of LAB in flavour modification and the optimal starter culture. Therefore, this study was conducted to evaluate the capability of different LAB strain to modify the flavour compound in fermented chilli mash using GC-MS static headspace.

Preparation of lactic acid fermented mash
Cilibangi T.M fruits were selected to make free from blemishes, defects, and insect damage. Fruits were cleaned and pericarp removed. The chillies were washed with tap water to remove any impurities then ground using a food blender (Panasonic) with 6% rock salt added. Fermentation was conducted using 150 ml Schott bottle each containing 100 g of pepper mash inoculated with log 10 cfu/mL 24 h cultures of LAB (1% v/v) adapting Shahidah et al. (2016) [8] method for 14 days at 30 o C. Three LAB strain were used; Lactobacillus plantarum Alo1 isolated from home-made yoghurt, Lactobacillus pentosus Alo2 isolated from raw cow milk and Lactobacillus plantarum Au2 isolated from fermented chili mash. Spontaneous fermentation with no LAB inoculated chilli mash served as positive control while the raw chilli served as the negative control.

Identification of volatile organic compounds
Five grams of each fermented chilli mash was introduced into a 10 ml headspace vial . VOC in fermented chilli mash samples were identified using Agilent-Technologies 7890A GC system equipped with Agilent-Technologies 5975C Inert MSD with triple-axis detector and Agilent-Technologies G1888 Network headspace sampler (Agilent-Technologies, Little Falls, CA, USA). GC/MS analysis of volatile compounds was performed using DB-WAX column (30 m × 0.25 mm × 0.25 μm) (J&W Scientific, Folsom, CA, USA). The volatile compounds were identified by comparing and matching mass spectra fragment with the NIST08 MS library (80-100 % similarity). The extraction protocol by direct injection method was modified by Aimi et al. (2013) [13] and simplified in Table 1. The static headspace GC-MS was carried out in duplicate. Table 1. Extraction protocol for GC and headspace condition. Table 2 shows the number of compound and the respective total peak area corresponding to each chemical class detected by GC-MS. The components were classed into 7 components consisting of alcohol, alkane, acid and hydrocarbon. The volatile profiles show diverse qualitative patterns. Alcoholic group was dominating the fermented chili mash in both spontaneous and LAB inoculated chili mash with total peak area around 35.50-40.92% as compared to raw chili. Vice versa, nitrogen containing compound was detected in high amount in raw chilli with 88.32% total peak. There was absence of alkane in fermented chili mash inoculated with Lactobacillus plantarum Alo1 and ether in fermented chili mash inoculated with Lactobacillus plantarum Alo1 and Lactobacillus plantarum Au2.

Results and discussions
Levels of individual volatile compounds related to the aroma profile were largely influenced by the presence of fermentation process and LAB (Table 2). Ethanol was the major compound present in all fermented chili mash but not in raw chili. Few types of methyl alcoholic compound such as 1-butanol, 3-methyl, 1-propanol, and 2-methyl alcoholic group present in fermented chilli mash inoculated with Lactobacillus plantarum Alo1. A compound of α-terpineol was only detected present in spontaneous fermentation with 9.91% total peak area (  [12] observed the presence of alcohols during fermentation resulted from carbohydrate metabolism by the yeast in the presence of LAB. Additionally, methyl alcohols could be originated from activities of endogenous enzymes and residual enzymes from microorganisms [13]. The compounds could be generated from reduction of propanal, 2-methyl-1-propanal, pentanal, 4methyl-1-pentanal [14]. The volatile acids detected present in LAB-inoculated fermented chilli mash were L-(+)-ascorbic acid, trans-13-octadecanoic acid, pterin-6-carboxylic acid, hydroxy acetic acid (Table 3). Ascorbic acid is a common volatile detected present in different variety of raw chilli cultivars quantified by GC-MS solid phase micro extraction [15]. However, this study did not detect any ascorbic acid in both raw chili and spontaneous fermentation (Table 3). Only fermented chili mash inoculated with L. plantarum Alo1 produced L-(+)-ascorbic acid with 5.16% peak area. The absent of the ascorbic acid in raw chilli might be due to incapability of static headspace GC-MS to detect the compound. The headspace condition during extraction may have resulted in the conversion of ascorbic acid to benzene through decarboxylation process [16].
Each of LAB isolates produced different compound of volatile organic compound. The compounds of L-(+)-ascorbic acid, cyclopropane tetradecanoic acid and 2-octyl-methyl ester were only detected present in Lactobacillus plantarum Alo1 inoculated fermented chilli mash with 5.16% and 0.48% total peak area respectively. On the other hand, 1,1'-bicyclopropyl-2-octanoic acid was only detected present in fermented chili mash inoculated with Lactobacillus plantarum Alo1 and Lactobacillus plantarum Au2 with 0.50% and 0.54% total peak area. The trans-13-octadecenoic acid only present in fermented chilli mash inoculated with Lactobacillus plantarum Au2. The variation be influenced by unique LAB interaction with the chili substrate. The bacterial can alter the enzyme that responsible for flavour generation [17].
For instance, pectin esterase is present in plant tissue which specifically affects methyl groups on linear chain of pectin producing the methyl ester compound group. LAB is believed to play important role to synthesise ester via esterification reaction in which fatty acyl groups from glycerides is transferred to alcohols [18]. LAB esterase producer such as Lactobacillus plantarum and Lactobacillus pentosus have the ability to hydrolyse and synthesise ester. Esterolytic activity liberated by the esterases contributed to wine aroma can increase in wine quality [19].    Hexadecanoic acid was found to be the major constituent in fresh chili but found to be decreased and disappeared once the chili achieves maturation [20]. However, this compound is not detected in this study as this compound was absent in raw chili and spontaneous fermented chili mash. It is believed that N-hexadecanoic acid are metabolically synthesised by the presence of LAB as this compound present in all LAB inoculated chili mash. This compound was found to be the common secondary metabolites produced by LAB in malolactic wine fermentation [21]. The presence of LAB will convert the complex carbohydrate into simple glucose which then become the main precursor for generation of acetyl-CoA. Consequently, Acetyl-CoA caters the generation of Hexadecanoic acid [22]. Hydrocarbon 1,6-octadie-3-ol,3,7-di methyl or commonly known as β-linalool was detected in all fermented chilli (Table 2). This compound is naturally present in different type of spices such as coriander, chili and ginger [23]. It contributes to flowery and spicy odour in herbs. The study conducted by Shahidah & Zaition (2015) [9] found out both yeast and LAB were present in LAB-inoculated and naturally fermented chili mash. The present of this compound in all fermented chili was assumed to be metabolically synthesised by single yeast or interaction between LAB and yeast. Carrau et al. (2005) [24] discovered that 1,6-octadie-3-ol,3,7-di methyl is a yeast metabolite in wine after alcoholic fermentation.
This study had detected a methyl ester; cyclopropane tetradecanoic acid 2-octyl-methyl ester, methoxyaceticacid, 2-tridecyl ester, 2,15-octadecadiynoic acid, methyl ester, cyclopropanedodecenic acid, 2-octyl-methyl ester, 9,12,15-octadecatrienoic acid, 2-(trimethylysily)doxyl-1 [(trimethylsilyl)oxy)-methyl-ethyl ester] compound in both LAB inoculated and spontaneously fermented chilli mash (Table 3). Ester can be derived from the raw chilli and from the chemical esterification of alcohols and acid during the fermentation process [25]. Ageing of fermented chilli mash during 14 days fermentation resulted in plant tissue breakdown. Pectin esterase is present in plant tissue which specifically affects methyl groups on linear chain of pectin producing the methyl ester compound group [26]. The common LAB esterase producers that has ability to convert the fatty acyl groups from glyceride to alcohol are Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum which can [19].