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Isolation, identification and characterization of enzyme-producing lactic acid bacteria from traditional fermented foods

Isolation, identification and characterization of enzyme-producing lactic acid bacteria from... Abstract Industrialization processes are prone to produce various forms of waste which can be utilized to produce silage. These wastes can be treated by using lactic acid bacteria (LAB), which are known to be potential enzyme producers. Seven strains of LAB were isolated from traditional fermented food, ‘tapai pulut’, ‘tempeh’, ‘tempoyak’ and ‘fu yu’ and were screened for amylase, cellulase and protease production in order to select for strains that could potentially be used industrially in silage production. All seven LAB isolates exhibited high protease production, and two of them also exhibited high amylase and cellulase production. The two isolates exhibiting high amylase and cellulase production were selected and identified as Pediococcus acidilactici FY2 and Enterococcus durans FY3 via biochemical profiling (API 50 CHL) and 16 s rDNA sequencing. E. durans was found to have the highest amylase (Vmax: 5.51 μmol/mL/min; Km: 0.300 g/100 mL) and cellulase (Vmax: 3.50 μmol/mL/min; Km: 0.006 g/100 mL) production, while exhibiting strong protease production (Vmax: 0.51 μmol/mL/min; Km: −0.287 g/100 mL). P. acidilactici was found to have strong amylase (Vmax: 4.43 μmol/mL/min; Km: 0.433 g/100 mL) and cellulase (Vmax: 2.66 μmol/mL/min; Km: 0.002 g/100 mL) production while exhibiting the highest protease production (Vmax: 2.14 μmol/mL/min; Km: −0.348 g/100 mL). These results suggest that E. durans is a better candidate for future industrial application as overall it has a higher enzyme reaction velocity when compared to P. acidilactici. Further studies should be carried out to confirm the Km value for protease production, to purify and characterize all three enzymes produced and to optimize the growth conditions of E. durans. lactic acid bacteria, amylase, cellulase, protease, Pediococcus acidilactici, Enterococcus durans Introduction In this modern era, the industrialization process is growing at an alarming rate. The continuous discovery and invention of state-of-the-art technology continually pushes the industrial world to evolve at a fast pace. Thus, prioritizing the usage of existing resources is important to ensure optimal production and prevent wastage. Various agricultural industrial processes and general food consumption produce large amounts of wastes, and some of these can be utilized in silage production as the waste are usually rich in sugars and proteins (Show and Guo, 2012). Ensilage is preserved forage crops that is generally fermented by lactic acid bacteria (LAB) under anaerobic conditions and is usually void of undesirable microorganisms due to the production of lactic acid by the LAB (Ni et al., 2015b). Thus, scientists are searching for alternative microbial sources to find better strains to process silage, as LAB are well-known producers of extracellular enzymes (Patel, Shah and Prajapati, 2013; Tosungnoen, Chookietwattana and Dararat, 2014; Ni et al., 2015a). LAB with amylase and cellulase activity to degrade complex sugars such as starch and cellulose are much sought after, as this would reduce the cost of substrate pretreatment (Shibata et al., 2007; Yitbarek and Tamir, 2014). LAB are a group of Gram-positive bacteria that produce lactic acid during fermentation and are important for food manufacturing, especially in the milk, vegetables and meat industries (Konings et al., 2000; González et al., 2010). Various LAB are generally regarded as safe and have been used for a very long time as starter cultures to produce fermented foods through traditional means, as they can produce lactic acid and bacteriocins that act as natural preservatives and thus can extend the shelf life of silage (Holzapfel, Geisen and Schillinger, 1995; Perez, Zendo and Sonomoto, 2014). Therefore, LAB should be abundantly found in fermented foods. In this current study, traditional fermented foods such as fermented glutinous rice ‘tapai pulut’, fermented soybean cake ‘tempeh’, fermented durian condiment ‘tempoyak’ and fermented soybean curd ‘fu yu’ were investigated. The aim of this study was to isolate and screen for putative LAB producers for enzymes amylase, cellulase and protease from traditional fermented foods for future industrial application. The putative enzyme producers were identified using biochemical and molecular techniques, and the enzyme kinetics were determined. Materials and methods Isolation of LAB from fermented foods Four different types of local fermented foods, fermented glutinous rice ‘tapai pulut’ (TAP), fermented soybean cake ‘tempeh’ (TEM), fermented durian condiment ‘tempoyak’ (TYK) and fermented soybean curd ‘fu yu’ (FY) were purchased from a grocery store in Selangor, Malaysia. A total of 10 g of the food samples were mixed with 90 mL of peptone-buffered water and incubated at room temperature for 30 min. A 10-fold serial dilution was carried out from 10−1 to 10−7. Volumes of 0.1 mL of diluted samples were spread on de Man, Rogosa and Sharpe (MRS) agar plates and incubated at 37°C for 48 h. MRS agar is a selective media for LAB growth. After incubation, colonies with different morphology were randomly selected, Gram-stained and examined for cell morphology under a microscope. Maintenance and preparation of bacterial isolates Each LAB isolate was preserved in 20% (v/v) glycerol and stored in a −20°C freezer till used. Initially, isolates were kept active via subculturing by pipetting 0.2 mL of active culture into 10 mL of MRS broth, followed by incubation at 37°C for up to 72 h before the next subculture. In the event of no growth, 0.2 mL of the glycerol stock was inoculated in 10 mL of MRS broth and incubated at 37°C for 24 h. Then, a four-point streak was carried out on MRS agar to ensure no contamination occurred. A single pure colony was selected and inoculated in 10 mL of MRS broth and incubated at 37°C for 24 h, followed by another subculture in 10 mL of MRS broth and incubation at 37°C for 24 h, forming active 24-h-old LAB. Two days prior to an assay, 0.2 mL of active LAB isolates were inoculated in 10 mL of MRS broth at 37°C for 24 h and is subcultured once more to form active 24-h-old LAB that are ready-to-use. Preparation of crude enzymes The crude enzymes produced by LAB isolates were prepared freshly prior to use. A total of 2% (v/v) of 24-h-old LAB (average OD600nm = 0.713) was inoculated in 10 mL of MRS broth and incubated at 37°C for 24 h. The cell-free supernatant (CFS) containing crude enzymes was collected by centrifugation of the bacterial culture at 10 000 g for 10 min at 4°C and was kept in ice till used. Screening for enzyme producers Amylase activity assay This assay was adapted from the method by Zhang and Zeng (2007) to evaluate the amylase activity of crude enzymes from LAB isolates. In this assay, 1-fold, 2-fold and 10-fold diluted CFS were used accordingly to obtain absorbance readings of less than 2.5. Starch (1 g/100 mL; Bendosen) was used as the substrate and α-amylase (16 units/mg solid; Sigma-Aldrich) was used as the positive control. The reaction mixtures were prepared according to Table 1. The reaction mixtures were incubated at 37°C for 30 min, and the enzymatic reaction was halted by using an ice bath. A total of 0.3 mL of 3,5-dinitrosalicyclic acid (DNS) was added to the reaction mixture and the mixture was heated for 5 min in a 90°C water bath. The reaction was then stopped using an ice bath. Then, 0.2 mL of the reaction mixture was pipetted into a 96-well plate and was measured for absorbance at 550 nm wavelength using Fluostar OPTIMA microplate reader. Table 1. Reaction mixtures for amylase, cellulase and protease activity assay Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Open in new tab Table 1. Reaction mixtures for amylase, cellulase and protease activity assay Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Open in new tab A maltose standard curve was generated to quantify the amount of reducing sugar in the reaction mixture. One amylase unit was defined as the enzymatic activity that liberates one microgram of maltose per minute per millilitre CFS. The initial velocity (V0) of the crude enzyme to liberate one micromole of maltose per minute per millilitre CFS was determined. Cellulase activity assay This assay was adapted from the method by Wood and Bhat (1988) to evaluate the cellulase activity of crude enzymes from LAB isolates. In this assay, 1 g/100 mL carboxymethyl cellulose (CMC; Sigma-Aldrich) was dissolved in 0.05 M glycine/NaOH buffer (pH 9) and used as a substrate. The reaction mixtures were prepared according to Table 1. The reaction mixtures were incubated at 37°C for 30 min, and the enzymatic reaction was halted by using an ice bath. A total of 0.36 mL of DNS were added to the reaction mixture and the mixture was heated for 5 min in a 90°C water bath. The reaction was then stopped using an ice bath. Then, 0.2 mL of the reaction mixture was pipetted into a 96-well plate and was measured for absorbance at 550 nm wavelength using Fluostar OPTIMA microplate reader. A glucose standard curve was generated to quantify the amount of reducing sugar in the reaction mixture. One cellulase unit was defined as the enzymatic activity that liberates one microgram of glucose per minute per millilitre CFS. The V0 of the crude enzyme to liberate one micromole of glucose per minute per millilitre CFS was determined. Positive control was not available due to limitation of resources in the laboratory. Protease activity assay This assay was adapted from the method by Kanekar et al. (2002) to evaluate the protease activity of crude enzymes from LAB isolates. In this assay, 0.6 g/100 mL casein (Sigma-Aldrich) was dissolved in 0.4 M Tris-HCl buffer (pH 9) and used as a substrate. The reaction mixtures were prepared according to Table 1. The reaction mixtures were incubated at 37°C for 30 min, and the enzymatic reaction was terminated by adding 0.5 mL of 5 g/100 mL tricloroacetic acid. Then, 0.25 mL of the reaction mixture was mixed with 0.25 mL of Bradford’s reagent in a 96-well plate and was allowed to react for 5 min at room temperature. A tryptophan standard curve was generated to quantify the amount of protein in the reaction mixture. One protease unit was defined as the enzymatic activity that liberates one microgram of tryptophan per minute per millilitre CFS. The V0 of the crude enzyme to liberate one micromole of tryptophan per minute per millilitre CFS was determined. Positive control was not available due to limitation of resources in the laboratory. Identification via carbohydrate metabolism tests using API® 50 CHL kit Before testing the LAB for carbohydrate fermentation, the two isolates were subjected to a catalase test. A sterile loop was used to place a small amount of LAB growth onto the base of a Petri dish, then one drop of hydrogen peroxide was added and the Petri dish was immediately covered with a lid. The development of bubbles (effervescence) indicates a positive result. The selected LAB isolated were then tested for their carbohydrate fermentation pattern using the API® 50 CHL system kit (BioMérieux). The identification procedure was conducted in accordance to the manufacturer’s instructions. Active 24-h-old LAB isolates were added into the API 50 CHL basal medium and were filled into wells on the API 50 CHL test strips. The strips were then incubated at 37°C for 48 h. The strips were visually examined at 24 and 48 h, and the indication of a positive or negative result was determined from the colour change from a scale of 1 to 5, from purple (1) to green (3) to yellow (5) at the 48-h mark, whereby a value of ≥3 was considered positive. The results were then cross-referenced to the API® databases using APIweb™. DNA extraction, polymerase chain reaction amplification and DNA sequencing of 16 s rDNA gene DNA extraction was carried out using Wizard® Genomic DNA Purification Kit (Promega) on 24-h-old LAB isolates. Polymerase chain reaction (PCR) was carried out in total volumes of 20 μL containing 5 U/μL DreamTaq polymerase, 10× DreamTaq Buffer, 2 mM deoxyribonucleotide phosphate, 10 mM U8 Forward primer (5′-AGA GTT TGA TCC TGG CTC AG-3′), 10 mM 1492 reverse primer (5′-CGG TTA CCT TGT TAC GAC TT-3′), sterile distilled water and 5 ng/μL LAB DNA samples. DNA amplification was performed for 30 cycles and the PCR cycle was set as the initial denaturation at 95°C for 4 min, annealing at 65°C for 1 min, elongation at 72°C for 1 min, and followed by another 29 cycles of denaturation at 95°C for 1 min, annealing at 65°C for 1 min and elongation at 72°C for 1 min. A total of 20 μL of the amplified PCR products and a negative control were electrophoresed on 1 g/100 mL agarose gel containing SYBR® Safe and 1× Tris-acetate–EDTA buffer (TAE) at 75 V for 40 min. The gel was visualized using Gel Doc™ EZ Gel Documentation System (Bio-Rad). The remaining amplified PCR products were then sent to First BASE Laboratories Sdn. Bhd. for sequencing using the U8F and 1492 R primers under the DNA Sequencing Service + Plus (SS1201) service. The DNA sequence obtained was then compared with sequences in the GenBank database using BLAST by the National Center for Biotechnology Information (NCBI). Determination of the enzyme kinetics by using Hanes–Woolf plot The Michaelis–Menten constant (Km) and maximal velocity (Vmax) of the crude enzymes were calculated using the Hanes–Woolf plot. The V0 of the enzyme reaction was determined according to methods described in Sections 3.4.1–3.4.3. In this assay, a range of substrate concentrations were used for amylase (ranging from 0 to 20 g/100 mL starch), cellulase (ranging from 0 to 1.3 g/100 mL CMC) and protease (ranging from 0 to 1.5 g/100 mL casein). The Hanes–Woolf graph was then plotted to determine the Km and Vmax values of the targeted crude enzymes. A graph of [S]/V0 (g min/100μmol) against substrate concentration, [S] (g/100 mL) was plotted. As the Hanes–Woolf equation is ‘[S]/V0 = (1/Vmax)([S]) + Km/Vmax’, the Vmax was calculated by taking the reciprocal value of the gradient of the graph. Km is equal to the negative of the x-intercept, thus was calculated by taking the negative of the [S] value when [S]/V0 = 0. Statistical analysis Statistical analyses were carried out using IBM SPSS Statistics 23. All experimental data were obtained in triplicates and were analysed using the analysis of variance (ANOVA) test followed by Duncan’s post hoc test at P ≤ 0.05. Correlation between amylase and cellulase assay was analysed using Pearson’s correlation coefficient test. Results and discussion Isolation of LAB from fermented foods A total of 12 LAB were isolated from the four fermented foods, two isolates from ‘tapai pulut’ (TAP1–2), four isolates from ‘tempeh’ (TEM1–4), three isolates from ‘tempoyak’ (TYK1–3) and three isolates from ‘fu yu’ (FY1–3). However, only 7 out of 12 were still viable after repeated subcultures, hence only TAP2, TEM1, TEM2, TYK2, TYK3, FY2 and FY3 were chosen for preliminary screening of enzyme producers. This loss of viability for the five isolates could be due to two factors, (i) the selective media (MRS) used was not providing the nutrients needed and (ii) the changes in growth environment. This finding was similar to those reported by Birollo, Reinheimer and Vinderola (2000) showing that MRS may show a lower viable count of bacteria and there are other suitable alternatives to grow LAB. Other than that, the other LAB isolates may be in the unique form of viable but non-culturable state, whereby they cannot be cultured on routine microbiological media but are still viable (Fakruddin, Mannan and Andrews, 2013). Thus, the LAB isolates were most likely unable to acclimatize and grow continuously in MRS broth. This may be due to unfavourable environmental factors such as starvation, non-optimal pH or others (Pinto, Santos and Chambel, 2015). Morphological and physiological characterization of LAB The seven LAB isolates tested were Gram-positive, and two isolates were rod-shaped and the remaining five were coccus-shaped. FY2 had showed a tetracoccus cell arrangement, TEM2 and TYK3 showed diplococcus cell arrangement, while TEM1 and FY3 had no distinct coccus arrangements. The two rod-shaped isolates, TAP2 and TYK2 tended to form short chains. A summary of the morphology of the LAB isolates can be seen in Table 2. Table 2. Colony and cell morphology of LAB isolates LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus Open in new tab Table 2. Colony and cell morphology of LAB isolates LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus Open in new tab Screening for enzyme producers The seven LAB isolates tested were able to produce amylase, cellulase and protease enzymes at different strengths. The enzyme activity of the LAB isolates was determined via the liberation of a specific substrate based on the designated enzyme assay are depicted in Table 3. Table 3. Amylase, cellulase and protease activity of LAB isolates LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a Values are mean ± standard deviation, n = 3 (amylase and protease), n = 4 (cellulase). abcdef Within a column, values with different superscripts are significantly different at P ≤ 0.05. Open in new tab Table 3. Amylase, cellulase and protease activity of LAB isolates LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a Values are mean ± standard deviation, n = 3 (amylase and protease), n = 4 (cellulase). abcdef Within a column, values with different superscripts are significantly different at P ≤ 0.05. Open in new tab The amylase activity of isolate FY3 was ranked highest, followed by FY2 and TEM1 (Table 3). This finding was interesting as these three isolates were not isolated from carbohydrate-rich food sources (‘fu yu’ and ‘tempeh’), whereas isolates from carbohydrate-rich food sources (‘tapai’ and ‘tempoyak’) such as TAP2, TYK2 and TYK3 did not show high level of amylase activity. Similarly, the cellulase activity of FY3 was also ranked highest, followed by FY2 and TEM1 (Table 3). Pearson’s coefficient test showed that there was a significant positive correlation (r2 = +0.979) between amylase and cellulase activity in all LAB isolates. This finding suggests that amylase and cellulase production could be correlated. However, to the author’s knowledge, no relevant published literature was found. All LAB isolates were good producers of proteases as seen in Table 3. The protease activity of all isolates was at least 2-folds higher than amylase and cellulase activities under the assay conditions. However, no significant difference in protease activity was observed between isolates. This finding was in accordance with the study by Shin et al. (2008), where LAB from the genera Pediococcus and Enterococcus displayed high amylase and cellulase activities but did not exhibit any significant difference of protease activity compared to other bacteria. Therefore, the two top producers for amylase and cellulase isolated from ‘fu yu’—FY2 and FY3 were selected for further identification and enzyme kinetic assay. Bacterial identification via carbohydrate metabolism tests using API® 50 CHL kit FY2 and FY3 which displayed the highest amylase and cellulase activities were identified by using the API 50 CHL system kit to test for their carbohydrate fermentation patterns (Table 4). A catalase test was performed to complement the API 50 CHL test, where both isolates were found to be catalase-negative. Table 4. Carbohydrate fermentation pattern of FY2 and FY3 using API 50 CHL system kit No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − (−) negative, (+) positive, (±) undetermined. Open in new tab Table 4. Carbohydrate fermentation pattern of FY2 and FY3 using API 50 CHL system kit No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − (−) negative, (+) positive, (±) undetermined. Open in new tab Based on the API 50 CHL results in Table 4, FY2 was initially identified with a low % ID value of 34.2% as P. acidilactici. The % ID for FY2 meant that 34.2% of LAB with this specific biochemical profile was found to be P. acidilactici. The low % ID was due to the discrepancies between the biochemical profile of P. acidilactici in the APIweb® system as compared to the API 50 CHL results of FY2. FY2 exhibited the inability to utilize l-arabinose, d-galactose, l-rhamnose, salicin, d-trehalose and d-tagatose, whereas P. acidilactici in the APIweb® system had the following biochemical profile: (l-arabinose 100%), (d-galactose 100%), (l-rhamnose 75%), (salicin 75%), (d-trehalose 75%) and (d-tagatose 100%), whereby the percentage represents the likelihood of that particular LAB strain to have the ability to ferment that sugar. On the other hand, based on Table 4, FY3 was initially identified as Pediococcus pentosaceus with a higher % ID value of 80.9% but also displayed biochemical discrepancies such as the inability to utilize l-arabinose, d-trehalose and d-tagatose as compared to the APIweb® system’s biochemical profile for P. pentosaceus (l-arabinose 100%), (d-trehalose 99%) and (d-tagatose 99%). Bacterial identification via 16s rDNA gene sequencing analysis Since the API carbohydrate fermentation test was unable to confirm the identity for FY2, both isolates were then subjected to 16 s rDNA gene sequencing analysis for further confirmation. Based on the gene sequences (Fig. 1A) and BLAST results for FY2, 16 s rDNA gene sequencing analysis showed a similarity of 94%, 94% and 92% with P. acidilactici, P. pentosaceus and P. stilesil, respectively. However, the query cover for P. acidilactici was at 97%, whereas P. pentosaceus was at 94%. Thus, it was highly suspected that FY2 was indeed P. acidilactici due to a higher similarity in the biochemical test compared to P. pentosaceus. Besides that, the morphology of the LAB to form tetracoccus also supported the evidence of FY2 to be under the genera Pediococcus. Figure 1. Open in new tabDownload slide 16 s rDNA sequence of FY2 (A) and FY3 (B). Based on the gene sequences (Fig. 1B) and BLAST results for FY3, a similarity of 95%, 95% and 94% with Enterococcus durans, E. faecium and E. mundtii was obtained respectively. The query cover for E. durans and E. faecium was the same at 80%, thus the biochemical properties were examined to assist in identifying the species. However, the APIweb™ database did not show LAB from the genera Enterococcus, hence other literature were used as a reference. According to Bergey's manual of Determinative Bacteriology, FY3 showed discrepancies for both Enterococcus LAB such as raffinose (+) and mellibiose (+) (Holt, 2000). However, based on Bergey’s manual FY3 was more likely to be E. durans due to its inability to utilize glycerol (−) and d-mannitol (−), as compared to E. faecium. The discrepancies between the biochemical and molecular identification techniques might be due to the fact that principles of both techniques are fundamentally different. API 50 CHL is used to classify LAB based on their phenotypical properties by comparing the fermentation pattern of 49 different types of carbohydrate with other bacteria that are registered in the APIweb™ database. One of the limitations of this is that the APIweb™ database has been found to be lacking in biochemical profiles of certain LAB (Boyd et al., 2005). Besides that, phenotypical characterization has been known to have poor reproducibility and that the whole information potential of the LAB genome is not always expressed, as gene expression is directly related to environmental conditions such as the growth conditions in the laboratory (Mohania et al., 2008). On the other hand, 16 s rDNA sequencing relies on the amplification and sequencing of the highly conserved 16 s ribosomal RNA gene, which is akin to a unique biosignature for any organism (Isenbarger et al., 2008). Janda and Abbott (2007) reported that 16 s rDNA sequencing is an alternative to provide identification of unknown bacteria with unrecognized biochemical profiles or a low likelihood. This sequencing technique can provide information about the genus and species of most unknown bacteria, with high levels of genus identification (>90%) and moderate levels of species identification (65–83%; Janda and Abbott, 2007). A study by Bağder et al. (2014) compared the results of the API 50 CHL test with 16 s rRNA results and found that the API test did not give reliable identification results, with only 71 out of 152 tested isolates were in agreement. Another study by Moraes et al. (2013) reported the possibility for high reliability rates in the API 50 CHL to diverge greatly from 16 s rDNA results, supporting the identification of FY3 which had a high API 50 CHL % ID for P. pentosaceus (80.9%) but was identified as E. durans (95%) through BLAST. However, one of the limitations of using 16 s rDNA sequencing is that LAB in the genera Enterococcus are difficult to differentiate due to the highly conserved nature of 16 s rDNA (Moraes et al., 2013). Thus, the API 50 CHL biochemical test results were used as supporting evidence to support the main identification technique, 16 s rDNA sequencing to help discern the LAB isolates based on their genome and carbohydrate metabolism patterns. The finding of the amylase producers—FY2 and FY3 from the species of P. acidilactici and E. durans were in accordance to a report by Velikova et al. (2016) in which their targeted strains P. acidilactici and E. durans were also found to have highest extracellular and intracellular amylase activities, respectively. However, this finding was in contrast with a study by Musikasang et al. (2009), as bacterium from the genera Pediococcus and E. durans that were isolated from chicken’s gastrointestinal tract did not exhibit the ability to digest starch. According to Velikova et al. (2016), amylase-producing LAB contain a basic pool of chromosomal genes that is responsible for starch hydrolysis. However, only the strains that are forced to survive in starchy environment are able to display these genetically determined properties. Hence, the gastrointestinal tract of chicken might not have an ideal environment for these LAB to grow. These findings suggest that LAB isolated from different sources such as plants or animals may exhibit different enzymatic properties. According to Bergey’s manual, P. acidilactici can grow at higher temperatures of up to 50°C, whereas E. durans can grow at 45°C. This may be beneficial as the two LAB could possibly produce thermostable amylases which are of special interest in the industrial field, especially for starch saccharification (Saxena et al., 2007). Hence, further tests should be carried out to evaluate the ability of FY2 and FY3 to produce potential thermostable amylases. On the other hand, the cellulase-producing potential of FY3 (E. durans) was similar to the finding by Shil et al. (2014) where they first discovered cellulase activity from an E. durans strain that was isolated from the gut of the phytophagous insect Oxya velox. However, this was in contrast to a study by Mazzucotelli et al. (2013), as the E. durans isolated from cheese whey did not exhibit the ability to degrade cellulose. These findings further support the previous suggestion that LAB isolated from various sources may exhibit different enzymatic properties. Besides that, FY2 also exhibited high cellulase activity, similar to a study reported by Ventorino et al. (2015), whereby P. acidilactici which was isolated from biomass piles of Eucalyptus camaldulensis was found to have high levels of azo-carboxymethylcellulase activity. The protease-producing potential of FY2 and FY3 was contrasted with a study by Tuncer (2009), whereby E. durans isolated from Turkish tulum cheese showed varied levels of protease activity, ranging from low to high based on the strain observed, and even more variation among different species. Besides that, Moslehishad et al. (2013) reported that P. acidilactici PTCC1424 from the Iranian Research Organization for Science and Technology exhibited moderate protease activity in supernatant form. However, Moslehishad et al. (2013) also reported that different LAB had varying levels of protease activity based on the culture conditions, such as in CFS form, anaerobic conditions and enriched CO2 conditions, whereby anaerobic conditions showed more favourable protease activity. Although the protease activity was about 2-fold higher than the amylase and cellulase activities, these findings suggest that LAB protease activity could be even higher, if the assay were to be optimized. Determination of the enzyme kinetics by using Hanes–Woolf plot According to Berg, Tymoczko and Stryer (2002), enzymes are important to enhance the rate of biochemical reactions, thus the kinetic description of their activity is required to understand about the enzyme’s function. Thus, a graph of [S]/V0 against [S] was plotted to calculate Vmax and Km. Vmax is the maximum reaction velocity achieved by an enzyme and can be used to indicate the maximum capacity of an enzyme to carry out enzymatic reactions, provided sufficient substrates are available. Km is equal to [S] at which reaction velocity is equal to half of Vmax and is independent of enzyme and substrate concentrations. Km shows the required [S] needed for significant catalysis to occur and determines the viability of the enzyme for industrial use. Km and Vmax of LAB isolates FY2 and FY3 were calculated by plotting graphs as seen in Figs 2–4. For Fig. 4, the graph not extrapolated the past y-axis due to the nature of the graph. Figure 2. Open in new tabDownload slide Hanes–Woolf plot for starch concentration, [S]/V0 against [S]. Figure 3. Open in new tabDownload slide Hanes–Woolf plot for CMC concentration, [S]/V0 against [S]. Figure 4. Open in new tabDownload slide Hanes–Woolf plot for casein concentration, [S]/V0 against [S]. Based on Fig. 2, the amylase V0 of FY3 is generally higher than FY2, as its Vmax value was slightly higher (5.51–4.43 μmol/mL/min) and Km value was lower (0.299–0.433 g/100 mL) than FY2. This result suggests that both LAB isolates are not very efficient at degrading starch, as a high substrate concentration (average 4.97 g/100 mL) would be required for its enzyme to achieve maximum capability. However, due to the lack of amylase-producing LAB, the two isolates may still be of industrial use during fermentation processes (Fossi and Tavea, 2013). The cellulase V0 of FY3 was also higher than FY2 as seen in Fig. 3. FY3 had a higher Vmax value (3.50–2.66 μmol/mL/min) but also had a higher Km value (0.006–0.002 g/100 mL) than FY2. Hence, these results suggest that FY3 is a better cellulase producer than FY2 and the cellulase produced by both isolates are efficient at degrading carboxymethylcellulose due to their low Km value. Based on both amylase and cellulase assay, FY3 would be a better option to be studied as an industrial enzyme producer as it can achieve higher enzymatic activity when substrate concentration is saturated. FY3 would have a lower requirement of substrate concentration to achieve optimum enzymatic activity for amylase while also having a low requirement of substrate concentration for cellulase. Interestingly, FY2 had a higher Vmax value than FY3 (2.14–0.51 μmol/mL/min) but also a higher negative Km (−0.348 to −0.287 g/100 mL) in terms of protease activity as seen in Fig. 4. This negative value is due to a drastic reduction in protease reaction activity at casein concentrations higher than 1.2 g/100 mL, causing the FY3 graph to have an R2 value of 0.27. Koka and Weimer (2000) had a similar observation, whereby Pseudomonas fluorescens was observed to have a sudden reduction in protease activity at casein concentrations greater than 100 mmol. This was suspected to be caused by either casein micelle formation or self-aggregration, which would reduce the number of available bonds for hydrolysis (Koka and Weimer, 2000). In terms of protease, FY2 would be a more suitable choice compared to FY3 due to its higher Vmax value albeit it has a higher Km, as the difference between Km values is smaller than the difference between Vmax values of the two isolates. Conclusion This study has demonstrated that traditional fermented foods, namely, ‘tapai pulut’, ‘tempeh’, ‘tempoyak’ and ‘fu yu’ are the potential resources for isolating LAB enzyme producers. All seven LAB isolates tested are strong producers of protease and two of them (FY2 and FY3) also exhibited the highest amylase and cellulase production. FY2 and FY3 were identified as P. acidilactici and E. durans based on biochemical profiling and 16 s rDNA sequencing. Furthermore, FY3 had a higher overall V0 than FY2, hence FY3 is a better candidate for future industrial application. However, further studies are essential to enhance the understanding on these producers and the enzymes produced. Future studies should be carried out to confirm the Km value for protease production, to purify and characterize all the three enzymes produced and to optimize the growth conditions of FY2 and FY3 for future application in silage treatment. Author Biography Jason Chen graduated from the University of Nottingham (Malaysia Campus) in the year 2017 with a First Class Honours BSc in Biotechnology. This research article was submitted in partial fulfilment of the requirements for Jason's degree in his final year. Having an interest in microorganisms and food, Jason is hoping to pursue a career related to the food and microorganism industry. J.K.H., C. designed the study, carried out the research and wrote the paper. He has primary responsibility for the paper. Y.S., L. contributed in designing the initial project scope and improving the quality of the paper. Acknowledgements I would like to express my gratitude to all those who provided me the chance to complete this report. Special thanks should be given to Dr Lim Yin Sze, my final year project supervisor, for her professional guidance, patience and useful critiques for this project. My grateful thanks are extended to the School of Biosciences, University of Nottingham Malaysia Campus, for providing the opportunity to carry out this project. I would like to extend my thanks to the technicians for their help in providing resources. Finally, I wish to thank my family and friends for their support throughout this project. References Bağder , E. S. , Tokatlı , M., Dursun , D. et al. . 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( 2007 ) Psychrotrophic amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases , World Journal of Microbiology and Biotechnology , 23 ( 11 ), 1551 – 1557 . Google Scholar Crossref Search ADS WorldCat Author notes Supervisor: Dr Lim Yin Sze, School of Biosciences, Faculty of Science, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia Author’s Biography: Jason Chen graduated from the University of Nottingham (Malaysia Campus) in the year 2017 with a first-class honours BSc in Biotechnology. This research article was submitted in partial fulfilment of the requirements for Jason’s degree in his final year. Having an interest in microorganisms and food, Jason is hoping to pursue a career related to the food and microorganism industry. He designed the study, carried out the research and wrote the paper. He has primary responsibility for the paper. Yin Sze Lim contributed in designing the initial project scope and improving the quality of the paper © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2018. Published by Oxford University Press. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BioScience Horizons Oxford University Press

Isolation, identification and characterization of enzyme-producing lactic acid bacteria from traditional fermented foods

BioScience Horizons , Volume 11 – Jan 1, 2018

Isolation, identification and characterization of enzyme-producing lactic acid bacteria from traditional fermented foods

BioScience Horizons , Volume 11 – Jan 1, 2018

Abstract

Abstract Industrialization processes are prone to produce various forms of waste which can be utilized to produce silage. These wastes can be treated by using lactic acid bacteria (LAB), which are known to be potential enzyme producers. Seven strains of LAB were isolated from traditional fermented food, ‘tapai pulut’, ‘tempeh’, ‘tempoyak’ and ‘fu yu’ and were screened for amylase, cellulase and protease production in order to select for strains that could potentially be used industrially in silage production. All seven LAB isolates exhibited high protease production, and two of them also exhibited high amylase and cellulase production. The two isolates exhibiting high amylase and cellulase production were selected and identified as Pediococcus acidilactici FY2 and Enterococcus durans FY3 via biochemical profiling (API 50 CHL) and 16 s rDNA sequencing. E. durans was found to have the highest amylase (Vmax: 5.51 μmol/mL/min; Km: 0.300 g/100 mL) and cellulase (Vmax: 3.50 μmol/mL/min; Km: 0.006 g/100 mL) production, while exhibiting strong protease production (Vmax: 0.51 μmol/mL/min; Km: −0.287 g/100 mL). P. acidilactici was found to have strong amylase (Vmax: 4.43 μmol/mL/min; Km: 0.433 g/100 mL) and cellulase (Vmax: 2.66 μmol/mL/min; Km: 0.002 g/100 mL) production while exhibiting the highest protease production (Vmax: 2.14 μmol/mL/min; Km: −0.348 g/100 mL). These results suggest that E. durans is a better candidate for future industrial application as overall it has a higher enzyme reaction velocity when compared to P. acidilactici. Further studies should be carried out to confirm the Km value for protease production, to purify and characterize all three enzymes produced and to optimize the growth conditions of E. durans. lactic acid bacteria, amylase, cellulase, protease, Pediococcus acidilactici, Enterococcus durans Introduction In this modern era, the industrialization process is growing at an alarming rate. The continuous discovery and invention of state-of-the-art technology continually pushes the industrial world to evolve at a fast pace. Thus, prioritizing the usage of existing resources is important to ensure optimal production and prevent wastage. Various agricultural industrial processes and general food consumption produce large amounts of wastes, and some of these can be utilized in silage production as the waste are usually rich in sugars and proteins (Show and Guo, 2012). Ensilage is preserved forage crops that is generally fermented by lactic acid bacteria (LAB) under anaerobic conditions and is usually void of undesirable microorganisms due to the production of lactic acid by the LAB (Ni et al., 2015b). Thus, scientists are searching for alternative microbial sources to find better strains to process silage, as LAB are well-known producers of extracellular enzymes (Patel, Shah and Prajapati, 2013; Tosungnoen, Chookietwattana and Dararat, 2014; Ni et al., 2015a). LAB with amylase and cellulase activity to degrade complex sugars such as starch and cellulose are much sought after, as this would reduce the cost of substrate pretreatment (Shibata et al., 2007; Yitbarek and Tamir, 2014). LAB are a group of Gram-positive bacteria that produce lactic acid during fermentation and are important for food manufacturing, especially in the milk, vegetables and meat industries (Konings et al., 2000; González et al., 2010). Various LAB are generally regarded as safe and have been used for a very long time as starter cultures to produce fermented foods through traditional means, as they can produce lactic acid and bacteriocins that act as natural preservatives and thus can extend the shelf life of silage (Holzapfel, Geisen and Schillinger, 1995; Perez, Zendo and Sonomoto, 2014). Therefore, LAB should be abundantly found in fermented foods. In this current study, traditional fermented foods such as fermented glutinous rice ‘tapai pulut’, fermented soybean cake ‘tempeh’, fermented durian condiment ‘tempoyak’ and fermented soybean curd ‘fu yu’ were investigated. The aim of this study was to isolate and screen for putative LAB producers for enzymes amylase, cellulase and protease from traditional fermented foods for future industrial application. The putative enzyme producers were identified using biochemical and molecular techniques, and the enzyme kinetics were determined. Materials and methods Isolation of LAB from fermented foods Four different types of local fermented foods, fermented glutinous rice ‘tapai pulut’ (TAP), fermented soybean cake ‘tempeh’ (TEM), fermented durian condiment ‘tempoyak’ (TYK) and fermented soybean curd ‘fu yu’ (FY) were purchased from a grocery store in Selangor, Malaysia. A total of 10 g of the food samples were mixed with 90 mL of peptone-buffered water and incubated at room temperature for 30 min. A 10-fold serial dilution was carried out from 10−1 to 10−7. Volumes of 0.1 mL of diluted samples were spread on de Man, Rogosa and Sharpe (MRS) agar plates and incubated at 37°C for 48 h. MRS agar is a selective media for LAB growth. After incubation, colonies with different morphology were randomly selected, Gram-stained and examined for cell morphology under a microscope. Maintenance and preparation of bacterial isolates Each LAB isolate was preserved in 20% (v/v) glycerol and stored in a −20°C freezer till used. Initially, isolates were kept active via subculturing by pipetting 0.2 mL of active culture into 10 mL of MRS broth, followed by incubation at 37°C for up to 72 h before the next subculture. In the event of no growth, 0.2 mL of the glycerol stock was inoculated in 10 mL of MRS broth and incubated at 37°C for 24 h. Then, a four-point streak was carried out on MRS agar to ensure no contamination occurred. A single pure colony was selected and inoculated in 10 mL of MRS broth and incubated at 37°C for 24 h, followed by another subculture in 10 mL of MRS broth and incubation at 37°C for 24 h, forming active 24-h-old LAB. Two days prior to an assay, 0.2 mL of active LAB isolates were inoculated in 10 mL of MRS broth at 37°C for 24 h and is subcultured once more to form active 24-h-old LAB that are ready-to-use. Preparation of crude enzymes The crude enzymes produced by LAB isolates were prepared freshly prior to use. A total of 2% (v/v) of 24-h-old LAB (average OD600nm = 0.713) was inoculated in 10 mL of MRS broth and incubated at 37°C for 24 h. The cell-free supernatant (CFS) containing crude enzymes was collected by centrifugation of the bacterial culture at 10 000 g for 10 min at 4°C and was kept in ice till used. Screening for enzyme producers Amylase activity assay This assay was adapted from the method by Zhang and Zeng (2007) to evaluate the amylase activity of crude enzymes from LAB isolates. In this assay, 1-fold, 2-fold and 10-fold diluted CFS were used accordingly to obtain absorbance readings of less than 2.5. Starch (1 g/100 mL; Bendosen) was used as the substrate and α-amylase (16 units/mg solid; Sigma-Aldrich) was used as the positive control. The reaction mixtures were prepared according to Table 1. The reaction mixtures were incubated at 37°C for 30 min, and the enzymatic reaction was halted by using an ice bath. A total of 0.3 mL of 3,5-dinitrosalicyclic acid (DNS) was added to the reaction mixture and the mixture was heated for 5 min in a 90°C water bath. The reaction was then stopped using an ice bath. Then, 0.2 mL of the reaction mixture was pipetted into a 96-well plate and was measured for absorbance at 550 nm wavelength using Fluostar OPTIMA microplate reader. Table 1. Reaction mixtures for amylase, cellulase and protease activity assay Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Open in new tab Table 1. Reaction mixtures for amylase, cellulase and protease activity assay Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Open in new tab A maltose standard curve was generated to quantify the amount of reducing sugar in the reaction mixture. One amylase unit was defined as the enzymatic activity that liberates one microgram of maltose per minute per millilitre CFS. The initial velocity (V0) of the crude enzyme to liberate one micromole of maltose per minute per millilitre CFS was determined. Cellulase activity assay This assay was adapted from the method by Wood and Bhat (1988) to evaluate the cellulase activity of crude enzymes from LAB isolates. In this assay, 1 g/100 mL carboxymethyl cellulose (CMC; Sigma-Aldrich) was dissolved in 0.05 M glycine/NaOH buffer (pH 9) and used as a substrate. The reaction mixtures were prepared according to Table 1. The reaction mixtures were incubated at 37°C for 30 min, and the enzymatic reaction was halted by using an ice bath. A total of 0.36 mL of DNS were added to the reaction mixture and the mixture was heated for 5 min in a 90°C water bath. The reaction was then stopped using an ice bath. Then, 0.2 mL of the reaction mixture was pipetted into a 96-well plate and was measured for absorbance at 550 nm wavelength using Fluostar OPTIMA microplate reader. A glucose standard curve was generated to quantify the amount of reducing sugar in the reaction mixture. One cellulase unit was defined as the enzymatic activity that liberates one microgram of glucose per minute per millilitre CFS. The V0 of the crude enzyme to liberate one micromole of glucose per minute per millilitre CFS was determined. Positive control was not available due to limitation of resources in the laboratory. Protease activity assay This assay was adapted from the method by Kanekar et al. (2002) to evaluate the protease activity of crude enzymes from LAB isolates. In this assay, 0.6 g/100 mL casein (Sigma-Aldrich) was dissolved in 0.4 M Tris-HCl buffer (pH 9) and used as a substrate. The reaction mixtures were prepared according to Table 1. The reaction mixtures were incubated at 37°C for 30 min, and the enzymatic reaction was terminated by adding 0.5 mL of 5 g/100 mL tricloroacetic acid. Then, 0.25 mL of the reaction mixture was mixed with 0.25 mL of Bradford’s reagent in a 96-well plate and was allowed to react for 5 min at room temperature. A tryptophan standard curve was generated to quantify the amount of protein in the reaction mixture. One protease unit was defined as the enzymatic activity that liberates one microgram of tryptophan per minute per millilitre CFS. The V0 of the crude enzyme to liberate one micromole of tryptophan per minute per millilitre CFS was determined. Positive control was not available due to limitation of resources in the laboratory. Identification via carbohydrate metabolism tests using API® 50 CHL kit Before testing the LAB for carbohydrate fermentation, the two isolates were subjected to a catalase test. A sterile loop was used to place a small amount of LAB growth onto the base of a Petri dish, then one drop of hydrogen peroxide was added and the Petri dish was immediately covered with a lid. The development of bubbles (effervescence) indicates a positive result. The selected LAB isolated were then tested for their carbohydrate fermentation pattern using the API® 50 CHL system kit (BioMérieux). The identification procedure was conducted in accordance to the manufacturer’s instructions. Active 24-h-old LAB isolates were added into the API 50 CHL basal medium and were filled into wells on the API 50 CHL test strips. The strips were then incubated at 37°C for 48 h. The strips were visually examined at 24 and 48 h, and the indication of a positive or negative result was determined from the colour change from a scale of 1 to 5, from purple (1) to green (3) to yellow (5) at the 48-h mark, whereby a value of ≥3 was considered positive. The results were then cross-referenced to the API® databases using APIweb™. DNA extraction, polymerase chain reaction amplification and DNA sequencing of 16 s rDNA gene DNA extraction was carried out using Wizard® Genomic DNA Purification Kit (Promega) on 24-h-old LAB isolates. Polymerase chain reaction (PCR) was carried out in total volumes of 20 μL containing 5 U/μL DreamTaq polymerase, 10× DreamTaq Buffer, 2 mM deoxyribonucleotide phosphate, 10 mM U8 Forward primer (5′-AGA GTT TGA TCC TGG CTC AG-3′), 10 mM 1492 reverse primer (5′-CGG TTA CCT TGT TAC GAC TT-3′), sterile distilled water and 5 ng/μL LAB DNA samples. DNA amplification was performed for 30 cycles and the PCR cycle was set as the initial denaturation at 95°C for 4 min, annealing at 65°C for 1 min, elongation at 72°C for 1 min, and followed by another 29 cycles of denaturation at 95°C for 1 min, annealing at 65°C for 1 min and elongation at 72°C for 1 min. A total of 20 μL of the amplified PCR products and a negative control were electrophoresed on 1 g/100 mL agarose gel containing SYBR® Safe and 1× Tris-acetate–EDTA buffer (TAE) at 75 V for 40 min. The gel was visualized using Gel Doc™ EZ Gel Documentation System (Bio-Rad). The remaining amplified PCR products were then sent to First BASE Laboratories Sdn. Bhd. for sequencing using the U8F and 1492 R primers under the DNA Sequencing Service + Plus (SS1201) service. The DNA sequence obtained was then compared with sequences in the GenBank database using BLAST by the National Center for Biotechnology Information (NCBI). Determination of the enzyme kinetics by using Hanes–Woolf plot The Michaelis–Menten constant (Km) and maximal velocity (Vmax) of the crude enzymes were calculated using the Hanes–Woolf plot. The V0 of the enzyme reaction was determined according to methods described in Sections 3.4.1–3.4.3. In this assay, a range of substrate concentrations were used for amylase (ranging from 0 to 20 g/100 mL starch), cellulase (ranging from 0 to 1.3 g/100 mL CMC) and protease (ranging from 0 to 1.5 g/100 mL casein). The Hanes–Woolf graph was then plotted to determine the Km and Vmax values of the targeted crude enzymes. A graph of [S]/V0 (g min/100μmol) against substrate concentration, [S] (g/100 mL) was plotted. As the Hanes–Woolf equation is ‘[S]/V0 = (1/Vmax)([S]) + Km/Vmax’, the Vmax was calculated by taking the reciprocal value of the gradient of the graph. Km is equal to the negative of the x-intercept, thus was calculated by taking the negative of the [S] value when [S]/V0 = 0. Statistical analysis Statistical analyses were carried out using IBM SPSS Statistics 23. All experimental data were obtained in triplicates and were analysed using the analysis of variance (ANOVA) test followed by Duncan’s post hoc test at P ≤ 0.05. Correlation between amylase and cellulase assay was analysed using Pearson’s correlation coefficient test. Results and discussion Isolation of LAB from fermented foods A total of 12 LAB were isolated from the four fermented foods, two isolates from ‘tapai pulut’ (TAP1–2), four isolates from ‘tempeh’ (TEM1–4), three isolates from ‘tempoyak’ (TYK1–3) and three isolates from ‘fu yu’ (FY1–3). However, only 7 out of 12 were still viable after repeated subcultures, hence only TAP2, TEM1, TEM2, TYK2, TYK3, FY2 and FY3 were chosen for preliminary screening of enzyme producers. This loss of viability for the five isolates could be due to two factors, (i) the selective media (MRS) used was not providing the nutrients needed and (ii) the changes in growth environment. This finding was similar to those reported by Birollo, Reinheimer and Vinderola (2000) showing that MRS may show a lower viable count of bacteria and there are other suitable alternatives to grow LAB. Other than that, the other LAB isolates may be in the unique form of viable but non-culturable state, whereby they cannot be cultured on routine microbiological media but are still viable (Fakruddin, Mannan and Andrews, 2013). Thus, the LAB isolates were most likely unable to acclimatize and grow continuously in MRS broth. This may be due to unfavourable environmental factors such as starvation, non-optimal pH or others (Pinto, Santos and Chambel, 2015). Morphological and physiological characterization of LAB The seven LAB isolates tested were Gram-positive, and two isolates were rod-shaped and the remaining five were coccus-shaped. FY2 had showed a tetracoccus cell arrangement, TEM2 and TYK3 showed diplococcus cell arrangement, while TEM1 and FY3 had no distinct coccus arrangements. The two rod-shaped isolates, TAP2 and TYK2 tended to form short chains. A summary of the morphology of the LAB isolates can be seen in Table 2. Table 2. Colony and cell morphology of LAB isolates LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus Open in new tab Table 2. Colony and cell morphology of LAB isolates LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus Open in new tab Screening for enzyme producers The seven LAB isolates tested were able to produce amylase, cellulase and protease enzymes at different strengths. The enzyme activity of the LAB isolates was determined via the liberation of a specific substrate based on the designated enzyme assay are depicted in Table 3. Table 3. Amylase, cellulase and protease activity of LAB isolates LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a Values are mean ± standard deviation, n = 3 (amylase and protease), n = 4 (cellulase). abcdef Within a column, values with different superscripts are significantly different at P ≤ 0.05. Open in new tab Table 3. Amylase, cellulase and protease activity of LAB isolates LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a Values are mean ± standard deviation, n = 3 (amylase and protease), n = 4 (cellulase). abcdef Within a column, values with different superscripts are significantly different at P ≤ 0.05. Open in new tab The amylase activity of isolate FY3 was ranked highest, followed by FY2 and TEM1 (Table 3). This finding was interesting as these three isolates were not isolated from carbohydrate-rich food sources (‘fu yu’ and ‘tempeh’), whereas isolates from carbohydrate-rich food sources (‘tapai’ and ‘tempoyak’) such as TAP2, TYK2 and TYK3 did not show high level of amylase activity. Similarly, the cellulase activity of FY3 was also ranked highest, followed by FY2 and TEM1 (Table 3). Pearson’s coefficient test showed that there was a significant positive correlation (r2 = +0.979) between amylase and cellulase activity in all LAB isolates. This finding suggests that amylase and cellulase production could be correlated. However, to the author’s knowledge, no relevant published literature was found. All LAB isolates were good producers of proteases as seen in Table 3. The protease activity of all isolates was at least 2-folds higher than amylase and cellulase activities under the assay conditions. However, no significant difference in protease activity was observed between isolates. This finding was in accordance with the study by Shin et al. (2008), where LAB from the genera Pediococcus and Enterococcus displayed high amylase and cellulase activities but did not exhibit any significant difference of protease activity compared to other bacteria. Therefore, the two top producers for amylase and cellulase isolated from ‘fu yu’—FY2 and FY3 were selected for further identification and enzyme kinetic assay. Bacterial identification via carbohydrate metabolism tests using API® 50 CHL kit FY2 and FY3 which displayed the highest amylase and cellulase activities were identified by using the API 50 CHL system kit to test for their carbohydrate fermentation patterns (Table 4). A catalase test was performed to complement the API 50 CHL test, where both isolates were found to be catalase-negative. Table 4. Carbohydrate fermentation pattern of FY2 and FY3 using API 50 CHL system kit No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − (−) negative, (+) positive, (±) undetermined. Open in new tab Table 4. Carbohydrate fermentation pattern of FY2 and FY3 using API 50 CHL system kit No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − (−) negative, (+) positive, (±) undetermined. Open in new tab Based on the API 50 CHL results in Table 4, FY2 was initially identified with a low % ID value of 34.2% as P. acidilactici. The % ID for FY2 meant that 34.2% of LAB with this specific biochemical profile was found to be P. acidilactici. The low % ID was due to the discrepancies between the biochemical profile of P. acidilactici in the APIweb® system as compared to the API 50 CHL results of FY2. FY2 exhibited the inability to utilize l-arabinose, d-galactose, l-rhamnose, salicin, d-trehalose and d-tagatose, whereas P. acidilactici in the APIweb® system had the following biochemical profile: (l-arabinose 100%), (d-galactose 100%), (l-rhamnose 75%), (salicin 75%), (d-trehalose 75%) and (d-tagatose 100%), whereby the percentage represents the likelihood of that particular LAB strain to have the ability to ferment that sugar. On the other hand, based on Table 4, FY3 was initially identified as Pediococcus pentosaceus with a higher % ID value of 80.9% but also displayed biochemical discrepancies such as the inability to utilize l-arabinose, d-trehalose and d-tagatose as compared to the APIweb® system’s biochemical profile for P. pentosaceus (l-arabinose 100%), (d-trehalose 99%) and (d-tagatose 99%). Bacterial identification via 16s rDNA gene sequencing analysis Since the API carbohydrate fermentation test was unable to confirm the identity for FY2, both isolates were then subjected to 16 s rDNA gene sequencing analysis for further confirmation. Based on the gene sequences (Fig. 1A) and BLAST results for FY2, 16 s rDNA gene sequencing analysis showed a similarity of 94%, 94% and 92% with P. acidilactici, P. pentosaceus and P. stilesil, respectively. However, the query cover for P. acidilactici was at 97%, whereas P. pentosaceus was at 94%. Thus, it was highly suspected that FY2 was indeed P. acidilactici due to a higher similarity in the biochemical test compared to P. pentosaceus. Besides that, the morphology of the LAB to form tetracoccus also supported the evidence of FY2 to be under the genera Pediococcus. Figure 1. Open in new tabDownload slide 16 s rDNA sequence of FY2 (A) and FY3 (B). Based on the gene sequences (Fig. 1B) and BLAST results for FY3, a similarity of 95%, 95% and 94% with Enterococcus durans, E. faecium and E. mundtii was obtained respectively. The query cover for E. durans and E. faecium was the same at 80%, thus the biochemical properties were examined to assist in identifying the species. However, the APIweb™ database did not show LAB from the genera Enterococcus, hence other literature were used as a reference. According to Bergey's manual of Determinative Bacteriology, FY3 showed discrepancies for both Enterococcus LAB such as raffinose (+) and mellibiose (+) (Holt, 2000). However, based on Bergey’s manual FY3 was more likely to be E. durans due to its inability to utilize glycerol (−) and d-mannitol (−), as compared to E. faecium. The discrepancies between the biochemical and molecular identification techniques might be due to the fact that principles of both techniques are fundamentally different. API 50 CHL is used to classify LAB based on their phenotypical properties by comparing the fermentation pattern of 49 different types of carbohydrate with other bacteria that are registered in the APIweb™ database. One of the limitations of this is that the APIweb™ database has been found to be lacking in biochemical profiles of certain LAB (Boyd et al., 2005). Besides that, phenotypical characterization has been known to have poor reproducibility and that the whole information potential of the LAB genome is not always expressed, as gene expression is directly related to environmental conditions such as the growth conditions in the laboratory (Mohania et al., 2008). On the other hand, 16 s rDNA sequencing relies on the amplification and sequencing of the highly conserved 16 s ribosomal RNA gene, which is akin to a unique biosignature for any organism (Isenbarger et al., 2008). Janda and Abbott (2007) reported that 16 s rDNA sequencing is an alternative to provide identification of unknown bacteria with unrecognized biochemical profiles or a low likelihood. This sequencing technique can provide information about the genus and species of most unknown bacteria, with high levels of genus identification (>90%) and moderate levels of species identification (65–83%; Janda and Abbott, 2007). A study by Bağder et al. (2014) compared the results of the API 50 CHL test with 16 s rRNA results and found that the API test did not give reliable identification results, with only 71 out of 152 tested isolates were in agreement. Another study by Moraes et al. (2013) reported the possibility for high reliability rates in the API 50 CHL to diverge greatly from 16 s rDNA results, supporting the identification of FY3 which had a high API 50 CHL % ID for P. pentosaceus (80.9%) but was identified as E. durans (95%) through BLAST. However, one of the limitations of using 16 s rDNA sequencing is that LAB in the genera Enterococcus are difficult to differentiate due to the highly conserved nature of 16 s rDNA (Moraes et al., 2013). Thus, the API 50 CHL biochemical test results were used as supporting evidence to support the main identification technique, 16 s rDNA sequencing to help discern the LAB isolates based on their genome and carbohydrate metabolism patterns. The finding of the amylase producers—FY2 and FY3 from the species of P. acidilactici and E. durans were in accordance to a report by Velikova et al. (2016) in which their targeted strains P. acidilactici and E. durans were also found to have highest extracellular and intracellular amylase activities, respectively. However, this finding was in contrast with a study by Musikasang et al. (2009), as bacterium from the genera Pediococcus and E. durans that were isolated from chicken’s gastrointestinal tract did not exhibit the ability to digest starch. According to Velikova et al. (2016), amylase-producing LAB contain a basic pool of chromosomal genes that is responsible for starch hydrolysis. However, only the strains that are forced to survive in starchy environment are able to display these genetically determined properties. Hence, the gastrointestinal tract of chicken might not have an ideal environment for these LAB to grow. These findings suggest that LAB isolated from different sources such as plants or animals may exhibit different enzymatic properties. According to Bergey’s manual, P. acidilactici can grow at higher temperatures of up to 50°C, whereas E. durans can grow at 45°C. This may be beneficial as the two LAB could possibly produce thermostable amylases which are of special interest in the industrial field, especially for starch saccharification (Saxena et al., 2007). Hence, further tests should be carried out to evaluate the ability of FY2 and FY3 to produce potential thermostable amylases. On the other hand, the cellulase-producing potential of FY3 (E. durans) was similar to the finding by Shil et al. (2014) where they first discovered cellulase activity from an E. durans strain that was isolated from the gut of the phytophagous insect Oxya velox. However, this was in contrast to a study by Mazzucotelli et al. (2013), as the E. durans isolated from cheese whey did not exhibit the ability to degrade cellulose. These findings further support the previous suggestion that LAB isolated from various sources may exhibit different enzymatic properties. Besides that, FY2 also exhibited high cellulase activity, similar to a study reported by Ventorino et al. (2015), whereby P. acidilactici which was isolated from biomass piles of Eucalyptus camaldulensis was found to have high levels of azo-carboxymethylcellulase activity. The protease-producing potential of FY2 and FY3 was contrasted with a study by Tuncer (2009), whereby E. durans isolated from Turkish tulum cheese showed varied levels of protease activity, ranging from low to high based on the strain observed, and even more variation among different species. Besides that, Moslehishad et al. (2013) reported that P. acidilactici PTCC1424 from the Iranian Research Organization for Science and Technology exhibited moderate protease activity in supernatant form. However, Moslehishad et al. (2013) also reported that different LAB had varying levels of protease activity based on the culture conditions, such as in CFS form, anaerobic conditions and enriched CO2 conditions, whereby anaerobic conditions showed more favourable protease activity. Although the protease activity was about 2-fold higher than the amylase and cellulase activities, these findings suggest that LAB protease activity could be even higher, if the assay were to be optimized. Determination of the enzyme kinetics by using Hanes–Woolf plot According to Berg, Tymoczko and Stryer (2002), enzymes are important to enhance the rate of biochemical reactions, thus the kinetic description of their activity is required to understand about the enzyme’s function. Thus, a graph of [S]/V0 against [S] was plotted to calculate Vmax and Km. Vmax is the maximum reaction velocity achieved by an enzyme and can be used to indicate the maximum capacity of an enzyme to carry out enzymatic reactions, provided sufficient substrates are available. Km is equal to [S] at which reaction velocity is equal to half of Vmax and is independent of enzyme and substrate concentrations. Km shows the required [S] needed for significant catalysis to occur and determines the viability of the enzyme for industrial use. Km and Vmax of LAB isolates FY2 and FY3 were calculated by plotting graphs as seen in Figs 2–4. For Fig. 4, the graph not extrapolated the past y-axis due to the nature of the graph. Figure 2. Open in new tabDownload slide Hanes–Woolf plot for starch concentration, [S]/V0 against [S]. Figure 3. Open in new tabDownload slide Hanes–Woolf plot for CMC concentration, [S]/V0 against [S]. Figure 4. Open in new tabDownload slide Hanes–Woolf plot for casein concentration, [S]/V0 against [S]. Based on Fig. 2, the amylase V0 of FY3 is generally higher than FY2, as its Vmax value was slightly higher (5.51–4.43 μmol/mL/min) and Km value was lower (0.299–0.433 g/100 mL) than FY2. This result suggests that both LAB isolates are not very efficient at degrading starch, as a high substrate concentration (average 4.97 g/100 mL) would be required for its enzyme to achieve maximum capability. However, due to the lack of amylase-producing LAB, the two isolates may still be of industrial use during fermentation processes (Fossi and Tavea, 2013). The cellulase V0 of FY3 was also higher than FY2 as seen in Fig. 3. FY3 had a higher Vmax value (3.50–2.66 μmol/mL/min) but also had a higher Km value (0.006–0.002 g/100 mL) than FY2. Hence, these results suggest that FY3 is a better cellulase producer than FY2 and the cellulase produced by both isolates are efficient at degrading carboxymethylcellulose due to their low Km value. Based on both amylase and cellulase assay, FY3 would be a better option to be studied as an industrial enzyme producer as it can achieve higher enzymatic activity when substrate concentration is saturated. FY3 would have a lower requirement of substrate concentration to achieve optimum enzymatic activity for amylase while also having a low requirement of substrate concentration for cellulase. Interestingly, FY2 had a higher Vmax value than FY3 (2.14–0.51 μmol/mL/min) but also a higher negative Km (−0.348 to −0.287 g/100 mL) in terms of protease activity as seen in Fig. 4. This negative value is due to a drastic reduction in protease reaction activity at casein concentrations higher than 1.2 g/100 mL, causing the FY3 graph to have an R2 value of 0.27. Koka and Weimer (2000) had a similar observation, whereby Pseudomonas fluorescens was observed to have a sudden reduction in protease activity at casein concentrations greater than 100 mmol. This was suspected to be caused by either casein micelle formation or self-aggregration, which would reduce the number of available bonds for hydrolysis (Koka and Weimer, 2000). In terms of protease, FY2 would be a more suitable choice compared to FY3 due to its higher Vmax value albeit it has a higher Km, as the difference between Km values is smaller than the difference between Vmax values of the two isolates. Conclusion This study has demonstrated that traditional fermented foods, namely, ‘tapai pulut’, ‘tempeh’, ‘tempoyak’ and ‘fu yu’ are the potential resources for isolating LAB enzyme producers. All seven LAB isolates tested are strong producers of protease and two of them (FY2 and FY3) also exhibited the highest amylase and cellulase production. FY2 and FY3 were identified as P. acidilactici and E. durans based on biochemical profiling and 16 s rDNA sequencing. Furthermore, FY3 had a higher overall V0 than FY2, hence FY3 is a better candidate for future industrial application. However, further studies are essential to enhance the understanding on these producers and the enzymes produced. Future studies should be carried out to confirm the Km value for protease production, to purify and characterize all the three enzymes produced and to optimize the growth conditions of FY2 and FY3 for future application in silage treatment. Author Biography Jason Chen graduated from the University of Nottingham (Malaysia Campus) in the year 2017 with a First Class Honours BSc in Biotechnology. This research article was submitted in partial fulfilment of the requirements for Jason's degree in his final year. Having an interest in microorganisms and food, Jason is hoping to pursue a career related to the food and microorganism industry. J.K.H., C. designed the study, carried out the research and wrote the paper. He has primary responsibility for the paper. Y.S., L. contributed in designing the initial project scope and improving the quality of the paper. Acknowledgements I would like to express my gratitude to all those who provided me the chance to complete this report. Special thanks should be given to Dr Lim Yin Sze, my final year project supervisor, for her professional guidance, patience and useful critiques for this project. My grateful thanks are extended to the School of Biosciences, University of Nottingham Malaysia Campus, for providing the opportunity to carry out this project. I would like to extend my thanks to the technicians for their help in providing resources. Finally, I wish to thank my family and friends for their support throughout this project. References Bağder , E. S. , Tokatlı , M., Dursun , D. et al. . 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( 2007 ) Psychrotrophic amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases , World Journal of Microbiology and Biotechnology , 23 ( 11 ), 1551 – 1557 . Google Scholar Crossref Search ADS WorldCat Author notes Supervisor: Dr Lim Yin Sze, School of Biosciences, Faculty of Science, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia Author’s Biography: Jason Chen graduated from the University of Nottingham (Malaysia Campus) in the year 2017 with a first-class honours BSc in Biotechnology. This research article was submitted in partial fulfilment of the requirements for Jason’s degree in his final year. Having an interest in microorganisms and food, Jason is hoping to pursue a career related to the food and microorganism industry. He designed the study, carried out the research and wrote the paper. He has primary responsibility for the paper. Yin Sze Lim contributed in designing the initial project scope and improving the quality of the paper © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2018. Published by Oxford University Press.

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Abstract

Abstract Industrialization processes are prone to produce various forms of waste which can be utilized to produce silage. These wastes can be treated by using lactic acid bacteria (LAB), which are known to be potential enzyme producers. Seven strains of LAB were isolated from traditional fermented food, ‘tapai pulut’, ‘tempeh’, ‘tempoyak’ and ‘fu yu’ and were screened for amylase, cellulase and protease production in order to select for strains that could potentially be used industrially in silage production. All seven LAB isolates exhibited high protease production, and two of them also exhibited high amylase and cellulase production. The two isolates exhibiting high amylase and cellulase production were selected and identified as Pediococcus acidilactici FY2 and Enterococcus durans FY3 via biochemical profiling (API 50 CHL) and 16 s rDNA sequencing. E. durans was found to have the highest amylase (Vmax: 5.51 μmol/mL/min; Km: 0.300 g/100 mL) and cellulase (Vmax: 3.50 μmol/mL/min; Km: 0.006 g/100 mL) production, while exhibiting strong protease production (Vmax: 0.51 μmol/mL/min; Km: −0.287 g/100 mL). P. acidilactici was found to have strong amylase (Vmax: 4.43 μmol/mL/min; Km: 0.433 g/100 mL) and cellulase (Vmax: 2.66 μmol/mL/min; Km: 0.002 g/100 mL) production while exhibiting the highest protease production (Vmax: 2.14 μmol/mL/min; Km: −0.348 g/100 mL). These results suggest that E. durans is a better candidate for future industrial application as overall it has a higher enzyme reaction velocity when compared to P. acidilactici. Further studies should be carried out to confirm the Km value for protease production, to purify and characterize all three enzymes produced and to optimize the growth conditions of E. durans. lactic acid bacteria, amylase, cellulase, protease, Pediococcus acidilactici, Enterococcus durans Introduction In this modern era, the industrialization process is growing at an alarming rate. The continuous discovery and invention of state-of-the-art technology continually pushes the industrial world to evolve at a fast pace. Thus, prioritizing the usage of existing resources is important to ensure optimal production and prevent wastage. Various agricultural industrial processes and general food consumption produce large amounts of wastes, and some of these can be utilized in silage production as the waste are usually rich in sugars and proteins (Show and Guo, 2012). Ensilage is preserved forage crops that is generally fermented by lactic acid bacteria (LAB) under anaerobic conditions and is usually void of undesirable microorganisms due to the production of lactic acid by the LAB (Ni et al., 2015b). Thus, scientists are searching for alternative microbial sources to find better strains to process silage, as LAB are well-known producers of extracellular enzymes (Patel, Shah and Prajapati, 2013; Tosungnoen, Chookietwattana and Dararat, 2014; Ni et al., 2015a). LAB with amylase and cellulase activity to degrade complex sugars such as starch and cellulose are much sought after, as this would reduce the cost of substrate pretreatment (Shibata et al., 2007; Yitbarek and Tamir, 2014). LAB are a group of Gram-positive bacteria that produce lactic acid during fermentation and are important for food manufacturing, especially in the milk, vegetables and meat industries (Konings et al., 2000; González et al., 2010). Various LAB are generally regarded as safe and have been used for a very long time as starter cultures to produce fermented foods through traditional means, as they can produce lactic acid and bacteriocins that act as natural preservatives and thus can extend the shelf life of silage (Holzapfel, Geisen and Schillinger, 1995; Perez, Zendo and Sonomoto, 2014). Therefore, LAB should be abundantly found in fermented foods. In this current study, traditional fermented foods such as fermented glutinous rice ‘tapai pulut’, fermented soybean cake ‘tempeh’, fermented durian condiment ‘tempoyak’ and fermented soybean curd ‘fu yu’ were investigated. The aim of this study was to isolate and screen for putative LAB producers for enzymes amylase, cellulase and protease from traditional fermented foods for future industrial application. The putative enzyme producers were identified using biochemical and molecular techniques, and the enzyme kinetics were determined. Materials and methods Isolation of LAB from fermented foods Four different types of local fermented foods, fermented glutinous rice ‘tapai pulut’ (TAP), fermented soybean cake ‘tempeh’ (TEM), fermented durian condiment ‘tempoyak’ (TYK) and fermented soybean curd ‘fu yu’ (FY) were purchased from a grocery store in Selangor, Malaysia. A total of 10 g of the food samples were mixed with 90 mL of peptone-buffered water and incubated at room temperature for 30 min. A 10-fold serial dilution was carried out from 10−1 to 10−7. Volumes of 0.1 mL of diluted samples were spread on de Man, Rogosa and Sharpe (MRS) agar plates and incubated at 37°C for 48 h. MRS agar is a selective media for LAB growth. After incubation, colonies with different morphology were randomly selected, Gram-stained and examined for cell morphology under a microscope. Maintenance and preparation of bacterial isolates Each LAB isolate was preserved in 20% (v/v) glycerol and stored in a −20°C freezer till used. Initially, isolates were kept active via subculturing by pipetting 0.2 mL of active culture into 10 mL of MRS broth, followed by incubation at 37°C for up to 72 h before the next subculture. In the event of no growth, 0.2 mL of the glycerol stock was inoculated in 10 mL of MRS broth and incubated at 37°C for 24 h. Then, a four-point streak was carried out on MRS agar to ensure no contamination occurred. A single pure colony was selected and inoculated in 10 mL of MRS broth and incubated at 37°C for 24 h, followed by another subculture in 10 mL of MRS broth and incubation at 37°C for 24 h, forming active 24-h-old LAB. Two days prior to an assay, 0.2 mL of active LAB isolates were inoculated in 10 mL of MRS broth at 37°C for 24 h and is subcultured once more to form active 24-h-old LAB that are ready-to-use. Preparation of crude enzymes The crude enzymes produced by LAB isolates were prepared freshly prior to use. A total of 2% (v/v) of 24-h-old LAB (average OD600nm = 0.713) was inoculated in 10 mL of MRS broth and incubated at 37°C for 24 h. The cell-free supernatant (CFS) containing crude enzymes was collected by centrifugation of the bacterial culture at 10 000 g for 10 min at 4°C and was kept in ice till used. Screening for enzyme producers Amylase activity assay This assay was adapted from the method by Zhang and Zeng (2007) to evaluate the amylase activity of crude enzymes from LAB isolates. In this assay, 1-fold, 2-fold and 10-fold diluted CFS were used accordingly to obtain absorbance readings of less than 2.5. Starch (1 g/100 mL; Bendosen) was used as the substrate and α-amylase (16 units/mg solid; Sigma-Aldrich) was used as the positive control. The reaction mixtures were prepared according to Table 1. The reaction mixtures were incubated at 37°C for 30 min, and the enzymatic reaction was halted by using an ice bath. A total of 0.3 mL of 3,5-dinitrosalicyclic acid (DNS) was added to the reaction mixture and the mixture was heated for 5 min in a 90°C water bath. The reaction was then stopped using an ice bath. Then, 0.2 mL of the reaction mixture was pipetted into a 96-well plate and was measured for absorbance at 550 nm wavelength using Fluostar OPTIMA microplate reader. Table 1. Reaction mixtures for amylase, cellulase and protease activity assay Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Open in new tab Table 1. Reaction mixtures for amylase, cellulase and protease activity assay Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Reagents . Mixture volume (mL) . Amylase . Cellulase . Protease . Blank . −ve control . +ve control . Sample . Blank . −ve control . Sample . Blank . −ve control . Sample . CFS 0.000 0.000 0.000 0.050 0.000 0.000 0.020 0.000 0.000 0.250 α-Amylase 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Water 0.075 0.050 0.000 0.000 0.120 0.020 0.000 0.500 0.250 0.000 1 g/100 mL Starch 0.000 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.4 M Tris-HCl buffer, pH 9 0.025 0.025 0.025 0.025 0.000 0.000 0.000 0.000 0.000 0.000 1 g/100 mL CMC 0.000 0.000 0.000 0.000 0.000 0.100 0.100 0.000 0.000 0.000 0.6 g/100 mL Casein 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.250 0.250 5 g/100 mL Tricloroacetic acid 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.500 0.500 0.500 Total volume 0.100 0.100 0.100 0.100 0.120 0.120 0.120 1.000 1.000 1.000 Open in new tab A maltose standard curve was generated to quantify the amount of reducing sugar in the reaction mixture. One amylase unit was defined as the enzymatic activity that liberates one microgram of maltose per minute per millilitre CFS. The initial velocity (V0) of the crude enzyme to liberate one micromole of maltose per minute per millilitre CFS was determined. Cellulase activity assay This assay was adapted from the method by Wood and Bhat (1988) to evaluate the cellulase activity of crude enzymes from LAB isolates. In this assay, 1 g/100 mL carboxymethyl cellulose (CMC; Sigma-Aldrich) was dissolved in 0.05 M glycine/NaOH buffer (pH 9) and used as a substrate. The reaction mixtures were prepared according to Table 1. The reaction mixtures were incubated at 37°C for 30 min, and the enzymatic reaction was halted by using an ice bath. A total of 0.36 mL of DNS were added to the reaction mixture and the mixture was heated for 5 min in a 90°C water bath. The reaction was then stopped using an ice bath. Then, 0.2 mL of the reaction mixture was pipetted into a 96-well plate and was measured for absorbance at 550 nm wavelength using Fluostar OPTIMA microplate reader. A glucose standard curve was generated to quantify the amount of reducing sugar in the reaction mixture. One cellulase unit was defined as the enzymatic activity that liberates one microgram of glucose per minute per millilitre CFS. The V0 of the crude enzyme to liberate one micromole of glucose per minute per millilitre CFS was determined. Positive control was not available due to limitation of resources in the laboratory. Protease activity assay This assay was adapted from the method by Kanekar et al. (2002) to evaluate the protease activity of crude enzymes from LAB isolates. In this assay, 0.6 g/100 mL casein (Sigma-Aldrich) was dissolved in 0.4 M Tris-HCl buffer (pH 9) and used as a substrate. The reaction mixtures were prepared according to Table 1. The reaction mixtures were incubated at 37°C for 30 min, and the enzymatic reaction was terminated by adding 0.5 mL of 5 g/100 mL tricloroacetic acid. Then, 0.25 mL of the reaction mixture was mixed with 0.25 mL of Bradford’s reagent in a 96-well plate and was allowed to react for 5 min at room temperature. A tryptophan standard curve was generated to quantify the amount of protein in the reaction mixture. One protease unit was defined as the enzymatic activity that liberates one microgram of tryptophan per minute per millilitre CFS. The V0 of the crude enzyme to liberate one micromole of tryptophan per minute per millilitre CFS was determined. Positive control was not available due to limitation of resources in the laboratory. Identification via carbohydrate metabolism tests using API® 50 CHL kit Before testing the LAB for carbohydrate fermentation, the two isolates were subjected to a catalase test. A sterile loop was used to place a small amount of LAB growth onto the base of a Petri dish, then one drop of hydrogen peroxide was added and the Petri dish was immediately covered with a lid. The development of bubbles (effervescence) indicates a positive result. The selected LAB isolated were then tested for their carbohydrate fermentation pattern using the API® 50 CHL system kit (BioMérieux). The identification procedure was conducted in accordance to the manufacturer’s instructions. Active 24-h-old LAB isolates were added into the API 50 CHL basal medium and were filled into wells on the API 50 CHL test strips. The strips were then incubated at 37°C for 48 h. The strips were visually examined at 24 and 48 h, and the indication of a positive or negative result was determined from the colour change from a scale of 1 to 5, from purple (1) to green (3) to yellow (5) at the 48-h mark, whereby a value of ≥3 was considered positive. The results were then cross-referenced to the API® databases using APIweb™. DNA extraction, polymerase chain reaction amplification and DNA sequencing of 16 s rDNA gene DNA extraction was carried out using Wizard® Genomic DNA Purification Kit (Promega) on 24-h-old LAB isolates. Polymerase chain reaction (PCR) was carried out in total volumes of 20 μL containing 5 U/μL DreamTaq polymerase, 10× DreamTaq Buffer, 2 mM deoxyribonucleotide phosphate, 10 mM U8 Forward primer (5′-AGA GTT TGA TCC TGG CTC AG-3′), 10 mM 1492 reverse primer (5′-CGG TTA CCT TGT TAC GAC TT-3′), sterile distilled water and 5 ng/μL LAB DNA samples. DNA amplification was performed for 30 cycles and the PCR cycle was set as the initial denaturation at 95°C for 4 min, annealing at 65°C for 1 min, elongation at 72°C for 1 min, and followed by another 29 cycles of denaturation at 95°C for 1 min, annealing at 65°C for 1 min and elongation at 72°C for 1 min. A total of 20 μL of the amplified PCR products and a negative control were electrophoresed on 1 g/100 mL agarose gel containing SYBR® Safe and 1× Tris-acetate–EDTA buffer (TAE) at 75 V for 40 min. The gel was visualized using Gel Doc™ EZ Gel Documentation System (Bio-Rad). The remaining amplified PCR products were then sent to First BASE Laboratories Sdn. Bhd. for sequencing using the U8F and 1492 R primers under the DNA Sequencing Service + Plus (SS1201) service. The DNA sequence obtained was then compared with sequences in the GenBank database using BLAST by the National Center for Biotechnology Information (NCBI). Determination of the enzyme kinetics by using Hanes–Woolf plot The Michaelis–Menten constant (Km) and maximal velocity (Vmax) of the crude enzymes were calculated using the Hanes–Woolf plot. The V0 of the enzyme reaction was determined according to methods described in Sections 3.4.1–3.4.3. In this assay, a range of substrate concentrations were used for amylase (ranging from 0 to 20 g/100 mL starch), cellulase (ranging from 0 to 1.3 g/100 mL CMC) and protease (ranging from 0 to 1.5 g/100 mL casein). The Hanes–Woolf graph was then plotted to determine the Km and Vmax values of the targeted crude enzymes. A graph of [S]/V0 (g min/100μmol) against substrate concentration, [S] (g/100 mL) was plotted. As the Hanes–Woolf equation is ‘[S]/V0 = (1/Vmax)([S]) + Km/Vmax’, the Vmax was calculated by taking the reciprocal value of the gradient of the graph. Km is equal to the negative of the x-intercept, thus was calculated by taking the negative of the [S] value when [S]/V0 = 0. Statistical analysis Statistical analyses were carried out using IBM SPSS Statistics 23. All experimental data were obtained in triplicates and were analysed using the analysis of variance (ANOVA) test followed by Duncan’s post hoc test at P ≤ 0.05. Correlation between amylase and cellulase assay was analysed using Pearson’s correlation coefficient test. Results and discussion Isolation of LAB from fermented foods A total of 12 LAB were isolated from the four fermented foods, two isolates from ‘tapai pulut’ (TAP1–2), four isolates from ‘tempeh’ (TEM1–4), three isolates from ‘tempoyak’ (TYK1–3) and three isolates from ‘fu yu’ (FY1–3). However, only 7 out of 12 were still viable after repeated subcultures, hence only TAP2, TEM1, TEM2, TYK2, TYK3, FY2 and FY3 were chosen for preliminary screening of enzyme producers. This loss of viability for the five isolates could be due to two factors, (i) the selective media (MRS) used was not providing the nutrients needed and (ii) the changes in growth environment. This finding was similar to those reported by Birollo, Reinheimer and Vinderola (2000) showing that MRS may show a lower viable count of bacteria and there are other suitable alternatives to grow LAB. Other than that, the other LAB isolates may be in the unique form of viable but non-culturable state, whereby they cannot be cultured on routine microbiological media but are still viable (Fakruddin, Mannan and Andrews, 2013). Thus, the LAB isolates were most likely unable to acclimatize and grow continuously in MRS broth. This may be due to unfavourable environmental factors such as starvation, non-optimal pH or others (Pinto, Santos and Chambel, 2015). Morphological and physiological characterization of LAB The seven LAB isolates tested were Gram-positive, and two isolates were rod-shaped and the remaining five were coccus-shaped. FY2 had showed a tetracoccus cell arrangement, TEM2 and TYK3 showed diplococcus cell arrangement, while TEM1 and FY3 had no distinct coccus arrangements. The two rod-shaped isolates, TAP2 and TYK2 tended to form short chains. A summary of the morphology of the LAB isolates can be seen in Table 2. Table 2. Colony and cell morphology of LAB isolates LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus Open in new tab Table 2. Colony and cell morphology of LAB isolates LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus LAB isolates . Colony morphology . Gram stain . Shape . TAP2 Circular, milky Positive Short chain rod TEM1 Circular, milky Positive Coccus TEM2 Circular, milky Positive Diplococcus TYK2 Circular, white Positive Short chain rod TYK3 Circular, white Positive Diplococcus FY2 Circular, milky Positive Tetracoccus FY3 Circular, white, pinprick sized Positive Coccus Open in new tab Screening for enzyme producers The seven LAB isolates tested were able to produce amylase, cellulase and protease enzymes at different strengths. The enzyme activity of the LAB isolates was determined via the liberation of a specific substrate based on the designated enzyme assay are depicted in Table 3. Table 3. Amylase, cellulase and protease activity of LAB isolates LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a Values are mean ± standard deviation, n = 3 (amylase and protease), n = 4 (cellulase). abcdef Within a column, values with different superscripts are significantly different at P ≤ 0.05. Open in new tab Table 3. Amylase, cellulase and protease activity of LAB isolates LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a LAB isolates . Amylase activity (mg/mL/min) . Amylase reaction velocity, V0 (μmol/mL/min) . Cellulase activity (mg/mL/min) . Cellulase reaction velocity, V0 (μmol/mL/min) . Protease activity (mg/mL/min) . Protease reaction velocity, V0 (μmol/mL/min) . TAP2 0.725 ± 0.053d 2.118 ± 0.155d 0.406 ± 0.012e 2.252 ± 0.064e 1.888 ± 0.347a 9.245 ± 1.700a TEM1 0.962 ± 0.029b 2.809 ± 0.085b 0.569 ± 0.014c 3.158 ± 0.080c 2.058 ± 0.399a 10.076 ± 1.956a TEM2 0.572 ± 0.009e 1.672 ± 0.026e 0.401 ± 0.012e 2.229 ± 0.064e 2.162 ± 0.471a 10.585 ± 2.304a TYK2 0.803 ± 0.032c 2.347 ± 0.093c 0.484 ± 0.019d 2.688 ± 0.105d 2.079 ± 0.164a 10.180 ± 0.804a TYK3 0.184 ± 0.008f 0.538 ± 0.022f 0.083 ± 0.007f 0.460 ± 0.039f 2.035 ± 0.271a 9.963 ± 1.328a FY2 1.027 ± 0.071b 3.000 ± 0.208b 0.655 ± 0.013b 3.638 ± 0.072b 2.150 ± 0.427a 10.529 ± 2.093a FY3 1.224 ± 0.007a 3.575 ± 0.021a 0.816 ± 0.007a 4.528 ± 0.040a 2.307 ± 0.044a 11.295 ± 0.216a Values are mean ± standard deviation, n = 3 (amylase and protease), n = 4 (cellulase). abcdef Within a column, values with different superscripts are significantly different at P ≤ 0.05. Open in new tab The amylase activity of isolate FY3 was ranked highest, followed by FY2 and TEM1 (Table 3). This finding was interesting as these three isolates were not isolated from carbohydrate-rich food sources (‘fu yu’ and ‘tempeh’), whereas isolates from carbohydrate-rich food sources (‘tapai’ and ‘tempoyak’) such as TAP2, TYK2 and TYK3 did not show high level of amylase activity. Similarly, the cellulase activity of FY3 was also ranked highest, followed by FY2 and TEM1 (Table 3). Pearson’s coefficient test showed that there was a significant positive correlation (r2 = +0.979) between amylase and cellulase activity in all LAB isolates. This finding suggests that amylase and cellulase production could be correlated. However, to the author’s knowledge, no relevant published literature was found. All LAB isolates were good producers of proteases as seen in Table 3. The protease activity of all isolates was at least 2-folds higher than amylase and cellulase activities under the assay conditions. However, no significant difference in protease activity was observed between isolates. This finding was in accordance with the study by Shin et al. (2008), where LAB from the genera Pediococcus and Enterococcus displayed high amylase and cellulase activities but did not exhibit any significant difference of protease activity compared to other bacteria. Therefore, the two top producers for amylase and cellulase isolated from ‘fu yu’—FY2 and FY3 were selected for further identification and enzyme kinetic assay. Bacterial identification via carbohydrate metabolism tests using API® 50 CHL kit FY2 and FY3 which displayed the highest amylase and cellulase activities were identified by using the API 50 CHL system kit to test for their carbohydrate fermentation patterns (Table 4). A catalase test was performed to complement the API 50 CHL test, where both isolates were found to be catalase-negative. Table 4. Carbohydrate fermentation pattern of FY2 and FY3 using API 50 CHL system kit No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − (−) negative, (+) positive, (±) undetermined. Open in new tab Table 4. Carbohydrate fermentation pattern of FY2 and FY3 using API 50 CHL system kit No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − No. . Substrates . LAB isolate code . No. . Substrates . LAB isolate code . FY2 . FY3 . FY2 . FY3 . 0 Control − − 25 Esculin ferric citrate + + 1 Glycerol − − 26 Salicin − + 2 Erythritol − − 27 d-Cellobiose + + 3 d-Arabinose − − 28 d-Maltose − + 4 l-Arabinose − − 29 d-Lactose (bovine origin) − + 5 d-Ribose + + 30 d-Mellibiose − + 6 d-Xylose + − 31 d-Saccharose (sucrose) − + 7 l-Xylose − − 32 d-Trehalose − − 8 d-Adonitol − − 33 Inulin − − 9 Methyl-βd-xylopyranoside − − 34 d-Melezitose − − 10 d-Galactose − + 35 d-Raffinose − + 11 d-Glucose + + 36 Amidon (starch) − − 12 d-Fructose + + 37 Glycogen − − 13 d-Mannose + + 38 Xylitol − − 14 l-Sorbose − − 39 Gentiobiose ± + 15 l-Rhamnose − + 40 d-Turanose − − 16 Dulcitol − − 41 d-Lyxose − − 17 Inositol − − 42 d-Tagatose − − 18 d-Mannitol − − 43 d-Fucose − − 19 d-Sorbitol − − 44 l-Fucose − − 20 Methyl-αd-Mannopyranoside − − 45 d-Arabitol − − 21 Methyl-αd-Glucopyranoside − − 46 l-Arabitol − − 22 N-Acetylglucosamine + + 47 Potassium gluconate − − 23 Amygdalin − + 48 Potassium 2-ketogluconate − − 24 Arbutin − + 49 Potassium 5-ketogluconate − − (−) negative, (+) positive, (±) undetermined. Open in new tab Based on the API 50 CHL results in Table 4, FY2 was initially identified with a low % ID value of 34.2% as P. acidilactici. The % ID for FY2 meant that 34.2% of LAB with this specific biochemical profile was found to be P. acidilactici. The low % ID was due to the discrepancies between the biochemical profile of P. acidilactici in the APIweb® system as compared to the API 50 CHL results of FY2. FY2 exhibited the inability to utilize l-arabinose, d-galactose, l-rhamnose, salicin, d-trehalose and d-tagatose, whereas P. acidilactici in the APIweb® system had the following biochemical profile: (l-arabinose 100%), (d-galactose 100%), (l-rhamnose 75%), (salicin 75%), (d-trehalose 75%) and (d-tagatose 100%), whereby the percentage represents the likelihood of that particular LAB strain to have the ability to ferment that sugar. On the other hand, based on Table 4, FY3 was initially identified as Pediococcus pentosaceus with a higher % ID value of 80.9% but also displayed biochemical discrepancies such as the inability to utilize l-arabinose, d-trehalose and d-tagatose as compared to the APIweb® system’s biochemical profile for P. pentosaceus (l-arabinose 100%), (d-trehalose 99%) and (d-tagatose 99%). Bacterial identification via 16s rDNA gene sequencing analysis Since the API carbohydrate fermentation test was unable to confirm the identity for FY2, both isolates were then subjected to 16 s rDNA gene sequencing analysis for further confirmation. Based on the gene sequences (Fig. 1A) and BLAST results for FY2, 16 s rDNA gene sequencing analysis showed a similarity of 94%, 94% and 92% with P. acidilactici, P. pentosaceus and P. stilesil, respectively. However, the query cover for P. acidilactici was at 97%, whereas P. pentosaceus was at 94%. Thus, it was highly suspected that FY2 was indeed P. acidilactici due to a higher similarity in the biochemical test compared to P. pentosaceus. Besides that, the morphology of the LAB to form tetracoccus also supported the evidence of FY2 to be under the genera Pediococcus. Figure 1. Open in new tabDownload slide 16 s rDNA sequence of FY2 (A) and FY3 (B). Based on the gene sequences (Fig. 1B) and BLAST results for FY3, a similarity of 95%, 95% and 94% with Enterococcus durans, E. faecium and E. mundtii was obtained respectively. The query cover for E. durans and E. faecium was the same at 80%, thus the biochemical properties were examined to assist in identifying the species. However, the APIweb™ database did not show LAB from the genera Enterococcus, hence other literature were used as a reference. According to Bergey's manual of Determinative Bacteriology, FY3 showed discrepancies for both Enterococcus LAB such as raffinose (+) and mellibiose (+) (Holt, 2000). However, based on Bergey’s manual FY3 was more likely to be E. durans due to its inability to utilize glycerol (−) and d-mannitol (−), as compared to E. faecium. The discrepancies between the biochemical and molecular identification techniques might be due to the fact that principles of both techniques are fundamentally different. API 50 CHL is used to classify LAB based on their phenotypical properties by comparing the fermentation pattern of 49 different types of carbohydrate with other bacteria that are registered in the APIweb™ database. One of the limitations of this is that the APIweb™ database has been found to be lacking in biochemical profiles of certain LAB (Boyd et al., 2005). Besides that, phenotypical characterization has been known to have poor reproducibility and that the whole information potential of the LAB genome is not always expressed, as gene expression is directly related to environmental conditions such as the growth conditions in the laboratory (Mohania et al., 2008). On the other hand, 16 s rDNA sequencing relies on the amplification and sequencing of the highly conserved 16 s ribosomal RNA gene, which is akin to a unique biosignature for any organism (Isenbarger et al., 2008). Janda and Abbott (2007) reported that 16 s rDNA sequencing is an alternative to provide identification of unknown bacteria with unrecognized biochemical profiles or a low likelihood. This sequencing technique can provide information about the genus and species of most unknown bacteria, with high levels of genus identification (>90%) and moderate levels of species identification (65–83%; Janda and Abbott, 2007). A study by Bağder et al. (2014) compared the results of the API 50 CHL test with 16 s rRNA results and found that the API test did not give reliable identification results, with only 71 out of 152 tested isolates were in agreement. Another study by Moraes et al. (2013) reported the possibility for high reliability rates in the API 50 CHL to diverge greatly from 16 s rDNA results, supporting the identification of FY3 which had a high API 50 CHL % ID for P. pentosaceus (80.9%) but was identified as E. durans (95%) through BLAST. However, one of the limitations of using 16 s rDNA sequencing is that LAB in the genera Enterococcus are difficult to differentiate due to the highly conserved nature of 16 s rDNA (Moraes et al., 2013). Thus, the API 50 CHL biochemical test results were used as supporting evidence to support the main identification technique, 16 s rDNA sequencing to help discern the LAB isolates based on their genome and carbohydrate metabolism patterns. The finding of the amylase producers—FY2 and FY3 from the species of P. acidilactici and E. durans were in accordance to a report by Velikova et al. (2016) in which their targeted strains P. acidilactici and E. durans were also found to have highest extracellular and intracellular amylase activities, respectively. However, this finding was in contrast with a study by Musikasang et al. (2009), as bacterium from the genera Pediococcus and E. durans that were isolated from chicken’s gastrointestinal tract did not exhibit the ability to digest starch. According to Velikova et al. (2016), amylase-producing LAB contain a basic pool of chromosomal genes that is responsible for starch hydrolysis. However, only the strains that are forced to survive in starchy environment are able to display these genetically determined properties. Hence, the gastrointestinal tract of chicken might not have an ideal environment for these LAB to grow. These findings suggest that LAB isolated from different sources such as plants or animals may exhibit different enzymatic properties. According to Bergey’s manual, P. acidilactici can grow at higher temperatures of up to 50°C, whereas E. durans can grow at 45°C. This may be beneficial as the two LAB could possibly produce thermostable amylases which are of special interest in the industrial field, especially for starch saccharification (Saxena et al., 2007). Hence, further tests should be carried out to evaluate the ability of FY2 and FY3 to produce potential thermostable amylases. On the other hand, the cellulase-producing potential of FY3 (E. durans) was similar to the finding by Shil et al. (2014) where they first discovered cellulase activity from an E. durans strain that was isolated from the gut of the phytophagous insect Oxya velox. However, this was in contrast to a study by Mazzucotelli et al. (2013), as the E. durans isolated from cheese whey did not exhibit the ability to degrade cellulose. These findings further support the previous suggestion that LAB isolated from various sources may exhibit different enzymatic properties. Besides that, FY2 also exhibited high cellulase activity, similar to a study reported by Ventorino et al. (2015), whereby P. acidilactici which was isolated from biomass piles of Eucalyptus camaldulensis was found to have high levels of azo-carboxymethylcellulase activity. The protease-producing potential of FY2 and FY3 was contrasted with a study by Tuncer (2009), whereby E. durans isolated from Turkish tulum cheese showed varied levels of protease activity, ranging from low to high based on the strain observed, and even more variation among different species. Besides that, Moslehishad et al. (2013) reported that P. acidilactici PTCC1424 from the Iranian Research Organization for Science and Technology exhibited moderate protease activity in supernatant form. However, Moslehishad et al. (2013) also reported that different LAB had varying levels of protease activity based on the culture conditions, such as in CFS form, anaerobic conditions and enriched CO2 conditions, whereby anaerobic conditions showed more favourable protease activity. Although the protease activity was about 2-fold higher than the amylase and cellulase activities, these findings suggest that LAB protease activity could be even higher, if the assay were to be optimized. Determination of the enzyme kinetics by using Hanes–Woolf plot According to Berg, Tymoczko and Stryer (2002), enzymes are important to enhance the rate of biochemical reactions, thus the kinetic description of their activity is required to understand about the enzyme’s function. Thus, a graph of [S]/V0 against [S] was plotted to calculate Vmax and Km. Vmax is the maximum reaction velocity achieved by an enzyme and can be used to indicate the maximum capacity of an enzyme to carry out enzymatic reactions, provided sufficient substrates are available. Km is equal to [S] at which reaction velocity is equal to half of Vmax and is independent of enzyme and substrate concentrations. Km shows the required [S] needed for significant catalysis to occur and determines the viability of the enzyme for industrial use. Km and Vmax of LAB isolates FY2 and FY3 were calculated by plotting graphs as seen in Figs 2–4. For Fig. 4, the graph not extrapolated the past y-axis due to the nature of the graph. Figure 2. Open in new tabDownload slide Hanes–Woolf plot for starch concentration, [S]/V0 against [S]. Figure 3. Open in new tabDownload slide Hanes–Woolf plot for CMC concentration, [S]/V0 against [S]. Figure 4. Open in new tabDownload slide Hanes–Woolf plot for casein concentration, [S]/V0 against [S]. Based on Fig. 2, the amylase V0 of FY3 is generally higher than FY2, as its Vmax value was slightly higher (5.51–4.43 μmol/mL/min) and Km value was lower (0.299–0.433 g/100 mL) than FY2. This result suggests that both LAB isolates are not very efficient at degrading starch, as a high substrate concentration (average 4.97 g/100 mL) would be required for its enzyme to achieve maximum capability. However, due to the lack of amylase-producing LAB, the two isolates may still be of industrial use during fermentation processes (Fossi and Tavea, 2013). The cellulase V0 of FY3 was also higher than FY2 as seen in Fig. 3. FY3 had a higher Vmax value (3.50–2.66 μmol/mL/min) but also had a higher Km value (0.006–0.002 g/100 mL) than FY2. Hence, these results suggest that FY3 is a better cellulase producer than FY2 and the cellulase produced by both isolates are efficient at degrading carboxymethylcellulose due to their low Km value. Based on both amylase and cellulase assay, FY3 would be a better option to be studied as an industrial enzyme producer as it can achieve higher enzymatic activity when substrate concentration is saturated. FY3 would have a lower requirement of substrate concentration to achieve optimum enzymatic activity for amylase while also having a low requirement of substrate concentration for cellulase. Interestingly, FY2 had a higher Vmax value than FY3 (2.14–0.51 μmol/mL/min) but also a higher negative Km (−0.348 to −0.287 g/100 mL) in terms of protease activity as seen in Fig. 4. This negative value is due to a drastic reduction in protease reaction activity at casein concentrations higher than 1.2 g/100 mL, causing the FY3 graph to have an R2 value of 0.27. Koka and Weimer (2000) had a similar observation, whereby Pseudomonas fluorescens was observed to have a sudden reduction in protease activity at casein concentrations greater than 100 mmol. This was suspected to be caused by either casein micelle formation or self-aggregration, which would reduce the number of available bonds for hydrolysis (Koka and Weimer, 2000). In terms of protease, FY2 would be a more suitable choice compared to FY3 due to its higher Vmax value albeit it has a higher Km, as the difference between Km values is smaller than the difference between Vmax values of the two isolates. Conclusion This study has demonstrated that traditional fermented foods, namely, ‘tapai pulut’, ‘tempeh’, ‘tempoyak’ and ‘fu yu’ are the potential resources for isolating LAB enzyme producers. All seven LAB isolates tested are strong producers of protease and two of them (FY2 and FY3) also exhibited the highest amylase and cellulase production. FY2 and FY3 were identified as P. acidilactici and E. durans based on biochemical profiling and 16 s rDNA sequencing. Furthermore, FY3 had a higher overall V0 than FY2, hence FY3 is a better candidate for future industrial application. However, further studies are essential to enhance the understanding on these producers and the enzymes produced. Future studies should be carried out to confirm the Km value for protease production, to purify and characterize all the three enzymes produced and to optimize the growth conditions of FY2 and FY3 for future application in silage treatment. Author Biography Jason Chen graduated from the University of Nottingham (Malaysia Campus) in the year 2017 with a First Class Honours BSc in Biotechnology. This research article was submitted in partial fulfilment of the requirements for Jason's degree in his final year. Having an interest in microorganisms and food, Jason is hoping to pursue a career related to the food and microorganism industry. J.K.H., C. designed the study, carried out the research and wrote the paper. He has primary responsibility for the paper. Y.S., L. contributed in designing the initial project scope and improving the quality of the paper. Acknowledgements I would like to express my gratitude to all those who provided me the chance to complete this report. Special thanks should be given to Dr Lim Yin Sze, my final year project supervisor, for her professional guidance, patience and useful critiques for this project. My grateful thanks are extended to the School of Biosciences, University of Nottingham Malaysia Campus, for providing the opportunity to carry out this project. I would like to extend my thanks to the technicians for their help in providing resources. Finally, I wish to thank my family and friends for their support throughout this project. References Bağder , E. S. , Tokatlı , M., Dursun , D. et al. . 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( 2007 ) Psychrotrophic amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases , World Journal of Microbiology and Biotechnology , 23 ( 11 ), 1551 – 1557 . Google Scholar Crossref Search ADS WorldCat Author notes Supervisor: Dr Lim Yin Sze, School of Biosciences, Faculty of Science, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia Author’s Biography: Jason Chen graduated from the University of Nottingham (Malaysia Campus) in the year 2017 with a first-class honours BSc in Biotechnology. This research article was submitted in partial fulfilment of the requirements for Jason’s degree in his final year. Having an interest in microorganisms and food, Jason is hoping to pursue a career related to the food and microorganism industry. He designed the study, carried out the research and wrote the paper. He has primary responsibility for the paper. Yin Sze Lim contributed in designing the initial project scope and improving the quality of the paper © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2018. Published by Oxford University Press.

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BioScience HorizonsOxford University Press

Published: Jan 1, 2018

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