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Optimization of Methionol Bioproduction by Saccharomyces Cerevisiae Using Response Surface Methodology

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Optimization of Methionol Bioproduction by Saccharomyces Cerevisiae Using Response Surface Methodology Ann Microbiol (2015) 65:197–205 DOI 10.1007/s13213-014-0850-y ORIGINAL ARTICLE Optimization of methionol bioproduction by Saccharomyces cerevisiae using response surface methodology Heng-Qian Lwa & Jingcan Sun & Shao-Quan Liu Received: 14 October 2013 /Accepted: 13 February 2014 /Published online: 18 March 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Methionol (3-methylthio-1-propanol) is an impor- Keywords Sulphur flavour . Methionol . L-methionine . tant volatile sulphur-containing alcohol that may significantly Saccharomyces cerevisiae . Response surface methodology impact food flavour. The purpose of this research is to inves- tigate the bioproduction of methionol from L-methionine catabolism by Saccharomyces cerevisiae EC-1118. The bio- Introduction transformation was carried out in coconut cream supplement- ed with L-methionine. Response surface methodology was Volatile sulphur flavour compounds (VSFCs) are essential for applied for the optimization of fermentation conditions to the aromas of many food products and beverages such as achieve a high yield of methionol. A second-order polynomial cheese, beer and wine (Janssens et al. 1992). Methionol is an model (R2=0.912) was established based on the experimental important VSFC that contributes significantly to the overall data obtained in this study using multiple regression analysis. aroma profile of cheeses such as Cheddar and Camembert To obtain more accurate prediction results, a reduced quadrat- (López del Castillo-Lozano et al. 2007; Tan et al. 2012). It has ic model was obtained through backward elimination. Based a low odour threshold of 1 to 3 ppm and imparts cauliflower- on the reduced model, the optimal conditions for maximum like, meaty, savoury or toasted cheese flavour notes. The methionol production were determined to be 0.30 % (w/v) of production of flavour compounds can be achieved by chem- L-methionine, 0.10 % (w/v) of yeast extract and zero level of ical synthesis, which can result in formation of racemic mix- diammonium phosphate. Under these optimal conditions, a tures that require further downstream separation and purifica- methionol concentration of 240.7±17.4 μg/mL was achieved. tion (Janssens et al. 1992; Vandamme and Soetaert 2002). This experimental result was in close agreement with the However, due to the increasing demand of consumers for predicted value of 243.5 μg/mL, indicating that this model natural products, alternative biotechnological methods are was adequate. These results indicate that fermentation by required (Hutkins 2006). Fermentation by microorganisms is S. cerevisiae in L-methionine-supplemented coconut cream one of the most important biotechnologies being applied for medium is an effective method for the production of the production of natural flavour compounds (Vandamme and methionol. Large-scale fermentation trials are needed to pro- Soetaert 2002). Methionol is found to be one of the main vide valuable information for industrial production. VSFCs produced by yeast metabolism of L-methionine (Etschmann et al. 2008;LiuandCrow2010). Biodegradation of the sulphur-carbon bond in L-methionine : : can lead to the formation of thiol intermediates and various H.<Q. Lwa J. Sun S.<Q. Liu VSFCs. Simplified pathways of L-methionine catabolism to Food Science and Technology Programme, produce VSFCs are presented in Fig. 1. Department of Chemistry, National University of Singapore, Science Drive 3, Singapore 117543 To obtain desirable flavour compounds, the bioconversion of specific substrates or precursors by fermentation has been S.<Q. Liu (*) gaining the interest of researchers in recent years. The sulphur- Advanced Food Research Laboratory, National University of containing amino acid L-methionine was frequently applied as Singapore (Suzhou) Research Institute, No. 377 Linquan Street, Suzhou Industrial Park, Suzhou, Jiangsu, China 215123 the precursor for the production of VSFCs. The reported e-mail: [email protected] yeasts that are able to biosynthesize VSFCs from L- 198 Ann Microbiol (2015) 65:197–205 Fig. 1 Simplified pathways of L-methionine catabolism to produce methionol and VSFCs in numerous microorganisms, including yeasts and bacteria. VSFCs, volatile sulphur flavour compounds; Atase: Aminotransferase; α-KG: alpha-ketoglutarate; Glu: glutamate: GDH: glutamate dehydrogenase; MGL: methionine γ-lyase; MOBA: 4-methylthio-2-oxobutyric acid; DMDS: dimethyl disulfide; DMTS: dimethyl trisulfide; DMQS: dimethyl tetrasulfide. (modified after Yvon and Rijnen 2001; López del Castillo-Lozano et al. 2007;Rauhut2009) methionine include Kluyveromyces lactis, Geotrichum bioproduction include glucose concentration, L-methionine candidum, Debaryomyces hansenii and Yarrowia lipolytica concentration and yeast extract level (Seow et al. 2010). To (Spinnler et al. 2001;YvonandRijnen2001;Kagklietal. optimize the fermentation conditions, an effective and effi- 2006; Cholet et al. 2008; Landaud et al. 2008). These yeasts cient experimental design is critical. The OVAT (one variable are dairy yeasts that are normally used for cheese production. at a time) method is frequently applied, but this approach The application of these dairy yeasts to the production of requires a large number of experiments, as more variables methionol was rarely reported. are involved. In addition to dairy yeasts, some wine yeasts such as Response surface methodology (RSM) is a multivariate Saccharomyces cerevisiae were also found to be capable statistic method that offers useful statistical information for of synthesizing VSFCs to modulate wine flavour improving and optimizing a process with only a small (Swiegers and Pretorius 2007). Saccharomyces cerevisiae number of experiments. RSM is based on a series of is known to metabolize L-methionine to methionol to- statistical and mathematical methods that serve to describe gether with other VSFCs via the Ehrlich pathway the relationships between one or more responses and a (Perpète et al. 2006; Etschmann et al. 2008). However, number of independent variables (Bezerra et al. 2008; the synthesis of methionol by Saccharomyces cerevisiae Sirisompong et al. 2011). As opposed to the OVAT ap- was mostly performed in synthetic media. Thus, it would proach, RSM is more efficient, and allows the interactive be more relevant to investigate methionol production effects between variables to be assessed (Hasan et al. from L-methionine by S. cerevisiae in food matrices so 2011). The Box-Behnken design (BBD) selects various that the fermented medium can be directly used as a points from the three-level factorial arrangements that allow flavouring ingredient. first-order and second-order coefficients of the model to be In the current study, coconut cream was chosen as the estimated, significantly reducing the number of experiments fermentation medium due to its unique characteristics, such (Bezerra et al. 2008). as low-cost, abundant availability in Southeast Asia, high fat Therefore, the objective of this study was to produce content for better retention of flavour compounds, and nutri- methionol through bioconversion of L-methionine by ents for the growth of yeasts. To improve production of S. cerevisiae in L-methionine-supplemented coconut cream. methionol in the coconut cream supplemented with L- Three nutritional factors, including L-methionine concentra- methionine, it is essential to identify the optimal fermentation tion, yeast extract level and diammonium phosphate conditions. The factors that play important roles in methionol level, were optimized using RSM with the BBD method, Ann Microbiol (2015) 65:197–205 199 because these were the significant factors that would affect L- The fermentation by S. cerevisiae EC-1118 was per- methionie metabolism by yeasts, while other factors such as formed by adding 1 % (v/v) of the pre-culture (initial temperature and pH were not significant within the ranges 107 CFU/mL) into the aseptically prepared medium and tested (Cholet et al. 2008; López del Castillo-Lozano et al. incubating at 25 ˚C for 48 h. Two uninoculated controls 2007, Sirisompong et al. 2011). were included: one consisting of only original coconut cream and the other of coconut cream supplemented with 0.30 % (w/v) of L-methionine. All the fermenta- Materials and methods tions were carried out in duplicate and the mean of methionol concentrations was used as the response. Yeast strain and pre-culture preparation The fermentations were stopped by adjusting the pH of fermentation medium to 2.5 with 1.0 M HCl. The The yeast used was S. cerevisiae EC-1118 from Lallemand samples were stored in 50-mL centrifuge tubes at −20 ˚Cuntil Inc., Ontario, Canada. Pure cultures were prepared in nutrient analysis. broth, which consisted of 0.25 % Bacto™ yeast extract (BD, Franklin Lakes, NJ, US), 0.25 % malt extract (Oxoid, Sample analysis Basingstoke, UK), 0.25 % peptone (Oxoid , Hampshire, England) and 2.0 % glucose (Glucolin®, GlaxoWellcome Analysis of all samples was conducted with headspace (HS) Ceylon Limited, Moratuwa, Sri Lanka). Initial pH of the solid-phase microextraction (SPME) coupled with gas chro- nutrient broth was adjusted to pH 5.0 with 1.0 M hydrochloric matography (GC)-mass spectrometer (MS) and flame ioniza- acid (HCl) (Merck, Darmstadt, Germany). Two loopfuls of tion detector (FID) (HS-SPME-GC-MS/FID). An 85 μm freeze-dried yeasts were added into 15 mL of sterilized nutri- carboxen-polydimethylsiloxane (CAR-PDMS) fibre ent broth and incubated at 25 ˚C for 28 h to obtain the pure (Supelco,
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