1 Introduction

Many microbial processes have been described able to produce interesting flavors, the number of industrial applications are limited. A reason for this in most cases is the low yield. The microbial flavors are often present only in low concentrations in the fermentation broths, resulting in high costs for upstream and down-stream processing. The development of Indigenous specific fermentation techniques and recovery methods is an important challenge for researchers in this field. The present investigation is a preliminary attempt to produce ingenious technology for the production of natural flavor.

γ-Decalactone (GDL; C10H18O2) is a well-known industrially important microbial aroma found in various foods and beverages (Iacazio et al., 2002). It is classified and generally recognized as a safe (GRAS) food additive by the U.S. Food and Drug Administration (New Hampshire, MD, USA), and used as a “natural” food flavoring additive when produced by biotechnological processes (Aguedo et al., 2004a). γ Lactones are widely distributed in nature; this moiety is present in around 10% of all natural compounds. Most display a broad biological profile including strong antibiotic, antihelmetic, antifungal, antitumour, antiviral, anti-inflammatory and cytostatic properties, which make them interesting lead structures for new drugs. An increasing demand for natural products has resulted in the use of biotechnological processes for the production of these lactones. This has led to numerous patents being taken out, and nowadays the biotechnologically produced lactone family is mainly represented by γ-Decalactone, but also to a smaller extent by γ-Dodecalactone and γ-Octalactone.

The world of flavors and fragrances has begun to industrialize with the increase in the demand of these substances. It can be easily obtained from hydroxyl fatty acids by microbial transformation and is also naturally present in many products fermented by yeasts. Most of the other lactones are also encountered in fermented products but are far more difficult to produce. Tahara et al. (1973) presented the first report on biotechnological GDL production using Sporidiobolus salmonicolor (Sporobolomyces odorus). Continuous investigations by researchers have increased the number of microorganisms that produce GDL, such as Pichia guilliermondii (Iacazio et al., 2002), Candida sp. (Aguedo et al., 2004b), Sporidiobolus salmonicolor (Lee et al., 1999), Sporobolomyces odorus (Berger & Drawert, 1985; Lee & Chou, 1994; Gatfield & Rabenhorst, 1999; Beney et al., 2001), Rhodotorula glutinous (Gatfield & Rabenhorst, 1999) and especially Yarrowia lipolytica (Waché et al., 2006). Previous studies focused mostly on three aspects of the production process: fermentation control and process optimization, including also optimization of nutritional and non-nutritional conditions (Berger & Drawert, 1985; Lee & Chou, 1994; Lin et al., 1996; Gatfield & Rabenhorst, 1999; Lee et al., 1999; Beney et al., 2001; Aguedo et al., 2004b), and ultra structural research with the objective to determine the metabolism responsible for bioconversion to GDL through peroxisomal or mitochondrial β-oxidation (Blin-Perrin et al., 2000; Feron et al., 2005); and the β-oxidation pathway and its optimization (Waché et al., 2001). Recently, Gomes et al. (2013) studied the impact of lipase hydrolysis of castor oil on GDL, providing a new way to improve GDL production and speed up the biotransformation process. During the bioconversion of ricinoleic acid to g-decalactone under controlled pH conditions, Sporidiobolus salmonicolor produced only the lactone form, while Sporidiobolus ruinenii produced both the lactone form and a precursor. The effects of some physical factors, including preculture time, media initial pH and bead concentrations, on the production of GDL by immobilized Sporidiobolus salmonicolor CCRC 21975 within calcium alginate beads were investigated. In the present study we have studied the development of UV mutant strain for the improved production of GDL Sporidiobolus salmonicolor (MTCC 485).

2 Materials and Methods

2.1 Microorganism

Sporidiobolus salmonicolor (MTCC 485) was obtained from the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology (IMTECH, Chandigarh), India and was maintained in YM Agar media slants at 25^°C. After three days growing yeast spores were stored in refrigerator (Figure 1).

2.2 Culture conditions

Pre cultures were prepared by transferring a loopful of microorganisms grown on YM agar slants to 100 ml preculturing glucose medium (glucose:15, tryptone:0.5, yeast extract:1 malt extract:1 casaminoacids:2 K2HPO4: 2 Cacl2: 0.13, Feso4: 0.01 and MgSO4: 3 g/l) in 250-ml Erlenmeyer flasks and then incubated in shaking incubator at 160 rpm and at 25°C for various cultivation periods (16, 24 or 42 h). To study the effects of operational parameters on GDL production with stationary phase cells, these were supplemented with 0.1% methyl Recinoleate (castor oil) in 250-ml screw-capped Erlenmyer flasks which were incubated at 25°C on a rotary shaker at 160 rpm. The medium initial pH was adjusted to 4.0, 5.0, 6.0, 7.0 and 8.0 by titration with sterilized 1 M HCl and 1 MNaOH.

2.3 Mutagenesis and mutant isolation

For the analysis of survival rates by UV mutagenesis, cells grown on YM agar slants for 18 hours at 25^°C. After incubation culture was collected and suspended in sterile distilled water. After that cell concentration was determined by counting cells was spread on YM agar plates. The plates were placed under a UV lamp at a distance of 55 cm and were irradiated for various periods of time (3, 5, 7, 10 min). Following irradiation, the plates were kept in dark for 1 hour before incubation at 25^°C for 3 days. The number of colonies on plates was then counted to determine survival rates. Mutant preculture was transferred into 250 ml screw-capped flask which contain 100 ml Glucose medium incubated with shaking at 160 rpm and at 25^°C. Cells were precultured in a Glucose medium for 49 hours to the logarithmic phase (Figure 2).

2.4 Fermentation studies

2% (v/v) of a Yeast suspension (from about 1 to 10 × 10 7 cells /ml) from the previously cultivated organism was inoculated to the 5 litre Fermentor which contain 2 litre fermentation media (Glucose medium). The cultures were agitated (or) stirred at 250 rpm. In the fermentor experiments, the cultures were aerated (1 vvm) with a KLa of 90/ hr. When cells reached the stationary phase, the desired volume of methyl ricinoleate (Castor oil) was added to the medium to initiate the bioconversion process. Gas was monitored during the entire fermentation: Oxygen and Carbon dioxide concentration was monitored using Servomen 1 100 gas analyser and URAS 3G analyser respectively (Figure 3). Fermentation process was carried out for various time periods such as 24, 48, 72, 96, 120 hours respectively. Fermentation process is carried out for various pH values like 7, 6, 5, 4, 3, by addition of NaOH (2.5N) or H2S04 (2.5N) in 500 mL culture flasks

3 Analytical procedures

3.1 Dry weight and viability

Viability 1 ml of the culture was diluted a million-fold in a sterile 0.85% NaCl solution. After vortex mixing, 100~1 of each dilution was plated onto Petridishes filled with the medium described above supplemented with agar (20 g/c) (Figure 4). Cell colonies were counted at different times. Dry weight 5 ml of the culture was filtered under vacuum and washed first with a mixture of ethanol/acetone (50:50, v/v) following by a second wash with distilled water only. The filtered biomass was put in small aluminum dishes and dried at 70°C until the weight became constant.

3.2 Extraction and aroma analysis

The fermentation liquid was micro filtered (0.2 µm) and the retentate (the residue containing the product and the biomass) was extracted batch-wise with eathanol. The product was neutralized by means of aqueous NaOH and NaCl was added under agitation .An upper phase (containing the product) was separated and was extracted with MTBE (methyl tri butyl either) After solvent evaporation, the extract was flash distilled under vacuum. The distillate consisted of 40% of γ–Decalactone. Gas chromatography (Agilent 7890) Volatile compounds were analyzed on a DB-FFAP bonded fused silica capillary column (30 m, 0.32 mm id, 0.25 pm film). Injection (1~1) was splitless/split (30 s). The temperature was raised from 40 to 240°C at 3°C/min. The hydrogen carrier gas velocity was 50 cm/s at room temperature. Integration of the lactone GC peaks was carried out using a chemstation software.

3.3 Growth behavior and production of GDL by S. Salmonoicolor T

The growth of S. salmonicolor in fermenter supplemented with castor oil hydrolysate containing 0.06% ricinoleic acid at the start of batch cultivation is shown in Figure 5. The population of S. salmonicolor increased from approximately 4.3 x 10⁴ CFU/ml at the start of cultivation to approximately 1.7 x 10⁸ CFU/ml at 72 h of cultivation at which time the culture entered the stationary growth phase. Thereafter, the population remained constant at approximately 1.8 x 10⁸ CFU/ml up to 120 h of cultivation in the batch culture.

The yield of γ-Decalactone increased rapidly after 24 h of cultivation and reached a maximum of 62.2 mg/l after 96 h up to 110 h. It was also observed that production rate will decreed after 110 h.

4 Effect of Mutation

After UV mutagenesis of Sporidiobolus salmonicolor for different time intervals (UV-1, UV-2, UV-3, UV-4) were used for production of γ-Decalactone in fermentation of glucose medium using castor oil. UV-3 producing 81.9 mg/l after 96 h, it was the maximum and high yield copared to wild and other mutants developed in the study. It was also observed that UV-1, UV-2mutants showed slight increment in production when compared to Wild type strain. And UV-4 mutant showed less GDL production when compared to all strains (Table 1; Figure 6).

5 Effects of PH

Among the various pH-controlled conditions we tested, the greatest yield of γ-decalactone for Wild type and mutant strain (UV-3). For Wild type strain 62.1 mg/l of decalactone was detected in the medium maintained at pH 4.0. However, pH 6 also showed favorable conditions for decalactone production. For mutant strain (UV-3) 80.1 mg/l of decalactone was detected in the medium maintained at pH 4.0. It was also observed pH 5, pH 6 also show favorable conditions for decalactone production (Table 2).

6 Conclusion

In this study the process standardization of microbial production of lactone and strain improvement for g- Decalactone production by UV mutagenesis. Comparing between mutant strains (UV-1, UV-2, UV-3, and UV-4) obtained from the UV treatments UV-4 ability was less than that of the mutant strain (UV-1, UV-2, UV-3). UV-1 and UV-2 was not much affected by the UV treatment and UV-3 show best result in the maximum production of the g -Decalactone.

Comparing between wild type strain and mutant strain, mutant strain ability was not much affected to the PH tolerance that the wild type. Although the UV treatment was not as a promising tool for improving PH tolerance at PH 7, 6, and 5. It might useful for the improving the production of the g -Decalactone. In this study even if the random mutagenesis method as UV show high efficiency for the general strain improvement, but not show tolerance at pH 7, 6, 5.

7 Discussion

The present paper deals with studies on upstream fermentation process for the production of optically active γ-hydroxydecanoic acid which may optionally be converted by lactonization to γ-Decalactone. Depending on the embodiment of the invention employed, the fermentation process involves culturing or incubating a S. salmonicolor capable of hydrolyzing castor oil and effecting β-oxidation of the resulting hydrolysate. The use of castor oil or castor oil hydrolysate as the substrate is determined by the S. salmonicolor employed in the process. S. almonicolor was subjected to UV Mutations in order to increase the yield of the process.

The metabolism of ricinoleic acid by some Candida strains was investigated by Okui et al. (J. Biochemistry, 54,536-540, 1963) who showed that γ-hydroxydecanoic acid was an intermediate in the oxidative degradation of ricinoleic acid. However, only trace amounts of γ-hydroxydecanoic acid were recovered from the fermentation medium due to the metabolysis of γ-hydroxydecanoic acid upon completion of the fermentation.

Acknowledgements

DMR is highly thankful to the authorities of SK University, Anantapur, APNLBT, IISC, Bangalore and IMTECH, Chandigarh for providing cultures.

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