Exploration of Potential Baker’s Yeast from Sugarcane Juice: Optimization and Evaluation

MATERIALS AND METHODS

Collection of samples: Sugarcane juice sample was collected in sterilized bottles from local market of Savar and allowed to ferment at room temperature for 2 days.

Isolation and culture media: Modified yeast extract malt extract agar (YMA) with combination of glucose (10 g L^-1), yeast extract (3 g L^-1), peptone (3 g L^-1), malt extract (3 g L^-1) and agar (15 g L^-1), pH 4.5 was used for isolation of yeasts while inhibiting other microorganisms. 0.1 ml dilutes of fermented sugarcane juice (diluted 10², 10⁴ and 10⁶ fold with autoclaved saline water) were plated onto modified YMA media and incubated for 48 h at 30°C to isolate morphologically well-formed single colonies (Qureshi et al., 2007). The yeast isolates were further sub-cultured (streak plated) on the respective medium to obtain pure culture.

Identification: The Gram stained yeast isolates were observed under the microscope (Hundwetzlar, Type-H600/12, Germany) to detect cellular shape and for bud and ascospore formation. Colony morphology was noted with respect to growth vigor, size, shape, margin, elevation, pigmentation, opacity and viscosity (Harrigan and McCance, 1982). Flocculent strains were selected by their capacity to sediment at the bottom of the tube at the end of fermentation as described by Verstrepen et al. (2001). The biochemical tests were based on the utilization of carbon and nitrogen sources (Harrigan and McCance, 1982; Vaughan-Martini and Martini, 1993; Sanni and Lonner, 1993), utilization of ethanol as sole carbon source and formation of ascospore (Harrigan and McCance, 1982).

Potency Assessment of identified S. cerevisiae for bread production
Production of undesirable compound: The six isolates were stab cultured at Bismuth Sulfite Agar, Kligler Iron Agar (KIA) and Sulfide Indole Motility (SIM) Agar and incubated at 30°C for 30 days and check every day for the capacity to produce H₂S. Butt that exhibit black color are H₂S producers (Jiranek et al., 1995).

Measurement of gas production: The production of gas was measured by a special apparatus made by attaching a vial in one end of a silicon adaptor and a 1 mL pipette containing 50 μL mercury at its tips on the other end. The vials were kept in a handmade rack where the pipettes were parallel to ground (to neglect gravitational movement) to confirm the movement of mercury by gas produced by the test microorganisms.

Optimum pH: Yeast strains were cultured in molasses broth of pH 3, 3.5, 4, 4.5, 5 and 5.5 containing 4% (w/v) molasses. Tubes containing 10 mL of these solutions were inoculated with same number of actively grown yeast cells (approximately 3.86x10⁵ cells mL^-1) and incubated at 30°C for 4 days. Optimum pH for growth and maximum biomass production was detected by measuring optical density at 550 nm using spectrophotometer (Shimadzu A-1075, Japan).

Optimum molasses concentration: Yeast strains cultured in molasses broth of pH 4.5 (previously determined as optimum) containing 2, 4, 8, 12, 16 and 20% (w/v) molasses, were inoculated with same number of actively grown yeast cells (approximately 3.86x10⁵ cells mL^-1) and incubated at 30°C for 4 days. Optimum molasses concentration for maximum growth and biomass production were detected by measuring optical density at 550 nm using spectrophotometer.

Optimum temperature: Yeast strains cultured in molasses broth of pH 4.5 containing 4% (w/v) molasses (previously determined as optimum), were inoculated with same number of actively grown yeast cells (approximately 3.86x10⁵ cells mL^-1) and incubated at 25, 30 and 37°C for 4 days. Optimum temperature for growth and maximum biomass production were determined by using spectrophotometer at 550 nm.

Incubation period: Yeast strains cultured in molasses broth of pH 4.5 containing 4% (w/v) molasses, were inoculated with same number of actively grown yeast cells and incubated at 30°C (previously determined as optimum) for 6 days. After incubation of each day, optical density was measured at 550 nm by using a spectrophotometer. The broth was vortex, serially diluted up to 10⁶ fold and spread plated to count viable cells (CFU mL^-1). After centrifugation pellets were weighted to determine cream cell mass and dried at 105°C in a heat dryer until constant weight to get dry cell mass.

Determination of agitation’s effect: Yeast strains cultured in molasses broth of pH 4.5 containing 4% (w/v) molasses, were inoculated with same number of actively grown yeast cells and incubated at 140 rpm and 30°C (previously determined as optimum). After 4 days centrifuged pellets were weighted to determine biomass production.

Preparation of bread from composite flour and cultivated yeast isolates: Bread was prepared with isolate S1 and S6 to observe the baking potency, using conventional method as described by Aboaba and Obakpolor (2010). Baking potency was determined by measuring the percentage of volume increment.

Statistical analysis: The experiments were performed in triplicate. Standard deviations were less than 3% in all experiments.

RESULTS

Six single colonies were selected on the basis of their morphological differences in YMA plates designated as S1, S2, S3, S4, S5 and S6, respectively. Among them, four (designated as S1, S2, S5 and S6) were identified as Saccharomyces cerevisiae and two as Saccharomyces rouxii by analyzing standard cultural, morphological and biochemical characteristics (Table 1-3).

Four identified S. cerevisiae strains were undergone several tests to determine their potency as baker’s yeast. None of them found to produce undesirable H₂S. In case of assessment of CO₂ production by the S. cerevisiae strains, S6 was found to generate highest amount of gas (226.67 mm³ mL^-1) whereas S5 produced about 62% lower CO₂ (Fig. 1).

Molasses broth was found to support better growth of yeast strains than sucrose broth. Highest OD for all the S. cerevisiae strains were found in molasses broth likely 1.81, 1.771, 1.803 and 1.864 for S1, S2, S5 and S6, respectively (Fig. 2) which were comparatively very low in sucrose broth (0.583, 0.607, 0.548 and 1.393, respectively).

Fig. 1:	Gas production by test isolates

Table 1:	Cultural (colony) characteristics of isolated yeast strains
Ci: Circular, Cr: Creamy, CW: Creamy white, O: Opaque, Co: Convex, Um: Umbonate, S: Smooth, E: Entire, L: Lobate, Un: Undulate, V: Viscid, ALG: Ring growth in Air-Liquid-Glass junction, SG: Sediment growth

Table 2:	Morphological characteristics of isolated yeast strains
‘+’ indicates present and ‘-’ indicates absent

Table 3:	Biochemical characteristics of test yeast isolates
“+” indicates highly positive, “-” indicates negative and “w” indicates weak acid and gas production and/or growth

Table 4:	OD at 550 nm of culture broth of different pH

Table 5:	OD at 550 nm of culture broth of different molasses concentration

Table 6:	OD after different incubation period

Table 7:	Cream cell mass mL-1 after different incubation period

Fig. 2:	Growth in different carbon sources, all the test isolates showed much better growth in molasses broth than sucrose broth (‘In’ for Inoculation, ‘OD3’ for Optical Density after 3 days, ‘SB’ for Sucrose Broth and ‘MB’ for Molasses Broth)

S. cerevisiae S1 strain showed the maximum cell density (2.451) at pH 4.5. The growth of other test isolates were somehow similar and found to be 2.301, 2.323 and 2.358 for S2, S5 and S6 in pH 4.5, 5.0, 4.5 and 4.0, respectively (Table 4). Very good growth patterns of yeast isolates were observed for S. cerevisiae strains at 2 to 4% of molasses broth. The best cell density was achieved for S5 strain (2.321) at 4% molasses broth. Among others, S5 had better cell density (2.173) at 2% of molasses broth (Table 5). Optimum temperature for growth was found 30°C for all the S. cerevisiae strains. It was found that 25 and 37°C suppress cellular growth significantly (Fig. 3). In case of optimization of incubation period, highest OD were found 2.591 and 2.498 for S1 and S5, respectively after 6 days of incubation at 30°C (Table 6). Cream cell mass mL^-1 (Table 7) was 33.14 and 27.96 mg for S1 and S2, respectively after 6 days and 29.12 and 29.68 mg for S5 and S6, respectively after 4 days of incubation. Dry cell mass mL^-1 (Table 8) was 4.94 and 4.16 mg for S1 and S2, respectively after 6 days and 4.20 and 4.54 mg for S5 and S6, respectively after 4 days of incubation. However, maximum CFU mL^-1 (Table 9) was found 6.76x10⁹, 5.28x10⁹, 1.528x10¹⁰ and 7.68x10⁹ for isolates S1, S2, S5 and S6, respectively after 4 days of incubation. All the test isolates showed much better production in shake culture than non-shake culture. S1, S2, S5 and S6 produced highest biomass of 84.2, 72.6, 81.4 and 68.8 mg mL^-1 in shake culture whereas it was 27.22, 27, 29.12 and 29.68 mg mL^-1, respectively in non-shake condition (Fig. 4).

Table 8:	Dry cell mass mL-1 after different incubation period

Table 9:	CFU mL-1 after different incubation period

Table 10:	Leavening profiles of dough using S. cerevisiae S1 and S6 strain

Fig. 3:	OD at 550 nm of culture broth incubated at different temperature

The strain S6 showed comparatively good baking potential than S1. S6 was relatively good in producing flavor and color of bread baked at 180°C for 1.5 h although bread texture, taste and mouth feeling were not uncourageous (Table 10).

Fig. 4:	Effect of shaking on biomass production by test isolates. Biomass was produced much better in shake culture than non-shake culture by all the test isolates

DISCUSSION

Successful commercial production of baker’s yeast depends on the availability of cheap suitable carbon source, good yeast strain, easily manageable fermentation process for maximum viable biomass production and the process development for preservation. Beside this the production efficiency of a strain depends on many factors such as nature, composition and concentration of substrate, temperature, pH, inoculums size, incubation period etc. (Supanwong et al., 1983). Selection of optimum cultural and fermentation conditions with maximum viable biomass production and later gas production at a moderate rate is mandatory for natural yeast isolates to be used as an effective commercial baker’s yeast.

Baker’s yeast act as a natural leavener, or rising agent of baked bread by producing CO₂ gas during the time of dough incubation. In our experiment, S. cerevisiae S6 strain was found to be the highest gas producer among four isolates. H₂S is an undesirable compound associated with an off-flavor and unpleasant taste that must be absent in processed foods (Ribeiro and Horii, 1999; Vicente et al., 2006). Production of H₂S by test isolates was checked and no strain found to produce H₂S.

The cost of baker’s yeast production depends mostly on the medium carbon source used. Molasses containing about 60% sucrose (Patrascu et al., 2009) was found to be better carbon source than pure sucrose by measuring the max cell biomass yielded in the respective culture media. It may be due to the presence of some important growth promoting substances likely vitamins, minerals and other natural molecules in molasses broth that enhance yeast growth significantly. This finding indicates the use of molasses as an effective cheap and widely available carbon source for baker’s yeast production. Qureshi et al. (2007) reported low pH to be helpful for inhibiting the growth of undesirable microorganisms in large scale fermentation. All the test Saccharomyces cerevisiae strains grown well at a pH range of 4.0 to 5.0 that is suitable to resists contamination by unwanted microbes.

Cream cell mass and dry cell mass were maximum for S1 and S2 after 6 days and S5 and S6 after 4 days of incubation. However, all the test isolates produced maximum CFU mL^-1 after 4 days of incubation and falls down drastically after 4 days. This might be due to accumulation of waste products, high cell concentration and depletion of nutrients that cause rapid demise of yeast cell after given time period. As maximum cell density was found before the death phase begins, fed-batch or continuous culture could be used effectively for the production of baker’s yeast instead of batch culture that initiates ethanol production. Four days of incubation seemed to be suitable/optimum period of incubation when highest number of viable cell with lowest cell weight was obtained.

Optimization of relevant growth parameters is important to obtain maximum production of yeast cell biomass. The biomass and viable cell production rate by S. cerevisiae S6 was significantly higher than the other yeast strains. Aeration and agitation are two important parameters influenced the production of cell mass reported by Ivanova et al. (2001). In all the test, S. cerevisiae stains showed two to three fold better biomass production in shake than non-shake culture. This might be happened as shaking ensures the availability of nutrients and oxygen for all the cells and thus confirms maximum biomass production within minimum period of time (Narang and Satyanarayana, 2001).

Aboaba and Obakpolor (2010) found increased dough volume with increased agitation time but only to a limited extent as longer period (40 min) caused a drastic drop in dough volume. The period for mixing therefore is important as it influences the size and quality of bread that enhances the ability of the dough to trap more air. About 111% increase in bread size was observed after mixing S. cerevisiae S6 strain followed by incubating the dough for 30 min. Baking at 180°C for 1.5 h found to increase the bread volume to about 128%. Although, the color and flavor of baked bread were somehow better, the texture, taste and mouth feeling were poor.

CONCLUSION

The present study found S. cerevisiae strain S6 as potent candidate to be used as baker’s yeast. Although in some instances it showed insignificant activity that can be overcome by adding some chemical food additives when producing bread. Further research should be carried out to modify S. cerevisiae strain S6 genetic level by radiation, chemical mutagen or genetic incorporation to overproduce the enzymes responsible for breaking down dough sugar into ethanol and carbon-dioxide and thereby increasing its bread making efficiency.

ACKNOWLEDGMENTS

The authors would like to thank the Microbiology and Industrial Irradiation Division (MIID), Institute of Food and Radiation Biology (IFRB), Atomic Energy Research Establishment, Ganakbari, Savar, Dhaka, Bangladesh for providing laboratory facilities.