Y = mannitol concentration.
x = optical density value.
The mannitol yield is shown in Table 1. The values ranged from 0.36 to 0.65 g/L. Among the screened ones, isolates
viz., L4, L6, L8 and L9 showed comparatively higher mannitol yield of 0.51, 0.54, 0.65 and 0.57 g/L respectively selected for further study. In a study conducted by
Lactobacillus reuteri CRL 1101 was shown to be capable of producing mannitol effectively by
Ortiz et al., (2017). According to
Von Weymarn et al., (2002) Leuconostoc, Oenococcus and Lactobacillus, can produce mannitol. Cell density shows that mannitol production differs amongst LAB species with identical growth rates (Table 1).
Helanto et al., (2005) observed maximum cell growth of
Leuconostoc pseudomesenteroides, along with mannitol production 30°C, pH 5 and 200 rpm.
Optimization of the cultivation conditions for maximum mannitol production
Effect of varying temperature, pH and non-agitation on mannitol yield
Mean mannitol production by selected isolates at 33°C and different pH was in Table 2. All isolates produced significantly higher mannitol at pH 7.0. In fact, increasing the pH from 6.0 to 7.0 resulted in a small increase in productivity. Likewise, at 37°C all the isolates produced maximum mannitol at pH 7.0. Among that L9 produced higher mannitol of 1.30± 0.27 g/L. Significantly no differences were observed in mannitol yield at different pH. While at 42°C, it is obvious from Table 3 that except L9, all other isolates showed a better productivity at pH 7.0. However, strain L4 showed significant difference in yield at different pH.
Yun and Kim (1998) reported maximum production of mannitol of 73 g and 26 g from
Lactobacillus species and
Leuconostoc species respectively, at 35°C and pH 8.0 and 6.0 respectively. In research conducted by
Weymarn et al., (2002), mannitol production from
Lactobacillus fermentum were estimated to be 86, 89 and 94 mol per cent at 25°C, 30°C and 35°C respectively. Volumetrically higher mannitol yield was detected at 35°C than at 25°C in
Lactobacillus and
Leuconostoc species. In a study conducted by
Ojamo et al., (2003), higher mannitol production of 20 g/L/h was reported by
Leuconostoc pseudomesenteroides ATCC 12291 strain at high cell density fermentation.
Effect of different temperature, pH and agitation on mannitol yield
When evaluating effect of agitation, pH 7.0 shown to be the best for all isolates, resulting in significantly higher mannitol production at 33°C (Table 4). The highest mannitol production was shown by isolate L8 (0.99±0.01 g/L) followed by L4, L9, and L6. Production of standard
Leuconostoc mesenteroides was 0.89±0.02 g/L. At 37°C (Table 5) isolate L8 produced significantly higher mannitol of 1.17±0.04 g/L at pH 6.5. Isolates didn’t show any significant variation at pH 6.5 and 7.0. At 42°C with agitation the values ranged from 1.12±0.09 g/L to 1.63±0.16 g/L, where peak mannitol production was by isolate L8 at pH 7.0.
Yun and Kim (1998) specified that partial aeration was necessary for higher mannitol yield.
Weymarn et al., (2002) observed an increase in mannitol production with
Lactobacillus fermentum from 1.33±0.02 to 1.65±0.06 (g/L/h) at semi anaerobic condition. Sugarcane molasses yielded the highest mannitol concentrations (38 and 41.5 g/L) when cultivated
Lactobacillus reuteri in agitated cultures at 37°C
(Ortiz et al., 2017). This finding was in harmony to the present exploration.
Effect of temperature, pH and agitation on bacterial cell density
Growth rate of all isolates was significantly different (P<0.05) in different conditions. While considering the non-agitated condition, isolate L8 showed significant difference in cell density on different pH at 33°C and 42°C. L6 showed significant difference in cell density at 33°C and 37°C, whereas L4 showed significant difference in cell density only at 42°C. With respect to condition of agitation at 100 rpm, significant difference was noted in cell density for isolate L9 and L6 at all temperatures studied. Isolate L4 showed significant difference at 33°C and 37°C, whereas L8 at 33°C and 42°C. Significantly higher growth rate was observed in agitated cultures than non-agitated. Hence by proper agitation, temperature and pH the yield of mannitol by different lactic acid bacteria can be improved.
Grobben et al. (2001) opined higher production of mannitol of 75 mM by
Leuconostoc pseudomesenteroides with high cell density at 30°C and pH 4.5.
Weymarn et al., (2002) observed that growth rate of all strains increased in semi anaerobic condition, in accordance to present findings.
Ojamo et al. (2003) reported higher mannitol production by
Leuconostoc pseudomesenteroides with high cell density.
Helanto et al., (2005) observed maximum cell growth of
Leuconostoc pseudomesenteroides, at 30°C temperature, pH 5 and 200 rpm agitation.
Selection of superior lab isolate by response surface methodology
A total of 540 combinations were obtained and statistically analyzed using response surface methodology
(Kumari et al., 2016). Dendrogram is shown in Fig 2. The 16
th cluster produced higher mannitol concentration of 1.6209 g/L with 0.2799 optical density of cell growth. Sixteenth cluster included 2 combination studies with agitation (100 rpm), of isolate L8 with 1.6067 g/L mannitol and 0.213767 OD value at pH 6.5, 42°C, later with1.635 g/L mannitol and 0.345967 OD at pH 7.0, 42°C. From these second one was selected as the best. Using cluster and dendrogram analysis, mannitol producer (9.46 0.27 g/l) strain
F. tropaeoli was discovered from fruits by
Ruiz Rodriguez et al., (2017). With a maximum volumetric productivity of 2.36 g/l h and the highest yield, of mannitol was obtained for
L. fructosum NRRL B-2041 according to
Carvalheiro et al. (2011). Also, different
Leuconostoc pseudomesenteroides strains were studied for its higher mannitol production potential at different growth conditions
(Bhatt et al., 2013).
Identification of lab isolates
Biochemical identification
Most of the phenotypical and biochemical characteristics of strains, L4, L6, L8 and L9 were explained in Fig 3. Sugar fermentation tests, growth condition studies and other biochemical studies proved that the isolate L4 was
Enterococcus sp. L8 was
Leuconostoc sp. and L6 and L9 belong to
Lactobacillus sp. according to Bergey’s Manual of Determinative Bacteriology.
In a study conducted by
Makanjuola and Springham (1984),
Leuconostoc sp. was able to produce gas from glucose, ammonia from arginine and also showed growth at 15°C and 45°C. Fermentation results of rhamnose and arabinose support the present study to characterize L8 isolate as
Leuconostoc sp.
Gancel et al. (1997) identified genus
Lactobacillus from vacuum packed fish meat by biochemical characterization with tolerance of high levels of NaCl, bile salts and CO2 production. But in present study all the isolates of LAB L4, L6, L8 and L9 were intolerant to higher concentration NaCl. In different studies (
Azadnia and Nazer, 2009;
Nair and Surendran, 2005) fermentation of specific sugars were observed for Lactobacillus species especially
L. plantarum. These observations are related to the results obtained for L9 isolate in the present study. Also,
Bhatt et al. (2012) used bile tolerance test in LAB identification. According to
Hassanzadazar et al. (2012) all LAB strains isolated could not tolerate the pH≤2 and the presence of bile salts, which agree with the existing results. Sugar fermentation assays and growth of LAB at different conditions in the above investigation were supportive to the present study.
Molecular characterization of LAB strains
Sequencing result confirmed L4 as
Enterococcus faecium strain Gr17, L6 as
Lactobacillus rhamnosus strain 6870, L8 as
Leuconostoc pseudomesenteroides culture IMAU: 11666 and L9 as
Lactobacillus plantarum subsp.
plantarum strain NMB8. Bacterial species identification by 16S rRNA-based technique is the most accepted, as large public-domain sequence databases are accessible in Gene Bank for comparison
(Morgan et al., 2009; Sharif et al., 2018).
