Screening of microbial isolates for extracellular fructosyltransferase production

Fructooligosaccharides are gaining importance by leaps and bounce every day due to their low caloric value and accompanying health benefits for the consumer. Hence, newer potential sources for the fructosyltransferase (Ftase) enzyme are thoroughly scrutinized. The present work was carried out for preliminary and secondary screening of the microbial isolates for determination of the Ftase producing potential. Three classes of microorganisms’ viz. molds, yeasts and bacterial isolates were qualitatively screened. The preliminary screening revealed molds as the most potent group exhibiting greater zone of hydrolysis in the range of 0.30 ± 0.10 to 2.58 ± 0.10 cm. The secondary screening of 20 selected mold isolates was performed in liquid batch culture. Four isolates identified as Aspergillus niger, Aspergillus flavus, Aspergillus stallus and Aspergillus versicolor exhibited higher fructosyltransferase : invertase ratio which is most critical for the synthesis of fructooligosaccharides due to higher transferase activity and low hydrolytic activity. A. niger exhibited highest Ftase activity, 36.88 ± 0.23 IU/mg. A. flavus produced 21.45 ± 0.33 IU/mg of Ftase. A. stallus produced 18.09 ± 0.14 IU/ mg and A. versicolor was found to produce 23.78 ± 0.12 IU/mg of Ftase without any cultural medium optimization.


INTRODUCTION
Functional foods like fructooligosaccharides are gaining prime importance in the health market because they provide multiple health benefits like low caloric value, promoting growth of Bifidobacteria in colon (prebiotic property), low glycemic index and low carcinogenicity in aid of basic nutrition (Dominguez et al., 2013;Moore et al., 2003 andSlevin et al., 2014).The most popular functional food coming into the lime light is fructooligosaccharides (FOS) obtained from sucrose.
The sucrose is biologically transformed into FOS either by using microorganism itself (Chien et al., 2001;Sanchez et al., 2010) or by enzymes derived from them (Fawkia et al., 2009).
Scientists are constantly attempting to isolate new strains of microorganisms having higher potential for production of the enzyme.Since screening is an elaborate time consuming and tedious process, very few attempts have been made to indigenously isolate the microbe from environment and subject them to screening (Madlova et al., 1999;Reddy et al., 2010;Fernandez et al., 2007).The objective of the present study was to screen the filamentous fungi, yeast and bacteria to enable their usage in industries.

Screening for yeast isolates
The modified method of Maugeri and Heernalsteens (2007) was followed in which the medium was replaced by the above defined basal medium at pH 5.50.Loop full of cells were inoculated into 10 ml of basal medium in test tubes and cultivated at 25°C for 24 h.After adequate development of the cultures, they were transferred into 500 ml flasks containing a further 100 ml of the same medium, and cultivated at 25°C for 72 h .The cells were removed by centrifugation at 6000 ×g (10 min, 4°C), and the cell-free supernatants were used to screen for transfructosylating activity by plate method containing selective medium.The conditions maintained were for support of fructosyltransferase production.

Screening for molds isolates
The molds were cultivated on basal medium at pH 5.5 with sucrose as the substrate.Screening was carried out under static conditions at 28°C for 72 h.All the experiments were conducted in triplicates.The supernatant was centrifuged at 6000 ×g (10 min, 4°C).The cell-free supernatant was used to screen for transfructosylating activity by plate method containing selective medium.

Preliminary screening of Ftase production by fungi
Wells were made aseptically by using cock borer in the Petri plate containing selective media (sucrose-30.0,NaNO 3-2.5, MgSO 4 .7H 2 O -0.5, KH 2 PO 4 -1.5 and Agar powder -20 at pH 5.50).Two hundred microliter of cell-free supernatants of the yeast and mold cultures were individually (crude enzyme) loaded into the well and kept for incubation at 28°C for 12 h.Staining was carried out by spraying triphenyltetrazolium chloride (TTC) reagent to the agar plate and kept for incubation of 20 min in the dark.After washing with the 0.1 M acetate buffer (pH-5), the extracellular production of fructosyltransferase was confirmed by the appearance of the red zone (measured in cm) around the well according to the protocol reported by Reddy et al. (2010).

Screening for bacteria
To select strains of bacteria, the isolates were plated in selective Belorkar et al. 731 media plates and incubated for 48 h and subjected to TTC staining directly to measure the zone of hydrolysis described by Kim et al. (2000).

Assay of fructosyltransferase activity
Quantitative assay of fructosyltransferase was based on the procedure used by Yun et al. (1997).The filterate was taken as a crude enzyme with 50% sucrose solution as a substrate at 5.50 pH (0.1 M sodium acetate buffer).The mixture was incubated for 1 h at 60°C.The reducing sugars were estimated by Dinitro-salicylic acid reagent.The enzymatic reaction was terminated by keeping the test tube at 100°C in a water bath for 10 min.One unit of enzyme activity was defined as the amount of enzyme producing 1 µmol of glucose under experimental conditions.

Assay of hydrolytic activity
Sucrose hydrolytic activity was measured according to the method described by Sangeetha et al. (2003).The filtrate was taken as a crude enzyme with 0.50% sucrose solution as a substrate at 5.50 pH (0.1 M sodium acetate buffer).The mixture was incubated for 1 h at 60°C.The reducing sugars were estimated by dinitro-salicylic acid reagent.The enzymatic reaction was terminated by keeping the test tube at 100°C in a water bath for 10 min.One unit of sucrose hydrolytic activity was considered as the amount of enzyme required to produce 1 µmol of glucose under experimental conditions.

Determination of protein concentration
The protein content was determined following Lowry et al. (1951).

Biomass concentration
Biomass concentration was determined in g/100 ml volume of fermentation medium.The biomass obtained by filtration of the fermentation broth was washed thrice with distilled water, air dried and weighed.

Statistical analysis
All the experiments were conducted in triplicate.The results were expressed in Mean ± SE.One-way ANOVA using SPSS 16.0 was performed to analyze the variance in preliminary screening.

Identification of the selected isolates
These four isolates were then identified morphologically by lactophenol cotton blue staining and found to be SSFM1 as Aspergillus niger NFCCI2736, SSFM2 as Aspergillus flavus NFCCI2734, SAFM30 as Aspergillus stallus, RSCDS48 as Aspergillus versicolor gr.

Preliminary screening
Thirty one (31) yeast isolates, forty two bacterial isolates  and 54 molds isolated during the previous study were subjected to prelimnary screening.The zone of hydrolysis for molds, yeasts and bacteria are presented in Figures 1A, 2 and 3, respectively.The mold isolates proved to be the most potential Ftase producers.The zone of hydrolysis developed after TTC staining is represented in Figure 1B The mold isolates exhibited highest zone of hydrolysis ranging from 0.30 ± 0.10 to 2.58 ± 0.10 cm.The largest zone of hydrolysis was recorded for isolate SSFM1 (2.58 ± 0.10 cm) and smallest zone was recorded for SAFM 27, SAFM 35, BM 37 and WY 44.The zone of hydrolysis recorded for molds was found to be similar to the findings of Reddy et al. (2010).Yeasts have been consistently reported to be potent producers of Ftase since the pioneering work of Bacon and Edelman (1950) investigating action of invertase on sucrose.Similarly, isolation of potent bacterial strains like Psuedomonas sp.No. 65 from soil has been reported by Kim et al. (1997).
The zone of hydrolysis for yeast isolates was found to be in the range of 0.15 ± 0.01 to 0.58 ± 0.01 cm as given in Figure 2. The maximum zone of hydrolysis was exhibited by RSCDS 24, the value being to 0.58 ± 0.01 cm.The zone of hydrolysis for bacterial isolates was found to be in the range of 0.12 ± 0.01 to 0.54 ± 0.10 cm.The maximum zone of hydrolysis exhibited was SSFB2, the value being 0.54 ± 0.10 cm as given in Figure 3.The bacterial and yeast isolates exhibited less potential for fructosyltransferase production as compared to results of Kim et al, (1997) and Maugeri and Hernalsteens (2007), respectively .
In this study, highest range of zone of hydrolysis was found to be of filamentous fungi among the three classes of microbes, similar to the results reported by Yun (1996).The filamentous fungi were therefore, selected for secondary screening in this study as well.

Secondary screening for determination of extracellular Ftase and invertase production
The comparison of zone of hydrolysis of the three microbial groups was subjected to preliminary screening directed in the present study towards selection of molds for secondary screening.Twenty (20) mold isolates were selected for secondary screening in liquid basal medium depending upon their zone of hydrolysis namely SSFM1, SSFM2, SSFM5, BM8, SSFM9, SSFM11, SSFM12, GDRM13, SAFM19, SAFM21, SAFM24, SAFM26, SAFM28, SAFM29, SAFM30, SAFM31, RSCDS36, SSFM39, RSCDS48 and RSCDS52.
The average values of biomass production, Ftase production and invertase (Inv) production by the isolates are presented in Table 1.The most vital parameter in screening experiments for Ftase production is the Ftase : Inv ratio (Hidaka et al., 1988;Hidaka 1988).The Ftase : Inv ratio was also determined for each mold and was found to be desirable in SSFM1 (1.63), SSFM2 (1.51), SAFM30 (1.36) and RSCDSM48 (1.04).In the earlier screening experiments, the ratio was reported between 0.3-15.9(Fernandez et al., 2007;Patel et al., 1994;Reddy et al., 2010).The ratio determines the efficacy of transferase activity for synthesis of FOS.The extracellular Ftase production has been reported to be in the range of 0.053 IU to 660 IU/ml (Maiorano et al., 2008).The four molds selected from the twenty molds subjected to secondary screening which exhibited high Ftase/Inv ratios was recorded for SSFM1 (1.63), SSFM2 (1.51), SAFM30 (1.36) and RSCDS48 (1.04).

Conclusion
As FOS has already been given GRAS status, the molds will be promising tool for production of Ftase enzyme for synthesis of FOS.The results of the present screening experiments revealed microbes especially molds to be source exhibiting potential for Ftase production which is gaining importance in food industry for synthesis of prebiotics.The urge of researchers to search for newer Ftase producing microbes can only be quenched with such screening experiments.The present study revealed higher Ftase : Inv ratio of the isolates which can be even enhanced upon optimization of cultural medium.Further optimization experiments will be carried out to boost the Ftase production levels for maximization of FOS synthesis.

Figure 1A .
Figure 1A.Zone of hydrolysis (cm) for mold isolates.Same legends represent the zone of hydrolysis of the isolates not significantly different.

Figure 1B .
Figure 1B.Zone of hydrolysis exhibited by identified isolates during preliminary screening.Same legends represent the zone of hydrolysis of the isolates not significantly different.

Figure 2 .
Figure 2. Zone of hydrolysis in cm for yeast isolates.Same legends represent the zone of hydrolysis of the isolates not significantly different.

Figure 3 .
Figure 3. Zone of hydrolysis in cm for bacterial isolates.Same legends represent the zone of hydrolysis of the isolates not significantly different.
All values presented in the table are mean ± SE. *Identified isolates (SSFM1 identified as Aspergillus niger; SSFM2 identified as A. flavus; SAFM 30 identified as A. stallus; RSCDS 48 identified as A. versicolor).