Recombinant GH-26 endo-mannanase from Bacillus sp. CFR1601: Biochemical characterization and application in preparation of partially hydrolysed guar gum

LWT - Food Science and Technology 64 (2015) 809e816

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Recombinant GH-26 endo-mannanase from Bacillus sp. CFR1601: Biochemical characterization and application in preparation of partially hydrolysed guar gum Praveen Kumar Srivastava, Mukesh Kapoor* Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru, 570020, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 April 2015 Received in revised form 16 June 2015 Accepted 17 June 2015 Available online 25 June 2015

Biochemical characterization studies of ManB-1601, a recombinant endo-mannanase from Bacillus sp. CFR1601, revealed that the enzyme was optimally active at moderately high temperature (50e55
C) and neutral pH (6e7). ManB-1601 showed excellent stability over a broad pH range of 6e10 by retaining more than 70% activity after 9 h of incubation at room temperature. The apparent Km, Vmax and Vmax/Km values of ManB-1601 were 6.5 mg/ml, 5000 IU/ml/min and 769.2 mmol/min/mg, respectively, using locust bean gum as the substrate. ManB-1601 was stable in the presence of organic solvents (DMSO, methanol, ethanol and acetone), heavy metals (Co2þ, Ni2þ, and Cu2þ) and displayed tolerance towards proteases (trypsin and chymotrypsin). The hydrolysis of guar gum (GG) by ManB-1601 led to production of partially hydrolyzed guar gum (PHGG) with high flow behaviour index (1.478 PHGG, 0.332 GG), markedly reduced average degree of polymerization (14 PHGG, 890 GG), average molecular weight (3814 PHGG, 239883 GG), apparent viscosity (2.8 mPas PHGG, 346 mPa GG) and consistency index (0.2 mPasn PHGG, 5734.7 mPasn GG). FTIR spectra of GG and PHGG indicated no major functional group transformations and similarity in the basic chemical structure. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Enzyme properties Guar galactomannan Enzymatic depolymerization Rheology Viscosity

1. Introduction In nature, hemicelluloses are the second most abundant biopolymers and found in plant cell wall in close association with cellulose and lignin. Mannans represent the major (15e20%) and minor (5%) hemicelluloses in softwood and hardwood, respectively (Moreira & Filho, 2008). The complete biodegradation of mannans requires concerted action of endo-b-mannanases (EC 3.2.1.78) which catalyse the random hydrolysis of b-mannosidic bonds (McCleary & Matheson, 1983) to produce manno-oligosaccharides of different degree of polymerization, b-D-mannosidases (EC 3.2.1.25) and b-D-glucosidases (EC 3.2.1.21) which act upon mannooligosaccharides and a-D-galactosidases (EC 3.2.1.22) and acetyl esterases (EC.3.1.1.6) which liberate the side group substituents (Dhawan & Kaur, 2007). A vast variety of wild-type and recombinant bacteria and fungi have been shown to produce mannan degrading enzymes (Chauhan, Puri, Sharma, & Gupta, 2012). Preparation of health-promoting prebiotic oligosaccharides and

* Corresponding author. E-mail address: [email protected] (M. Kapoor). http://dx.doi.org/10.1016/j.lwt.2015.06.059 0023-6438/© 2015 Elsevier Ltd. All rights reserved.

dietary fibres from guar and locust bean gum, bleaching of paper pulp, production of biofuels and fabric cleaning detergents represents some of the recent exciting applications of mannanases (Chauhan et al., 2012; Srivastava & Kapoor, 2014). Now-a-days industries are demanding mannanases with well-defined biochemical characteristics with respect to pH, temperature, additives (metal ions, surfactants and organic solvents) and proteolytic stability. Therefore, it is quintessential to understand the biochemical properties of mannanases for their optimal industrial utility. Guar gum (GG), obtained from the seeds of cluster bean (Cyamopsis tetragonoloba), is utilized in food products as a source of dietary fibre apart from providing hydration, thickening and stabilizing properties. However, high viscosity of GG hampers its incorporation in food products besides affecting protein efficacy and nutrient digestion (Yoon, Chu, & Juneja, 2008). PHGG like GG offers multiple health benefits in humans like reduction of serum cholesterol, free fatty acid, and glucose concentrations and help in addressing constipation issues by increasing the defecating frequency. Moreover, PHGG can be easily incorporated in food products due to high solubility, less viscosity and stabilizing properties and therefore represents a feasible alternative to GG for food industry (Yoon et al., 2008).

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P.K. Srivastava, M. Kapoor / LWT - Food Science and Technology 64 (2015) 809e816

Physical (hydrothermal, hydrodynamic cavitation, ultrasonication, microwave and gamma irradiation) and chemical (acid hydrolysis) methods for producing PHGG require high energy inputs and are not environment friendly (Gupta, Shah, Sanyal, Variyar, & Sharma, 2009; Gupta, Saurabh, Variyar, & Sharma, 2015; Hongbo, Min, Yanping, & Siqing, 2013; Miyazawa & Funazukuri, 2006; Prajapat & Gogate, 2015a, b; Sarkar, Gupta, Variyar, Sharma, & Singhal, 2012). Instead, low-cost and mild enzymatic depolymerization of GG represents a plausible alternative. Earlier, few workers have carried out depolymerization of GG using specific and non-specific enzymes (Gupta et al., 2015; Hussain et al., 2015; Mahammad, Prud'homme, Roberts, & Khan, 2006; Mudgil, Barak, & Khatkar, 2012a, Mudgil, Barak, & Khatkar, 2012b; Mudgil, Barak, & Khatkar, 2014; Shobha, Vishnu Kumar, Taranathan, Koka & Gaonkar, 2005; Wientjes, Duits, Bakker, Jongschaap, & Mellema, 2001) but rheological behaviour of endomannanase treated GG solutions still lack understanding especially with respect to intrinsic viscosity, yield stress, average degree of polymerization and spectral changes. We have reported previously thermal stability (Srivastava, Appu Rao, & Kapoor, 2014a; Srivastava, Rao, & Kapoor, 2014b) of recombinant endo-mannanase (ManB-1601) from Bacillus sp. CFR1601 (Srivastava & Kapoor, 2013, 2014). In the present study, ManB-1601 belonging to glycoside hydrolase family 26 has been characterized for its biochemical properties and used successfully in the production of PHGG. 2. Materials and methods 2.1. Preparation of ManB-1601 ManB-1601 was produced and purified according to the method reported elsewhere (Srivastava et al., 2014a, b). Briefly, gene specific primers were used to obtain amplicon and the amplified fragment was cloned and heterologously expressed. ManB-1601, after purification by Phenyl-Sepharose chromatography, was extensively dialysed with 20 mM (pH 6.0) phosphate buffer prepared in Milli Q (Milli-Q ultrapure water purification systems, MA, USA) water for 12 h with 4 intermittent changes of buffer. 2.2. Biochemical characteristics of purified ManB-1601 The optimum pH of ManB-1601 was estimated by assaying the enzyme activity over the pH range of 4e10 using different buffers [50 mM; acetate (pH 4), citrate phosphate (pH 5), phosphate (pH 6e8), glycine NaOH (pH 9, 10)] at 55
C while pH stability was studied by estimating the residual activity after pre-incubating the enzyme for 9 h in different buffers (pH 4e10, 20 mM) at room temperature. The optimum temperature of ManB-1601 was estimated by assaying the enzyme activity at pH 7 over the temperature range of 45e70
C. To study the effect of metal ions (1 mM) (Jiang et al., 2006; Kim et al., 2011; Ma et al., 2004) and surfactants (0.2% w/v/v/v) (Panwar, Srivastava, & Kapoor, 2014) on enzyme activity, suitably diluted enzyme was assayed in the presence of respective chemicals under standard conditions (pH 7.0, 55
C). To determine the effect of organic solvents (dimethyl sulfoxide, methanol, ethanol, acetone, ethyl acetate, acetonitrile) on the stability of ManB-1601, suitably diluted enzyme was pre-incubated with individual solvents at 25% and 50% (v/v) for 3 h at room temperature (Chauhan, Sharma, Puri, & Gupta, 2014). Thereafter, residual enzyme activity was measured under standard conditions. ManB-1601 without any additive (metal ion/surfactant/organic solvent) was used as the control. Kinetic parameters (Km, Vmax and Vmax/Km) were determined by measuring ManB-1601 activity at different concentrations of locust bean gum (1e5 mg/ml) in 50 mM sodium

phosphate buffer (pH 7). The data obtained were fitted to the Lineweaver-Burk plot. In order to understand the protease resistance of ManB-1601, enzyme (18 mg, 10,461.5 IU/mg) was treated in independent reactions with trypsin (0.4 mg, 10,800 IU/mg, pH 7.6) and chymotrypsin (0.4 mg, 53.5 IU/mg) at pH 7.6 and 7.4 in 50 mM sodium phosphate buffer, respectively (Sunna, 2010). The reaction was carried out for 30 min at 37
C. Thereafter, residual enzyme activity was estimated under standard conditions. All the experiments on biochemical properties were carried out in triplicate. 2.3. Physicochemical and rheological attributes of GG hydrolysed by ManB-1601 2.3.1. Hydrolysis of GG by ManB-1601 GG solution was prepared by sprinkling 1 g of guar gum powder in 100 ml of 50 mM phosphate buffer pH 7 under stirring conditions (600 rpm). The prepared solution was subjected to hydrolysis by ManB-1601 (10 IU/ml, pH 7) for 4 h under shaking conditions (100 rpm) at 50
C (Srivastava & Kapoor, 2014). The resulting solution was sterilized (121
C, 5 min) to inactivate the enzyme and filtered using Whatman No. 1 filter paper. The filtrate was lyophilized to get PHGG powder and stored at 20
C until further characterization. 2.3.2. Analysis of physicochemical and rheological properties A controlled stress rheometer with coaxial cylinder (Z20) attachment (Model # RS6000 Haake RheoWin, Thermo Scientific, Germany) was used to study the flow behaviour of the GG (1% w/v) and PHGG (1% w/v) dispersions (Dhanalakshmi & Bhattacharya, 2014). All the rheological measurements were carried out in triplicate at 25 ± 0.1
C by employing a circulating water bath for controlling temperature. Apparent viscosity was determined by calculating the ratio of shear-stress and shear-rate when the latter was taken as 100 s1. Yield stress was determined by shearing at 5 s1 for 30 s followed by stopping the spindle and allowing the sample to rest for 2 min. Shear-stress and shear-rate data obtained were fitted to the power law (Eq. (1)) and Herschel-Bulkley (Eq. (2)) models. The model parameters like the flow behaviour index (n) and consistency index (k) were estimated by using the software provided with the instrument. In the power law and Herschel-Bulkley models, s was the shearstress, g: was the shear-rate, k was the consistency index, n was the flow behaviour index (FBI) (dimensionless) and to was the yield stress. The correlation coefficient (r) was used to determine the suitability of power law model, and the significance of r-values was judged at P  0.01.

s ¼ kðg: Þ

n

s ¼ to þ kðg: Þ

(1) n

(2)

2.3.3. Determination of intrinsic viscosity, average molecular weight and average degree of polymerization The intrinsic viscosity was determined according to Mudgil et al., 2012a with slight modifications. Ostwald's capillary viscometer was used to determine the intrinsic viscosity of GG (0.2e1 mg/ ml) and PHGG (1e5 mg/ml) at room temperature. The solutions were prepared by dispersing GG/PHGG in Milli Q water at high vortexing followed by mixing for 2 h under shaking conditions (200 rpm) at 25
C. Thereafter, solutions were allowed to hydrate completely for 12 h at 25
C and then filtered through Whatman no. 1 filter paper. Relative viscosity (hr) was calculated as the ratio of time of flow of a given volume of the sample (GG/PHGG, t) and

P.K. Srivastava, M. Kapoor / LWT - Food Science and Technology 64 (2015) 809e816

811

solvent (MQ water, to). Relative viscosity values were used to determine specific viscosities (hsp ¼ hr1). Reduced viscosity values were obtained using the equation hred ¼ hsp/C, where C is the concentration of sample. Plot of reduced viscosities values versus respective sample concentrations was extrapolated to zero concentration in order to deduce intrinsic viscosity [h]. The concentration dependence of viscosity was represented by the following relationship (Reddy & Tammishetti, 2004).

SigmaeAldrich (St. Louis, MO, USA). All other chemicals and reagents procured commercially had the purity of highest available grade.

hsp ¼ ½h þ K½h2 C C

pH influences enzyme function as the formation of enzymesubstrate complex and catalysis are often dependent on the charge distributed on enzyme and substrate. ManB-1601 exhibited optimum activity at pH 6e7 and retained 80% of its activity at pH 5 but registered a sharp fall at pH 4 (<20% activity) (P  0.01). ManB1601 was more active in alkaline pH range by retaining at least 40% activity till pH 9 (Fig. 1A) (P  0.0007). Very low activity of ManB1601 in acidic pH range (
Where K is the Huggins constant. Average molecular weight of GG and PHGG was estimated using Mark-Houwink's equation ([h] ¼ kMa), where a and k were taken as 0.732 and 3.8  104, respectively (Robinson, Ross-Murphy, & Morris, 1982) and M denotes average molecular weight. Average degree of polymerization of GG and PHGG was calculated by the following equation:

Average degree of polymerization ¼

Average molecular weight of polymer Molecular weight of monomer

Molecular weight of monomer was taken as 270 (Mahammad et al., 2006). 2.3.4. Fourier transform-infrared spectroscopy IR-spectral studies for GG and PHGG samples were performed on FTIR spectrometer (IFS-25, Bruker, Germany) under dry air at room temperature using KBr pellets. Sample pellets were prepared by blending ~2e4 mg of sample with ~100 mg of solid KBr followed by pressing in the sampling unit. Spectra were scanned between 4000 and 500 cm-1and the reproducibility of the data was verified with three independent preparations (Mudgil et al., 2012b). Auto base line correction was done on the GG and PHGG FTIR spectra using OMNIC software version 7.2a. 2.4. Statistical analysis All the experimental results are reported as the arithmetic mean ± standard deviation (SD). For biochemical characterization experiments and intrinsic viscosity studies, analysis of variance (ANOVA) of data was performed with Origin Pro 7 (Origin Lab Corporation, MA, USA) statistical software. P values 0.05 were considered as statistically significant. Duncan's multiple range test (DMRT) was conducted on the rheological parameters of the samples to ascertain the statistical difference for the group of data at a statistical significance of P  0.01. The statistical significance of the correlation coefficient (r) was examined at a probability (P) of 0.01 (Little & Hills, 1978).

3. Results and discussion 3.1. Effect of pH and temperature on ManB-1601

3.2. Effect of metal ions on ManB-1601 As compared to ManB-1601 without added metal ion, Ni2þ, Ca2þ and Mn2þ moderately inhibited ManB-1601 activity to 96.4%, 95.9% and 77.11%, respectively while Hg2þ drastically inhibited the activity to 12% (P  0.02). Mg2þ, Kþ and Cu2þ did not affect the activity, while, Ba2þ, Fe3þ, Li2þand Co2þ marginally stimulated ManB1601 by 6%, 8% and 16%, respectively (Fig. 1C) (P  0.03). Co2þ has been shown earlier to enhance the activity of other mannanases from Bacillus nealsonii PN-11 (Chauhan et al., 2014), Bacillus subtilis WY34 (Jiang et al., 2006) and Pantoea agglomerans (Wang et al., 2010). The significant inhibition exerted by metal ions such as Cu2þ and Fe2þ on the activities of ManA from Bacillus sp. N16-5 (Ma et al., 2004) and ManK from Cellulosimicrobium sp. strain HY-13 (Kim et al., 2011) was not found for ManB-1601. In fact, similar to ManB-1601, activity of cold-active mannanase from Cryptopygus antarcticus, was enhanced by 1.5-fold in the presence of Cu2þ (Song et al., 2008). We understand from our previous studies on endomannanase thermo-stability (Srivastava et al., 2014b) that -SH group is not participating in enzyme catalysis and this finding has been re-confirmed in the present work as heavy metal ions like Li2þ, Co2þ, Cu2þ, Ni2þ which can interact with -SH group were not able to significantly affect ManB-1601 activity. 3.3. Effect of surfactants and organic solvents on ManB-1601

2.5. Analytical procedures ManB-1601 activity was determined by estimating the release of reducing sugars by the dinitrosalicylic acid method (Miller, 1959) at 55
C. Locust bean gum (0.5% w/v) was used as substrate in 50 mM sodium phosphate buffer (pH 7). One unit of endo-mannanase activity was defined as the amount of enzyme that liberates 1.0 mmol mannose per minute under the standard assay conditions. 2.6. Chemicals Locust bean gum (LBG), 3, 5-dinitrosalicylic acid, Tweens (20, 40, 60, and 80, pluronic F-68 and SDS) were purchased from

The activity of ManB-1601 in the presence of various non-ionic detergents (0.2% w/v/v/v) like pluronic F-68, Tween (20, 40, 60, 80) and PEG-3350 (Fig. 1D) was marginally enhanced by up to 7% (P  0.03) which might be due to amelioration in enzyme-substrate affinity (Singh, Van Hamme, & Ward, 2007) or decrease in the medium surface tension leading to high contact between enzyme and substrate (Kapoor, Khalil, Bhushan, Dadhich & Hoondal, 2000). The addition of anionic detergent sodium dodecyl sulphate (SDS) (0.2% w/v) and cationic detergent cetyltrimethylammonium bromide (CTAB) (0.2% w/v) led to decrease in enzyme activity by 16 and 78% (P  0.0005), respectively. Similar to our results, mannanase isolated from Bacillus sp. N16-5 and B. subtilis WY34 showed 88 and

A

100 80 60 40 20

90 80 70 60

7 pH

8

9

10

60 40 20

25% v/v

50 % v/v

l Contro

Metalion ions mM) Metal (1 (1 mM)

SDS

0 PEG-3 350

3+

a 2+ Li 2 + H C g 2+ on tr ol

80

C

2+

2+

n M g C o Fe

M

+

2+

75

100

C-TAB

20

2+

70

D

0

40

K

65

120

Pluron ic-F-68

60

2+

60

140

0

80

2+

55

Tween-8

100

u Ba N i

50

0

120

120

45

Temperature (0C)

C

C

40

11

Tween-6

6

Tween-4

5

Tween-2 0

4

% Relative ManB-1601 activity

3

0

Surfactant (0.2% v/v/ w/v)

E

1.60E-03

100

F

1.40E-03

80

1.20E-03

1/V (IU/ml)

% Residual ManB-1601 activity

B

100

50

0

140

% Relative ManB-1601 activity

% Relative ManB-1601 activity

P.K. Srivastava, M. Kapoor / LWT - Food Science and Technology 64 (2015) 809e816

% Relative/residual ManB-1601 activity

812

60 40 20

1.00E-03 y = 0.0013x + 0.0002 R² = 0.9732

8.00E-04

6.00E-04 4.00E-04

Solvent

l

2.00E-04

Contro

itrile Aceton

Ethyl a cetate

e Aceton

Ethano l

l Methan o

DMSO

0

0.00E+00

-0.5

0

0.5

1/S (mg/ml)

1

1.5

Fig. 1. Biochemical characteristics of ManB-1601. (A) pH (stability , optimum C) (B) Temperature optimum, (C) Effect of metal ions, (D) Effect of surfactants, (E) Effect of organic solvents and (F) Lineweaver-Burk double reciprocal plot using locust bean gum. 100% ManB-1601 activity was equivalent to 2900 IU/ml.

55% activity, respectively in presence of SDS (Jiang et al., 2006; Lin et al., 2007) while b-mannanase from B. nealsonii PN-11was inhibited by CTAB (Chauhan et al., 2014). The effect of various organic solvents (DMSO, methanol, ethyl acetate, ethanol, acetone and acetonitrile) on the stability of ManB1601 was examined. ManB-1601 retained almost 100% (P  0.00001) activity after pre-incubation for 3 h in the presence of all the tested solvents (25% v/v) except ethyl acetate where it lost 70% of its activity. However, high (50% v/v) level of acetonitrile, acetone and ethyl acetate reduced ManB-1601 activity by up to 84% (P  0.00001). No loss of ManB-1601 activity was observed with 50% (v/v) DMSO and methanol (P  0.00001) (Fig. 1E). The technological utility of hemicellulases like xylanases and mannanases for waste and kraft pulp bleaching would be higher if they are

stable and active in the presence of organic solvents (Kapoor, Kapoor, & Kuhad, 2007). Mannanase from B. nealsonii PN-11 retained more than 80% activity after its incubation with different solvents (Chauhan et al., 2014) while mannanase from Bacillus licheniformis THCM 3.1 showed 15e25% reduction in activity after incubation with acetone, toluene, benzene, dimethyl sulphoxide, 2propanol, acetonitrile or cyclohexane (Kanjanvas et al., 2009). 3.4. Kinetic parameters and protease resistance of ManB-1601 The Km, Vmax and Vmax/Km of ManB-1601 were found to be 6.5 mg/ml, 5000 IU/ml/min, and 769.2 mmol/min/mg, respectively (Fig. 1F). The Vmax of ManB-1601 was found to be much higher than earlier reported mannanases from Paenibacillus cookii (Yin, Tai, &

P.K. Srivastava, M. Kapoor / LWT - Food Science and Technology 64 (2015) 809e816

3.5.1. Flow behavior index (FBI) and consistency index GG solutions are known to exhibit shear thinning nature (Morris, Cutler, Ross-Murphy, Rees, & Price, 1981). GG solutions (FBI 0.332 ± 0.002) after treatment with ManB-1601 displayed remarkable increase in FBI (1.478 ± 0.068) (significant at P  0.01) which could be due to the ManB-1601 led depolymerization of guar galactomannan (Table 1). FBI values, well below 1 for GG dispersions corroborated its shear thinning characteristic while a value of more than 1 for hydrolyzed sample (PHGG) confirmed the shear thickening behavior. Thus, ManB-1601 hydrolysis of GG caused a change from shear thinning to shear thickening behaviour. Mudgil et al., 2012a reported FBI of guar gum and cellulase hydrolysed partially hydrolysed guar gum as 0.3094 and 1.701, respectively. Other researchers have used gamma radiation to depolymerize guar gum and reported increase in FBI values of depolymerized gum with an increase in radiation dose (Dogan, Kayacier, & Ic, 2007; Gupta et al., 2009). The consistency index reflects the overall status of consistency of the system. GG and PHGG dispersions were evaluated for their consistency index in order to understand the impact of ManB-1601 led depolymerization. A high value of the consistency index was obtained for GG samples (5734.7 ± 895.7 mPasn) whereas a very low value (0.2 ± 0.0 mPasn) was found for PHGG samples (Table 1). Similarly, (Mudgil et al., 2012a) showed significant differences in the consistency index of guar gum (4.04) and cellulase treated partially hydrolysed guar gum (0.071). PHGG produced using gamma radiation was also found to be low in consistency (Dogan et al., 2007; Gupta et al., 2009).

10.00

GG

PHGG

Yield stress (mPa) Apparent viscosity (mPa) Flow behaviour index Consistency index (mPasn) Correlation coefficient Intrinsic viscosityx (dL/g) Average molecular weightx Average degree of polymerizationx

8.21 ± 2.28a 346.3 ± 4.8b 0.332 ± 0.002a 5734.7 ± 895.7b 0.998e0.999* 3.306 ± 0.3c 239883 ± 200e 890 ± 10g

11.05 ± 3.34a 2.8 ± 0.1a 1.478 ± 0.068b 0.2 ± 0.0a 0.988e0.997* 0.159 ± 0.01d 3814 ± 100f 14 ± 1.5h

Values in the same row with different superscripts (aeb) are statistically different at P  0.01 according to Duncan's multiple range test (DMRT) (for rheological parameters). * Significant at P  0.01. x One way ANOVA analysis: intrinsic viscosity, average molecular weight and average degree of polymerization. Values in the same row with different superscripts (ceh) are statistically different at P  0.00005.

1

10

100

1000

Shear rate (S-1) Fig. 2. Sample rheogram for GG (1% w/v) (Ο) and PHGG (:) (1% w/v).

formation of new entanglements, molecules are aligning in the direction of flow, and there is a reduction in viscosity. The HerschelBulkley model appears to suit well in case of GG dispersion as reflected by very high correlation coefficient of more than 0.998. On the other hand, the PHGG sample followed power law model in an efficient manner (correlation coefficient 0.988) (significant at P  0.01). Further, the apparent viscosity, reported at a shear rate of 100 s1, was markedly and significantly higher for GG samples (346 ± 4.8 mPas) when compared with PHGG sample, which was found to be a very thin liquid, with apparent viscosity of only 2.8 ± 0.1 mPas (Table 1). The rheological parameters showed that the yield stress for both the systems has low magnitude and were

8

(A)

6

4 2

y = 49.05x + 3.30 R² = 0.96

0

-0.1

-0.05

0 0.05 -2 Concentration (mg/ml)

0.1

0.8

(B)

0.6

ŋsp\C (mg/ml)

Parameters

0.10

0.01

3.5.2. Rheological analysis The flow characteristics of GG and PHGG dispersion were studied, and results are presented in Fig. 2. The sample flow curve for GG dispersion showed a typical shear thinning while; PHGG sample displayed a shear thickening behavior (Fig. 2). The flow curve obtained for PHGG was in reasonable agreement with the earlier reports (Hussain et al., 2015). Moreover, by increasing the shear rate it is possible that the disruption is dominating over the

Table 1 Rheological characteristics of GG and PHGG dispersions.

1.00

ŋsp\C (mg/ml)

3.5. Application of ManB-1601 in generation of PHGG

100.00

Shear stress (Pa)

Jiang, 2012), B. nealsonii PN-11 (Chauhan et al., 2014) and Talaromyces leycettanus JCM12802 (Wang et al., 2015). ManB-1601 was found to display tolerance towards trypsin and chymotrypsin by retaining 92.5 and 95% (P  0.05) activity, respectively, after 30 min of treatment. Proteases resistance is one of the most desirable attribute for industrial enzymes required in the feed and textile industries (Shi et al., 2011).

813

-1

0.4 y = 0.43x + 0.15 R² = 0.99

0.2 0 -0.5

-0.2

0

0.5

1

1.5

-0.4 Concentration (mg/ml) Fig. 3. Intrinsic viscosity plot for (A) GG (0.2e1 mg/ml) and (B) PHGG (1e5 mg/ml).

814

P.K. Srivastava, M. Kapoor / LWT - Food Science and Technology 64 (2015) 809e816

4000

3000

1661.2 1627.3

2500 2000 Wavenumbers (cm-1)

1500

1000

677.9 608.0 527.0 465.3

500

3500

3000

2500

2000

1500

523.8

1082.8 1028.0

1160.0

940.0 870.5 816.5

1661.5 1637.7 1427.3 1371.3

2900.0

2940.0

2348.0

2444.0

PHGG

3381.9

100 95 90 85 80 75 70 65 60 55 50 45 40 35 30

3500

3736.8

%Transmittance

2919.7 2896.0

3332.1

-0

1428.2 1384.0 1320.0 1264.0 1228.0 1160.0 1077.2 1028.0

2172.0

40 30 20 10

3736.6

%Transmittance

80 70 60 50

904.0 871.4 814.2 772.0

GG

90

1000

500

Wavenumbers (cm-1) Fig. 4. IR spectra of GG and PHGG.

not statistically different at P  0.01 (Table 1). This could be due to poor ability of ManB-1601 to attack the small insoluble fractions present in the GG. 3.5.3. Intrinsic viscosity and average degree of polymerization The hydrolysis of GG with ManB-1601 resulted in extensive decrease in intrinsic viscosity from 3.306 to 0.159 dL/g (Table 1, Fig. 3A and B) (P  0.00005). The average degree of polymerization, which is the ratio of molecular weight of polymer and molecular weight of monomer, also witnessed drastic decrease (890e14) after ManB-1601 attack on GG (Table 1). ManB-1601 by hydrolyzing the susceptible glycosidic linkages (Daas, Schols, & de Jongh, 2000; Srivastava & Kapoor, 2014) between adjacent mannose residues in soluble fractions of GG lead to reduction in the chain length of GG which eventually led to lower values of intrinsic viscosity and average degree of polymerization in the resulting PHGG. 3.5.4. Fourier transform-infrared spectroscopy (FTIR) The FTIR spectra of GG and PHGG (Fig. 4) were carried out in order to analyse their chemical structure. The characteristic IR wave numbers observed are summarized in Table 2. The region between 3000 and 2800 cm1 in FTIR spectra shows the CeH stretching

modes (Kacurakova, Belton, Wilson, Hirsch, & Eblingerova, 1998). The 3300 cm1 peak recorded in spectra can be assigned to OeH stretching vibration of polysaccharide apart from involvement of water molecules in H-bonding (Fringant, Tvaroska, Mazeau, Rinaudo, & Desbrieres, 1995). The band around 2930 cm1 and 3330 cm1 exhibited significant change in shape and intensity suggesting differences in arrangement in between native and depolymerized gums, including decrease in the molecular size and solubility (Shobha et al., 2005). Furthermore, sharpening of the absorption near 1640 cm1 in PHGG indicated its proximate association with water and hence provides evidence for its improved solubility as compared to GG. The bands around 1400 cm1 region were due to CH2 deformation (Kacurakova et al., 1998). Bands at 1150 cm-1 and 1077 cm1 could be assigned to the stretching vibrations of CeOeC bonds of glycosidic bridges and complex vibrations involving the stretching of the C6eOeC1 bonds, linking the galactose residue to the main chain. Further, 871 and 813 cm1 were signature bands of guar gum (Daniela, Mariella, & Vittorio, 2005; Jana, Andriy, Marcela, Jitka, & Miroslava, 2001). The spectral region between 800 and 1200 cm1 showed peaks representing highly coupled CeCeO, CeOH and CeOeC stretching modes of the polymer backbone (Kacurakova et al., 1998). It has been

Table 2 Characteristic IR wave numbers of GG and PHGG. Characteristic group

GG

PHGG

OeH stretching vibrations CeH stretching modes of CH2 groups CH2 deformation CeOeC stretching, C6eOeC1 stretching eCH2 twisting vibration Signature bands of guar gum (galactose and mannose) CeCeO, CeOH and CeOeC stretching

3332 cm1 2896, 2919 cm1 1428.2 cm1 1077, 1160 cm1 1028 cm1 814.2, 871.4 cm1 800e1200 cm1

3381 cm1 2900, 2940 cm1 1427.3 cm1 1160, 1182 cm1 1028 cm1 816.5, 870.5 cm1 800e1200 cm1

P.K. Srivastava, M. Kapoor / LWT - Food Science and Technology 64 (2015) 809e816

reported earlier that crystallinity of the product is determined by the region between 700 and 500 cm1 (Tul'chinsky, Zurabyan, Asankozhoev, Kogan, & Ya, 1976). In the present study, change in PHGG spectra in this region indicated that there were conformational changes and more crystallinity which could be due to the small size of PHGG. Overall, comparison of FTIR spectra of PHGG with GG indicated no major functional group transformation. 4. Conclusions The biochemical properties of ManB-1601 like stability in presence of detergents, organic solvents and alkaline pH along with tolerance to proteases make it a good candidate for varied industrial applications. ManB-1601 was able to significantly alter the physicochemical and rheological characteristics of GG in order to produce PHGG without affecting the basic chemical structure. The PHGG produced may have potential in food and related sector. Acknowledgements The authors gratefully acknowledge The Director, CSIR-CFTRI, Mysuru for his support and encouragement. Authors sincerely thank Dr. Suvendu Bhattacharya, Food Engineering Department for helping in rheological experiments. Authors also thank Mr. Deepesh Panwar, JRF, for his help in statistical analysis of data. PKS thanks University Grants Commission (UGC), New Delhi, India for the grant of Junior (JRF) and Senior Research Fellowship (SRF). References Chauhan, P. S., Puri, N., Sharma, P., & Gupta, N. (2012). Mannanases: microbial sources, production, properties and potential biotechnological applications. Applied Microbiology and Biotechnology, 93, 1817e1830. Chauhan, P. S., Sharma, P., Puri, N., & Gupta, N. (2014). Purification and characterization of an alkali-thermostable b-mannanase from Bacillus nealsonii PN11 and its application in mannooligosaccharides preparation having prebiotic potential. European Food Research Technology, 238, 927e936. Daas, P. J., Schols, H. A., & de Jongh, H. H. (2000). On the galactosyl distribution of commercial galactomannans. Carbohydrate Research, 329(3), 609e619. Daniela, R., Mariella, D., & Vittorio, C. (2005). Guar gum methyl ethers. Part I. Synthesis and macromolecular characterization. Polymer, 46, 12247e12255. Dhanalakshmi, K., & Bhattacharya, S. (2014). Agglomeration of turmeric powder and its effect on physico-chemical and microstructural characteristics. Journal of Food Engineering, 120, 124e134. Dhawan, S., & Kaur, J. (2007). Microbial mannanases: an overview of production and applications. Critical Reviews in Biotechnology, 27, 197e216. Dogan, M., Kayacier, A., & Ic, E. (2007). Rheological characteristics of some food hydrocolloids processed with gamma irradiation. Food Hydrocolloids, 21(3), 392e396. Fringant, C., Tvaroska, I., Mazeau, K., Rinaudo, M., & Desbrieres, J. (1995). Hydration of a-maltose and amylose: molecular modeling and thermodynamics study. Carbohydrate Research, 278(1), 27e41. Gupta, S., Saurabh, C. K., Variyar, P. S., & Sharma, A. (2015). Comparative analysis of dietary fiber activities of enzymatic and gamma depolymerized guar gum. Food Hydrocolloids, 48, 149e154. Gupta, S., Shah, B., Sanyal, B., Variyar, P. S., & Sharma, A. (2009). Role of initial apparent viscosity and moisture content on post irradiation rheological properties of guar gum. Food Hydrocolloids, 23(7), 1785e1791. Hongbo, T., Min, S., Yanping, L., & Siqing, D. (2013). Preparation and properties of partially hydrolyzed cross-linked guar gum. Polymer Bulletin, 70(12), 3331e3346. Hussain, M., Bakalis, S., Gouseti, O., Zahoor, T., Anjum, F. M., & Shahid, M. (2015). Dynamic and shear stress rheological properties of guar galactomannans and its hydrolyzed derivatives. International Journal of Biological Macromolecules, 72, 687e691. Jana, C., Andriy, S., Marcela, C., Jitka, K., & Miroslava, N. (2001). Application of FTIR spectroscopy in detection of food hydrocolloids in confectionery jellies and food supplements. Czech Journal of Food Sciences, 19, 51e56. Jiang, Z., Wei, Y., Li, D., Li, L., Chai, P., & Kusakabe, I. (2006). High-level production, purification and characterization of a thermostable b-mannanase from the newly isolated Bacillus subtilis WY34. Carbohydrate Polymers, 66, 88e96. Kacurakova, M., Belton, P. S., Wilson, R., Hirsch, J., & Eblingerova, A. (1998). Hydration properties of xylan-type structure on FTIR study of xylooligosaccharides. Journal of Science of Food and Agriculture, 77, 38e45. Kanjanvas, P., Khawsak, P., Pakpitcharoen, A., Areekit, S., Sriyaphai, T.,

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