^{1}

^{1}

^{1}

^{2}

^{3}

Paper was presented at the 7th International Forum on Industrial Bioprocessing - IFIBiop 2017, May 21-24, 2017, Wuxi, PR China

Inulinases are an important class of industrial enzymes which are used for the production of high-fructose syrup and fructooligosaccharides. Inulin, a polyfructan, is generally employed for the production of inulinase, which is a very expensive substrate. A number of agroindustrial residues have been used for cost-effective production of inulinases. In the present study, carrot pomace was selected as a substrate for the production of inulinase by _{4}H_{2}PO_{4}, 0.2% NaNO_{3}, 0.2% KH_{2}PO_{4}, 0.05% MgSO_{4}·7H_{2}O and 0.001% FeSO_{4}·7H_{2}O was used for the production of inulinase in solid-state fermentation at 30 °C. Inulinase production (322.10 IU per g of dry substrate) was obtained under the optimized conditions,

Inulinases are important industrial hydrolysing enzymes which belong to glycoside hydrolase (GH) family 32. They act on β-2,1 linkages of inulin to produce high-fructose syrup (HFS) or fructooligosaccharides (FOSs). On the basis of their degrading action on inulin, inulinases are classified as exoinulinases or endoinulinases. Exoinulinase (EC 3.2.1.80, β-2-1-

Fermentation technique, type of microorganism and medium constituents are some key factors which play a vital role in enzyme production. Conventionally, inulinases are produced by submerged fermentation (

Inoculum was prepared by growing a stock culture of ^{4} spore/mL with presterilized distilled water, under aseptic conditions.

Fresh carrots were procured from the local market, Patiala, India. After thorough washing, they were crushed properly in a juice extraction machine (Philips India Ltd., Gurgaon, India) to obtain juice. Carrot residue obtained after the extraction was pressed and dried in an oven (Narang Scientific Works Pvt. Ltd, New Delhi, India) at 50 °C, until it dehydrated completely. The dried carrot pomace was ground using mortar and pestle to uniform powder and passed through a 150-µm sieve.

Solid-state fermentations (SSF) were carried out in Erlenmeyer flasks (250 mL) containing 10 g of carrot pomace moistened with distilled water containing (in %, mass per volume): inulin 0.5, NaNO_{3} 0.2, KH_{2}PO_{4} 0.2, KCl 0.1, MgSO_{4}·7H_{2}O 0.05, FeSO_{4}·7H_{2}O 0.001 and NH_{4}H_{2}PO_{4} 0.2 (all from HiMedia Laboratories Pvt. Ltd.). Three variables optimized for inulinase production using statistical experimental model are A: moisture content (70-90%), B: incubation time (4-6 days) and C: pH (5-7), while the other above-mentioned medium constituents were kept constant during the progression of the study. The Erlenmeyer flasks were sealed with hydrophobic cotton and autoclaved at 121 °C for 30 min. Preliminary studies showed no changes in the moisture content of the substrate after autoclaving. After cooling, the flasks were inoculated with previously prepared 2 mL of spore suspension (10^{4} spore/mL) and mixed uniformly with a sterile glass rod, under aseptic conditions. Subsequently, flasks were incubated in an environmental chamber (model CHM-10 Plus; REMI Lab World, Mumbai, India) with temperature and humidity control.

Optimization of fermentation conditions is an important factor in developing feasible bioprocess to obtain desirable product. Optimal processing conditions can be constituted using an efficacious statistical experimental model. Central composite rotatable design (CCRD) used in response surface methodology (RSM) is a collection of several statistical models and has been employed to study the influence of three different variables and their combined interactions on inulinase production by ^{3} factorial design. The five coded levels used to study three independent variables were -1.414, -1, 0, 1 and 1.414 (

Independent |
Code | Actual level of coded factors | ||||
---|---|---|---|---|---|---|

-1.414 | -1 | 0 | 1 | 1.414 | ||

A | 65.86 | 70 | 80 | 90 | 94.14 | |

B | 3.59 | 4 | 5 | 6 | 6.41 | |

pH | C | 4.59 | 5 | 6 | 7 | 7.41 |

Inulinase production and I/S ratio were the two responses on which the influence of three chosen independent variables was anticipated. A multiple regression analysis of experimental data obtained from an empirical model was accomplished using least square method, to fit into the following second-order polynomial equation:_{0}+∑β_{i}X_{i}+∑β_{ii}X_{i}^{2}+∑ β_{ij}X_{i}X_{j} /1/_{0} is the intercept term, β_{i} is linear coefficient, β_{ii} is quadratic coefficient, β_{ij} is an interaction coefficient and X_{i} and X_{j} represent coded independent variables. The statistical implication of the model was revealed through analysis of variance (ANOVA) for polynomial model with 95% confidence level. Student’s ^{2}), adjusted R^{2} and adequate precision. The value of R^{2} is important to explain the variability in the observed responses with respect to experimental factors and interactions between them. Moreover, 2D contour plots were also analysed to study interaction between each variable and its corresponding effect.

After successful fermentations, the extracellular enzyme was extracted by adding 100 mL of sodium acetate buffer (0.1 M, pH=5.0; HiMedia Laboratories Pvt. Ltd) to the fermented substrate in Erlenmeyer flasks. The content of each flask was kept under agitation (150 rpm) on a rotary incubator shaker (model CIS-24 BL; REMI Lab World) for 2 h at 30 °C for enzyme extraction. Thereafter, the extract was filtered through Whatman No. 1 filter paper and centrifuged at 3000×

Crude enzyme (100 µL) obtained after extraction was mixed with 900 µL of substrate solution (2% inulin; HiMedia Laboratories Pvt. Ltd) in 0.1 M sodium acetate buffer, pH=5.0; HiMedia Laboratories Pvt. Ltd). Reaction mixture was incubated at 55 °C for 10 min. Then, the reaction was stopped by heating the reaction mixture in boiling water bath (model NSW-133; Narang Scientific Works Pvt. Ltd) for 10 min. After that, reducing sugar content in the mixture was determined by 3,5-dinitrosalicylic acid (DNSA) method (

The reaction mixture (1.0 mL) comprising crude enzyme extract (100 µL) and substrate solution (2% sucrose; HiMedia Laboratories Pvt. Ltd) in 0.1 M sodium acetate buffer (pH=5.0; HiMedia Laboratories Pvt. Ltd) was incubated at 55 °C for 10 min. After incubation, the reaction was terminated by degrading the enzyme in the reaction mixture at 100 °C for 10 min. The resultant hydrolysate was analysed for reducing sugars by DNSA method (

Activity of the enzyme on inulin or sucrose as a substrate is crucial for its characterization, because of the participation of its single active site in both fructan and sucrose hydrolysis. Generally, the ratio of activity on inulin

Moisture content, incubation time and pH were considered as significant variables for statistical optimization of inulinase production from carrot pomace in SSF. The experimental design matrix was calculated using CCRD. Linear, 2F1, quadratic and cubic models were analysed to find the regression equation of the experimental data. The experimental design and the results obtained after statistical optimization of the two responses, ^{2}+9.59·B^{2}+2.84·C^{2} /2/^{2}–0.25·B^{2}– 0.18·C^{2} /3/

Run
Factor*
Experimental results
Predicted results
A
B
C
Inulinase production
I/S
Inulinase production
I/S
1
80.00
5.0
6.0
275.00
4.09
274.55
4.11
2
80.00
3.6
6.0
309.80
3.63
310.25
3.39
3
80.00
5.0
6.0
274.00
4.04
274.55
4.11
4
80.00
5.0
6.0
275.30
3.94
274.55
4.11
5
80.00
5.0
6.0
275.20
4.11
274.55
4.11
6
90.00
4.0
7.0
322.10
3.38
321.61
3.33
7
80.00
5.0
7.4
280.60
3.68
281.18
3.54
8
80.00
5.0
4.6
279.50
3.83
280.08
3.42
9
70.00
6.0
7.0
291.80
3.94
291.13
4.35
10
90.00
6.0
5.0
272.70
3.74
272.03
4.27
11
70.00
4.0
5.0
286.20
3.03
285.71
3.24
12
65.86
5.0
6.0
281.70
3.92
282.28
4.11
13
80.00
6.4
6.0
278.30
5.63
279.01
4.84
14
80.00
5.0
6.0
274.40
4.08
274.55
4.11
15
94.14
5.0
6.0
286.20
4.11
286.78
4.11

*Symbols A, B and C are the same as in

The statistical relevance of Eqs. 2 and 3 for response surface quadratic model was established by ANOVA. Statistical data given in ^{2}, B^{2} and C^{2} are the significant model terms for inulinase production. Amongst them, B, AB, BC, A^{2}, B^{2} and C^{2} were extremely significant (p>0.0001). For I/S ratio, A, B, C, AB, AC, BC, A^{2}, B^{2} and C^{2} were also significant, but A^{2}, B^{2} and C^{2} were the most significant terms (p>0.0001). The degree of freedom for pure error for both, inulinase production and I/S ratio, was 4, which once more manifests the authenticity of the models. Probability>F and F values of the lack-of-fit for inulinase production were 0.7075 and 0.16, while for I/S ratio they were 0.2441 and 1.86, respectively. This implies that the lack-of-fit is not significant compared to the pure error, which justifies the fitness of the quadratic model for the present study.

Source* | Inulinase production/(IU/g) | I/S | ||||
---|---|---|---|---|---|---|

Sum of squares | df | Probability>F | Sum of squares | df | Probability>F | |

Model | 2744.12 | 9 | <0.0001 | 1.51 | 9 | <0.0001 |

A | 10.13 | 1 | 0.0015 | 0.15 | 1 | 0.0002 |

B | 96.13 | 1 | <0.0001 | 8.450·10^{-3} |
1 | 0.0703 |

C | 5.000·10^{-3} |
1 | 0.8951 | 0.031 | 1 | 0.0070 |

AB | 298.40 | 1 | <0.0001 | 0.093 | 1 | 0.0006 |

AC | 3.45 | 1 | 0.0148 | 0.18 | 1 | 0.0001 |

BC | 33.77 | 1 | <0.0001 | 0.11 | 1 | 0.0004 |

A^{2} |
158.80 | 1 | <0.0001 | 0.33 | 1 | <0.0001 |

B^{2} |
709.03 | 1 | <0.0001 | 0.49 | 1 | <0.0001 |

C^{2} |
62.09 | 1 | <0.0001 | 0.24 | 1 | <0.0001 |

Residual | 1.30 | 5 | 8.031·10^{-3} |
5 | ||

Lack-of-fit | 0.051 | 1 | 0.7075 | 2.551·10^{-3} |
1 | 0.2441 |

Pure error | 1.25 | 4 | 5.480·10^{-3} |
4 | ||

Cor total | 2745.42 | 14 | 1.52 | 14 |

*Symbols A, B and C are the same as in

Source* | Inulinase production/(IU/g) | I/S | ||||
---|---|---|---|---|---|---|

Coefficient |
Standard error | F-value | Coefficient |
Standard error | F-value | |

Intercept | 274.81 | 0.22 | 1173.87 | 4.08 | 0.017 | 104.75 |

A | 1.59 | 0.25 | 38.98 | -0.19 | 0.020 | 90.77 |

B | -11.14 | 0.25 | 1910.08 | -0.046 | 0.020 | 5.26 |

C | 0.035 | 0.25 | 0.019 | -0.088 | 0.020 | 19.45 |

AB | -12.21 | 0.36 | 1148.82 | -0.22 | 0.028 | 58.03 |

AC | 1.31 | 0.36 | 13.28 | -0.30 | 0.028 | 114.66 |

BC | -4.11 | 0.36 | 130.01 | -0.24 | 0.028 | 70.78 |

A^{2} |
4.54 | 0.18 | 611.36 | -0.21 | 0.014 | 208.26 |

B^{2} |
9.59 | 0.18 | 2729.76 | -0.25 | 0.014 | 301.94 |

C^{2} |
2.84 | 0.18 | 239.05 | -0.18 | 0.014 | 148.33 |

*Symbols A, B and C are the same as in

The goodness of fit of the model is shown in ^{2} and predicted R^{2} were 0.9987 and 0.9973 for inulinase production, and 0.9852 and 0.8134 for I/S ratio, respectively. A minute difference between the adjusted R^{2} and predicted R^{2} showed reasonable agreement between the two values. Adequate precision measures the signal to noise ratio. A ratio greater than 4 is advisable, therefore, the ratio of 118.71 and 32.61 for inulinase production and I/S ratio, respectively, indicates an adequate signal for the present model and suggests that the model can be used to navigate the design space.

Source | Value | Source | Value | ||
---|---|---|---|---|---|

Inulinase production |
I/S | Inulinase production |
I/S | ||

S.D. | 0.51 | 0.04 | R^{2} |
0.99 | 0.99 |

Mean | 283.85 | 3.74 | Adjusted R^{2} |
0.99 | 0.98 |

CV/% | 0.18 | 1.07 | Predicted R^{2} |
0.99 | 0.81 |

Press | 7.36 | 0.28 | Adeq. precision | 118.71 | 32.61 |

I/S=inulinase/invertase, S.D.=standard deviation, CV=coefficient of variance

The quadratic models in Eqs. 2 and 3 have nine terms comprising three linear terms, three quadratic terms and three two-factorial interactions. Out of these terms, the insignificant terms (p>0.05) were found only in Eq. 2, which were neglected, resulting in the following equation:

^{2}+ 9.59·B

^{2}+2.84·C

^{2}/4/

The effect of three independent variables on inulinase production and I/S ratio was studied. _{2} or acidic metabolites released by aerobic microbial cells accumulate in the medium, which makes it slightly acidified (

Response surface contour plots of: a) moisture content and incubation time, b) moisture content and pH, and c) incubation time and pH for inulinase production by

Response surface contour plots of: a) moisture content and incubation time, b) moisture content and pH, and c) incubation time and pH for inulinase and invertase ratio (I/S) produced by

Many microbial sources have been reported to possess remarkable invertase activity along with inulinase activity. This mainly occurs due to the specificity of single active site of an enzyme for both inulin and sucrose as substrates. Hence, I/S ratio is used to distinguish the catalytic activity of the enzyme and to determine its inulinase or invertase activity. I/S ratio greater than 10^{-2} determines inulinase activity, while ratio lower than 10^{-4} demonstrates invertase activity (

The interactive effect of moisture content and pH on inulinase production and I/S ratio is shown in

The predicted values of inulinase production and I/S ratio obtained from the designed model at optimized conditions (moisture content 90%, incubation time 4 days and pH=7.0) were 321.61 IU per g of dry substrate and 3.33, respectively. To testify the validation of the model for predicting inulinase production and I/S ratio, experimental trial was conducted in triplicates using optimized conditions. The performed experiments yielded 322.10 IU per g of dry substrate and 3.38 for inulinase production and the corresponding I/S ratio, respectively. Comparison between these values indicates good agreement between experimental and predicted data, which verifies the validity of current model and existence of an optimal point.

Carrot pomace has been found to be a suitable substrate for the production of inulinase by

Authors are thankful to Head, Department of Biotechnology, Punjabi University, Patiala, India, for providing the necessary laboratory facilities to execute the present work.