Application of statistical designs strategy to improve cellulase production using agro-waste residue by a novel isolate Bacillus licheniformis strain-MA1 and assessing the enzyme effect on apple juice quality.

Abstract

Background: Cellulose is the major part of lignocellulosic biomass. It can be hydrolyzed into glucose units via specific enzymes called cellulases that have been applied in many commercial fields. There are several studies illustrate the influence of enzymes on apple juice clarification. However, to the best of our knowledge, the effect of microbial cellulase on volatile compounds of apple juice is not well known. The present study aimed to assess the effect of cellulase from a new bacterial isolate on the physicochemical properties of apple juice as well as volatile compounds. The hydrolysis of some polysaccharides (cellulose, hemicellulose, pectin) and polyphenols during apple juice production is necessary to reduce cloud sedimentation or color deterioration and increase the yield of juice. So, enzymes from new microbial isolates serve as processing aids to obtain clear juice with a high yield.

Results: Cellulase-producing bacterium was isolated, characterized and molecularly identified as Bacillus licheniformis strain-MA1 with an accession number of ON840115. Optimization of medium parameters was implemented using Plackett-Burman design (PBd) followed by Box-Behnken design (BBd) of response surface methodology (RSM). The PBd revealed the three most important (significant) variables including carboxymethyl cellulose (CMC), corn cob, and peptone that had positive impact on cellulase production. Additionally, using the agricultural residue (corn cob) by the bacterial strain as a carbon source helps in reducing the costs of enzyme production, recycling the by-products, and preserving the environment. The optimized medium using PBd and BBd enhanced cellulase production from B. licheniformis strain-MA1 by 6.8-fold. A remarkable increase was observed in juice yield in enzyme treated-juice sample (88.2 ± 0.15%) in comparison with control juice (75.4 ± 0.09%). The total phenolic contents in cloudy and clarified apple juices were 0.957 ± 0.09 and 0.412 ± 0.03 mg/mL, respectively. Also, DPPH and FRAP assays showed a remarkable increase in antioxidant activity (Low IC₅₀) in the control sample compared to enzyme treatment. Twenty-seven volatile compounds were extracted using headspace solid-phase microextraction-gas and analysis was performed by GC-MS. The identified volatile constituents belonged to several chemical classes: 15 esters; 6 alcohols; 4 aldehydes and 2 acids. The predominant class in apple juice volatile fraction was esters with a sweet and fruity odor.

Conclusion: The crude cellulase obtained from the novel bacterial isolate B. licheniformis strain-MA1 was successfully applied as a clarifying agent in apple juice.