Elsevier

Process Biochemistry

Volume 111, Part 1, December 2021, Pages 102-108
Process Biochemistry

An optimization approach to the bioconversion of flour mill waste to α-amylase enzyme by Aspergillus oryzae

https://doi.org/10.1016/j.procbio.2021.07.019Get rights and content

Highlights

Abstract

Alpha-amylase is one of the most employed enzymes in the food industry because of its capacity to degrade starch, improving the organoleptic and nutritional properties of food products. Thus, it is very important to develop novel industrial production processes for its production. A submerged fermentation process using Aspergillus oryzae was optimised to modulate the most important factors affecting alpha-amylase production. Flour mill waste, an abundant worldwide agro-industrial residue, was used as substrate due to its starch-rich composition. This residue was able to avoid catabolite repression during the fermentation, acting as a slow-release substrate. The process optimization led to a maximum yield of alpha-amylase production of 14076 ± 2346 U/L. The secretome of the fungi in the tested conditions was analysed by LC-MS, showing that two isoforms of alpha-amylase (amy-1 and amy-3) were produced. Finally, a two-step process was developed to purify alpha-amylase, consisting of fractional precipitation using (NH4)2SO4 followed by hydrophobic interaction chromatography. The purification allowed a four-time concentration of alpha-amylase, with an eleven-time purification factor and 65 % recovery and a reduction of the proteolytic activity to 14%.

Introduction

Alpha-amylase (AMY) is a glycoprotein present in most living organisms, including prokaryotes such as Bacillus species and eukaryotic organisms, from fungi to plants and mammals [1,2]. This enzyme hydrolyses the glycosidic bonds of amylose and amylopectin molecules to render a mixture of dextrins and fermentable sugars such as D-glucose [3]. Because of its capacity to degrade starch, AMY is widely used in several industrial areas to produce enzymatic detergents and bioethanol, among other products. Moreover, in the food industry, this enzyme is used to produce fermented foods and beverages such as bread and beer to improve their organoleptic properties by increasing the fermentable sugar content [4,5].

The production of AMY using filamentous fungi has been studied both in solid-state and submerged fermentations using inducing sugars alone or combined with wheat bran [6,7]. Nevertheless, flour mill waste (FMW), a starch-rich residue from the production of wheat flour that is produced in large quantities in China, India, Argentina, USA and other wheat producers, has scarcely been employed as substrate. As the carbon source may represent 80% of the cost of the culture medium, employing agro-industrial wastes as substrates can drastically reduce the production costs. In addition, it might act as a slow-release substrate [8], helping to reduce catabolite repression exerted by simple sugars [9].

Aspergillus oryzae is considered one of the best producers of AMY and the substances resulting from its culture are considered GRAS (generally recognized as safe). The AMY from A. oryzae (E.C. 3.2.1.1) is a globular, monomeric protein belonging to the glycoside hydrolase family 13 [10]. Its molecular weight is 55 kDa and its isoelectric point is around 4.50. Its optimum temperature may varies from 35 °C to 55 °C (depending on the strain) and the range of pH of stability is between 5.00 and 8.00 [11].

We have previously tested the application of combinations of soybean husk and FMW as substrate for solid-state fermentation production of α-amylase by A. oryzae [12]. Although being a promising strategy, the number of factors to be controlled increase exponentially when dealing with its optimization for industrial application in solid state, compared to submerged fermentation [13].

In the present study we use flour mill waste (FMW) as substrate for submerged fermentation of A. oryzae, a system that has less factors and is easier handling for optimization purposes. To the best of our knowledge, there are no studies on submerged fermentation using flour mill waste (FMW) as substrate. We aimed to develop a process to produce and purify AMY based on submerged fermentation of A. oryzae using FMW as a low-cost and slow-release substrate. For purification, we propose to use fractional precipitation and hydrophobic interaction chromatography, taking advantage of the high superficial hydrophobicity of AMY [14].

Section snippets

Microorganism, inoculum preparation and substrate

Aspergillus oryzae NRRL695 was used to produce AMY in submerged fermentation, the strain was purchased from the Agricultural Research Service Culture Collection. The inoculum was prepared by culturing A. oryzae in papa dextrose agar at 30 °C for 7 days. Then, the culture was harvested by adding 10 mL of 20% (w/w) glycerol solution and magnetically stirred for 30 min. Finally, 1 mL aliquots of the spore suspension were stocked at −20 °C until using. The concentration of spores in the inoculum

Screening of the factors involved in the production of AMY by A. oryzae using submerged fermentation on FMW

To develop an AMY production process from the broths of A. oryzae in FMW-based cultures, the experimental factor conditions were screened. An experimental design allowed for the analysis of independent factors and their interactions. The initial values for the factors intervals were based on fermentations using other substrates as carbon source [22,23]. In the screening phase, the aim was to maximise AMY production and minimize protease production, since proteases could degrade and destabilize

CONCLUSION

This is the first report about production of alpha-amylase by A. oryzae submerged fermentation using low-cost flour mill waste medium. Experimental design led to an increase in AMY production. A novel two-step purification protocol was developed to separate AMY after fermentation, including fractional precipitation and hydrophobic interaction chromatography. This technique was applied for the first time for the isolation of AMY from proteases. Previous dialysis step was not required.

Flour mill

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgement

Ignacio Cabezudo and Pablo Anselmi thank CONICET for their fellowships; Mauricio Braia, María-Rocío Meini and Diana Romanini are CONICET researchers. The authors would like to thank Dra. Silvia Moreno from CEQUIBIEM for performing the analysis of protein samples by LC-MS. This work was supported by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2016-4426, PICT 2016-1170 and BIO566). The founding sources were not involved in the study and interpretation of data; in the writing or

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