Available online at http://www.jabonline.in DOI: 10.7324/JABB.2019.70607 Sensitive and stable ethanol biosensor development based on Acetobacter aceti biofilm for halal detection of food and beverages Ninik Triayu Susparini 1 , Zaenal Abidin 1 , Dyah Iswantini 1,2 *, Novik Nurhidayat 3 1 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Agatis Kampus IPB Dramaga, Bogor, Indonesia 2 Biopharmaca Research Center, Bogor Agricultural University, Jalan Taman Kencana No. 3, Bogor, Indonesia 3 Division of Microbiology Research Center for Biology, The Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor Km 46 Cibinong, Bogor, Indonesia ARTICLE INFO Article history: Received on: October 17, 2018 Accepted on: April 14, 2019 Available online: November 12, 2019 Key words: Ethanol, Biosensor, A. aceti, Biofilm, PQQ-ADH, Halal ABSTRACT A highly sensitive ethanol biosensor based on Acetobacter aceti bioflm, which contains pyrroloquinoline quinone-alcohol dehydrogenase enzyme for halal detection in food and beverages, has been developed. In this study, A. aceti cells were purifed by determining the conditions of growth time, incubation temperature, and pH to suppress the yeast growth. Selection of optimum conditions for bioflm formation was also carried out to obtain a stable sensor operation. The optimum bioflm formation was 14 days. The performance of biosensor was investigated by the cyclic voltammetry technique. Linear range, sensitivity, limit of detection (LOD), and limit of quantifcation (LOQ) were found to be 5 × 10 −5 –3 × 10 −4 %, 43,076 µA % −1 , 2.32 × 10 −5 , and 7.03 × 10 −5 %, respectively. The repeatability of biosensor was tested in 1 × 10 −5 − 1% ethanol concentration and the relative standard deviation was 1.08% (for n = 7). The stability studies have shown that the cyclic voltammetry response of biosensor to ethanol leave the act of 98.99% at the 7 week. Thus, the developed biosensor is promising to be used for simple analysis of halal products and has been tested on real sample. 1. INTRODUCTION The use of ethanol as a solvent in various industrial sectors, especially food and beverages, has become a major controversy in halal product analysis for the countries with the majority of muslim consumers. Every ethanol produced by anaerobic fermentation with concentration more than 1% is considered as Haram (prohibited), while ethanol produced naturally and less than 1% of its halal status is tolerable [1,2]. The presence of high levels of ethanol compounds in foods can cause poisoning, depression, mutation in tumor suppressant genes, hence the main trigger of the risk of colon and esophageal cancer [2]. Therefore, a proper technique is needed to detect its halal. The technique commonly used to measure ethanol content in food and beverages includes infrared spectroscopy [3], gas chromatography- mass spectroscopy [4], and fuorometry [5]. Although quantitatively those methods are quite accurate for practical reasons, they are less supportive in samples preparation and tools operation. Biosensor application is one of the solutions for developing ethanol detection devices that requires easily sample preparation, to use, sensitive, fast, and inexpensive. The selection of bioreceptor components are a very crucial in making biosensors. Biosensor is a device that uses biological components as their bioreceptors [6]. Microbial cells are promising bioreceptors because there is no need to isolate enzymes, coenzyme, and cofactors already in the system, so enzymes are more stable in their natural environment within the cell [7]. Some microbes used as ethanol bioreceptors have been reported, including Pichia methanolica [8], Candida tropicalis [9], and Pichia angusta [10] as the producer of alcohol oxidase (AOX) enzymes. In addition, Saccharomyces cerevisiae [11], Saccharomyces ellipsoideus [12], and Gluconobacter oxydans [13,14] as a producer of alcohol dehydrogenase (ADH) enzymes. Acetobacter aceti is one of the microbes that has the potential to be used as an ethanol bioreceptor, because it has an ADH enzyme bound to pyrroloquinoline quinone (PQQ) which can convert ethanol to acetaldehyde [15]. In this study, used bacterial culture with K5 code obtained from the Indonesian Institute of Sciences (LIPI) collection. The culture has been isolated *Corresponding Author Dyah Iswantini, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Agatis Kampus IPB Dramaga, Bogor, Indonesia. E-mail: dyahprado@yahoo.co.id © 2019 Susparini, et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlike Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). Journal of Applied Biology & Biotechnology Vol. 7(06), pp. 40-47, Nov-Dec, 2019