The biosensors, integrated into a low-cost telemetry system, represents a new ... Biosensor designs (glucose, lactate and ethanol) were obtained by exposing a ...
Donatella Farina1, Sergio Scognamillo1, Manuel Zinellu2, Mauro Fanari1, Maria Cristina Porcu3, Giulia Puggioni1, Gaia Rocchitta4, Pier Andrea Serra4, Luca Pretti1 1 Porto
Conte Ricerche Srl, Località Tramariglio, 07041 Alghero (SS), Italia 2 Primo Principio COOP, Tramariglio-Alghero (SS) 07041, Italia 3 Istituto di Chimica Biomolecolare (ICB), CNR, Traversa La Crucca, 3 Regione Baldinca, 07100 Li Punti, Sassari, Italia 4Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Università di Sassari, V.le San Pietro 43B, 07100 Sassari, Italia
DEVELOPMENT OF A BIOSENSOR TELEMETRY SYSTEM APPLIED TO MICROBREWERIES IN SARDINIA INTRODUCTION Beer is currently the most consumed alcoholic beverage in the world. In the last years the craft beer production has had a rapid growth and gained great success also in the countries that are not traditional beer producers [1]; more than 900 craft breweries are known just in Italy. A plethora of studies suggest that moderate drinking of beer is associated with lower rates of cardiovascular disease, neurodegeneration, cancer and osteoporosis [2]. The positive association between moderate intake of beer and low risk for these diseases is due to several substances with antioxidant properties present in the barley and in the hop [3,4]. However, pasteurization and filtration in industrial productions degrade and separate important components. Beneficial properties of beer remain intact in handicraft production but the final product is more susceptible to alteration. So, it is important to monitor various critical parameters during the brewing process. Spectrophotometric and chromatographic analytical techniques are widely used for monitoring the changes that take place under the manufacturing process conditions but they are time-consuming and laborious and, in some of them, the sample pre-treatment is required [5]. Methods based on biosensors could be a good alternative to these classical methods. They offer fast analysis, easy and direct analytes detection and low cost production [6]. Fig. 1
AIM The goal of this study is to monitor glucose, ethanol and lactate levels in the real fermenting wort by means of amperometric biosensors, integrated to a wireless telemetric system [7] in order to evaluate the start of fermentation, the trend of ethanol production and to identify a possible contamination by lactic acid bacteria, independently of external laboratories (Fig. 1).
MATERIALS AND METHODS Biosensor Fabrication and Characterization Biosensor designs (glucose, lactate and ethanol) were obtained by exposing a cylindrical platinum/iridium (Pt/Ir) electrode (1 mm length and 125 μm diameter) by cutting away the Teflon insulation. Then, the electrode was covered by the electro-deposition of a polyortho-phenylenediamine (PPD) nanometer-thick membrane. Glucose and lactate biosensors were prepared by five immersion of PPD-covered Pt/Ir, by means of a quick dip, into polyethylenimine (PEI) and oxidase enzyme solutions [glucose oxidase from Aspergillus niger (GOx),and lactate oxidase from Pediococcus species (LOx), respectively]. The ethanol biosensor was made in the same way by changing the oxidase enzyme [alcohol oxidase from Pichia Pastoris (AOx)], and by dipping biosensors in a PEI and glycerol (Glyc) solution. Finally, the biosensors were quickly dipped in a Polyurethane solution (PU). The electrochemical studies were performed in a cell consisting of three biosensors as working electrodes (WE), one dummy (made as WE but without enzyme), an Ag/AgCl (NaCl, 3M) electrode as reference electrode (RE), and a large surface area Pt wire as auxiliary electrode (AE). Measurements were carried out in phosphate buffer solution at pH 7.4 at room temperature. The detection of hydrogen peroxide (H2O2) produced in enzymatic reactions was performed using the constant potential amperometry (CPA) by setting the oxidation potential for the H2O2 to + 700 mV against an Ag/AgCl (Fig.1). Different designs were characterized in vitro in terms of Michaelis−Menten kinetics (VMAX and KM), sensitivity [linear region slope, limit of detection (LOD), and limit of quantification (LOQ)], and electroactive interference blocking. The same parameters were monitored in selected designs up to 28 days after fabrication in order to quantify their stability over time. Finally, the best performing biosensor design (Tab. 1) was selected and coupled with a previously developed telemetric device for the monitoring of beer fermentation. When not in use biosensors were stored at -20 C.
Wort Samples and Microfermentations Wort [India Pale Ale (IPA) beer style] was supplied from the Sardinia Microbrewery P3 Brewing Company (Sassari). Experiment was organized in order to monitor TOP and BOTTOM fermentation using different yeasts strain (Fermentis): Safale US-05 at 22 C (top fermentation) and Saflager W34-70 at 10 C (bottom fermentation). Yeast was propagated in wort for 24 hours at 28 C, washed twice in sterile water, counted by using a Thoma chamber and inoculated in 800 ml of wort at pitching size of 10*106 cell/ml and 20*106 cell/ml respectively for top and bottom fermentation (Tables 2 and 3). Each experiment was conducted in duplicate. Monitoring was started from inoculum of yeast, for the first day every 2 hours until the beginning of the reduction of the glucose, and later every 24 hours until the end of fermentation. Cells grow was measured by a spectrophotometer at 600 nm (OD600). Plato degrees was made by equation (3b) proposed by Anthony J. Cutaia et al. [8]. The control of all parameters (glucose, ethanol, lactate, pH and temperature) was extended to all phases of the production process.
Validation Measurements In order to verify the reliability of the amperometric biosensors developed, spectrophotometric kit (510nm) for D-glucose (K-GLUC, GOPOD format, Megazyme International, Bray, Ireland) and modular measuring system (Anton-Paar for beer analysis Model PBA-B Generation M) for ethanol and Plato degrees were used to perform analyses in parallel with biosensor measurements.
RESULTS and DISCUSSION Results of the in vitro characterization of biosensors (Table 1) show a high sensitivity and a good linearity that remain relatively stable for the duration of the experiments. The high sensitivity and stability of biosensors allowed the use of small quantities of untreated sample (10 µl wort or beer in 10mL of PBS), avoiding fouling of the catalytic surfaces. The electrochemical assay of real samples was performed by means of the standard addition method to avoid any matrix effect. Sample dilution was required in order to fit the expected concentration value with the biosensor linear range. The biosensors have a good response time and an excellent agreement with our validation methods.
Table 1. In Vitro Characterization of three different biosensor designs at Day 1 (n = 6 for each group)
Analytical parameters measured in wort after yeast inoculation and at the end of fermentation are summarized in Table 2 and 3 (TOP and BOTTOM FERMENTATION). Lactic acid measured in the sample before and at the end of fermentation (0.040 0.008g/L), derived from a technological process (mash acidification). This concentration was below the organoleptic threshold values as reported in literature [9].
TOP FERMENTATION IPA
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BOTTOM FERMENTATION IPA
Figures 2 and 3 (panel A) show the results of the biosensoristic glucose, ethanol and degree Plato monitoring in top and bottom fermentation, respectively; these biosensoristic devices provide a tight control of the fermentation process and are able to locate the beginning and the end of both fermentations. Consistently with the temperature and yeast adopted in the two types of experiment, the course of the top fermentation is significantly faster than the bottom fermentation. Specifically, in the top fermentation the levels of glucose were reduced by 98.5% in the first 24 hours while the same percentage reduction was recorded at 96 hours in the bottom fermentation. The end of the fermentation was recorded at 168 hours and 240 hours for the top and the bottom respectively. In panels B of figures 2 (top fermentation) and 3 (bottom fermentation) the yeast growth curves (OD600) showing a correlation between cell growth and fermentation progress are reported . Biosensor results were compared with those obtained with the commercial kit (for glucose) and the Anton Paar (AP) (for ethanol) in order to validate them. The results obtained by our glucose and ethanol biosensor showed a good agreement with those obtained with the spectrophotometric method (Figure 2, panel C) and the AP (panel C of Figure 3) thus validating the applicability of the developed biosensors for glucose and ethanol determination in beer samples.
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Fig. 2
Fig. 3
CONCLUSIONS According to the results of this study, we can affirm that the developed set biosensors is a reliable device for short-time monitoring of the basic parameters in fermentation and could act as an indicator of bacterial contamination. The biosensors, integrated into a low-cost telemetry system, represents a new generation of analytical tools available for brewers.
REFERENCES [1] V. Sanna and L. Pretti. International Journal of Food Science & Technology (2014); 50 (3), 700-707;[2] V.R. Preedy. Beer in Health and Disease Prevention (2009); 1248 pages, ISBN: 978-0-12-3738912; [3] M.N. Maillard and C Berset. Journal of Agricultural and Food Chemistry (1995); 43 (7), 1789–1793; [4] J. De Keukeleire. Journal of Agricultural and Food Chemistry (2003); 51 (15), 4436-4441; [5] S. Pérez and E. Fàbregas. Analyst (2012); 137, 3584-3861; [6] B. Kuswandi , T. Irmawati , M.A. Hidayat , J. and M. Ahmad. Sensors (2014); 14, 2135-2149; [7] A. Barberis, Y. Spissu, G. Bazzu, Serra PA. Analytical Chemistry (2014); 86(17):8727-34; [8] A. J. Cutaia, A.-J. Reid, and R. A. Speers. Journal of the Institute of Brewing (2009), 115(4), 318-327. [9] S. Engan. J. Inst. Brew. (1973); 80, 162-163.
GS 2015 - Parma, 15-17 giugno 2015
