Electrochemical Detection of Lactate Produced by Foodborne Presumptive Lactic Acid Bacteria

Electrochemical Detection of Lactate Produced by Foodborne Presumptive Lactic Acid Bacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Electrochemical Detection of Lactate Produced by Foodborne Presumptive Lactic Acid Bacteria Ozum Ozoglu, Aytekin Uzunoglu, Mehmet Altay Unal, Mehmet Gumustas, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1175499/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Feb, 2023 Read the published version in Journal of Bioscience and Bioengineering → Version 1 posted You are reading this latest preprint version Abstract Background: The detection of lactate is an important indicator of freshness, stability, and storage stability of products as well as degree of fermentation in food industry. In addition, it can be used as a diagnostic tool in patients’ healthcare since it is known that the lactate level in blood increases in some pathological conditions including septic shock, tissue hypoxia, and sepsis. Thus, determination of lactate level plays an important role in not only food industry but also health fields. At this point, biosensor has become important to detect lactate due to having many advantages like rapid, cheap and easy to use. Methods and Results: In the current study, amperometric lactate biosensors based on lactate oxidase immobilization (with nafion 5% wt) were designed and limit of detection, linear range and sensitivity values were determined, which were found as 31 μM, 50-350 μM and 0.04 μA.μM -1 .cm -2 , respectively. Then, it was used for the measurement of lactic acid produced by 6 different and morphologically identified presumptive lactic acid bacteria (LAB) which were isolated from different naturally fermented cheese samples for this study. Then, the biosensors were used to perform lactate measurements successfully within 3 minutes for each sample even few of them were out of the limit of detection. Conclusion: Electrochemical biosensors should be alternative and quick solutions for measurement of lactate metabolites instead of using classical methods which are required long working time. Besides, it is the first study to measure lactate produced by foodborne LAB as real sample with a biosensor. Molecular Biology Lactate amperometric biosensor lactic acid bacteria (LAB) electrochemical detection Figures Figure 1 Figure 2 Figure 3 Introduction Lactate (lactic acid) can be produced naturally by lactic acid bacteria (LAB) and have two enantiomers which are L (+) and D (-). While L lactate is an intermediate in mammalian metabolism, D lactate is produced by microorganisms, algae, and plants, and utilization of D-lactate by humans is limited [ 1 , 2 ]. Besides, lactate is found in fermentative food products like pickled vegetables such as Korean Kimchi and sauerkraut, fermented dairy products like yogurt and cheese, wine, cured meat, and fish. Additionally, lactate is supplied to foods and beverages as an acidulant or food preservative or to modify the flavor (E270). Apart from these, D (-) enantiomer shows that reduced freshness in vacuum-packed meat products, a racemic mixture of bacterial contamination of tomatoes, tomato paste, and tomato juice, and spoilage of wine. In fruit juice, a higher amount of LAB is referred to as that organoleptic characteristic of the juice has damaged. Thus, detection of both D (-) and L (+) form is an important indicator for the determination of food quality and in other words, at least the total lactic acid amount should be monitored for food safety which is required in terms of sustainable agriculture [ 1 – 6 ]. Lactate is an important metabolite of the anaerobic metabolic pathway [ 2 , 7 ]. Blood lactate level is a significant parameter for diagnosis of patient conditions during intensive care and surgical operation process, multiple system organ failure and death of septic shock patients, trauma, performance level, fatigue, hydration, and sports medicine. Besides, increasing lactate levels of blood should have come from congestive heart failure, hypoxia, lactic acidosis, and diabetic ketoacidosis [ 2 , 6 – 8 ]. At this point, biosensor has gained importance for measuring or observed of lactate amount by the reason of their reliability, accessibility and quickness [ 4 , 9 – 11 ]. Biosensors are analytical devices that are comprised of a biological recognition element (bioreceptor) and a physical transducer. They are classified by their bioreceptors (enzyme, whole-cell, and affinity biomolecule based) and physical transducer (electrochemical, optical, piezoelectric, and thermal). Biosensors, can measure the concentration of biological materials through a signal which is produced from a transducer obtained in proportion to the specificity and the concentration of the bioreceptor [ 12 – 14 ]. Using electrochemical transducers especially amperometric ones on biosensors very common because, having well linear range, high sensitivity, and great accuracy with various bioreceptor [ 15 – 18 ]. They are powerful alternatives instead of common analytical methods which are expensive, need a long time and trained personal like spectrophotometer, HPLC, and chromatography due to the fact that, biosensors are rapid, accurate, sensitive, easy to use, and cheap equipment [ 12 , 19 ]. Thus, biosensors have been used for traceability and detection of target analytes such as microorganisms, enzymes, nucleic acids, pesticides, etc. in food, health, biotechnology, environment, and military fields [ 19 – 23 ]. Especially enzymatic amperometric types of biosensors have been used commonly for the detection of lactate in food and health sciences [ 4 , 9 , 11 , 24 ]. Enzyme-based amperometric biosensor for lactate determination is designed by immobilized lactate dehydrogenase (LDH) or lactate oxidase (LOx) enzymes as bioreceptors. Lactate oxidase is widely used in lactate biosensors due to the fact that, its simple reaction and easy biosensor design configuration. Thus, the detection of lactate concentration is based on the catalytic action of lactate oxidase on the oxidation of lactate to pyruvate that is shown in the following equations [ 16 , 25 , 26 ]: (1) (2) Present paper describes the design of an enzyme-based amperometric lactate biosensor for the detection of lactate produced by 6 different and morphologically identified presumptive lactic acid bacteria (LAB). In the first part of the study, LOx-based electrochemical lactate biosensors were constructed by drop-casting a certain amount of enzyme on a mirror-like Pt electrode surface. The electrochemical performance of the sensors was evaluated using cyclic voltammetry (CV) and chronoamperometry (CA) methods. In the second part of the study, the constructed sensors were exposed to 6 different lactic acid environments produced by presumptive LAB, and the level of lactate in those environments was determined. To the best of our knowledge, there is no other study in the literature regarding the detection of lactate production by foodborne LAB by constructing a biosensor. Material And Methods Chemicals and Reagents Lactate oxidase enzyme (LOx) (L0638-100UN Lactate Oxidase from Pediococus sp., CAS:9028-72-2), Phosphate Buffered Saline (PBS) solution (pH 7.4) and lithium lactate were purchased from Sigma Aldrich. Then, the media (M17 and MRS) used for isolation and growth of LAB were obtained from Merck. Besides, the Gram stain Kit used for characterization of LAB was acquired from Norateks Kimya. Isolation and Morphological Characterization of LAB LAB strains were isolated from different naturally fermented cheese samples which were provided in Ankara-Turkey. MRS (De Man Rogosa Sharpe) medium and M17 medium were used for the isolation of lactic acid bacteria from the samples. M17 medium was preferred for further analysis due to bacterial growth was slightly better than MRS. The strains were inoculated M17 media concerning pour plate technique and incubated at 30°C at 24-48 hours [ 27 ]. To exhibit morphological properties and Gram reaction of the strains, the Gram staining method was applied to the strains by Gram stain Kit [ 28 ]. Then, Gram (+) ones were stored at -20°C in an M17 broth medium containing %15 glycerol for further experiments. Acquirement of Metabolites from LAB Morphologically identified 6 presumptive LAB isolates (shown in Table 2 ) were activated at 30°C for 24 hours in M17 broth. Then, supernatants of the active cultures were collected after centrifuging at 6000 rpm for 10 minutes. The supernatants were filtered through a 0.45 µm syringe tip filter (Sartorius, Germany) [ 29 ]. Designing Amperometric Lactate Biosensor Enzyme Immobilisation Lactate oxidase enzyme (LOx) was used as a bioreceptor and lithium lactate was preferred as substrate. Furthermore, a thin Nafion® layer was created on the surface of the sensors to circumvent any possible interference effect of interfering molecules and enzyme leakage out of the sensor surface. The amount of the polymer layer drop cast on the surface was also experimented to achieve maximum efficiency [ 11 , 30 – 32 ]. Preparation of Biosensors Three-electrode-system was utilized in the electrochemical performance experiments, compromising of Ag / AgCl, Pt electrode and Pt wire as a reference, working and counter electrodes, respectively. Electrochemical sensors were prepared by a drop-casting method. Before the construction of the sensors, the surface of the Pt electrode was polished with alumina solutions (1 µm, 0.3 µM, and 0.05 µm). After that, it was rinsed with deionized water and ethanol mixture to remove residues from the surface. 10 µL PBS solution which contained 2U LOx enzyme was dropped on the surface of the dried electrode and left room temperature to dry for 2 hours. After 2 hours, 8 µL Nafion solution (5 wt%) was dropped on the modified enzyme electrode surface and then dried at room temperature [ 33 , 34 ]. Performance Analyses Performance analysis of the biosensor was realized with cyclic voltammetry (CV) and chronoamperometry (CA) methods. CV and CA studies were done with an Ivium Vertex One-100 mA potentiostat and IviumSoft software package. Sensor performance assays were performed at room temperature in PBS solution (0.01 M, 8 mL, pH 7.4) which was in a glass cell with added lactic acid (Lithium lactate). The prepared sensors were kept at 4°C when not in use [ 34 ]. Detection of Lactate Produced by Foodborne Lactic acid bacteria After specified biochemical properties and limit of detection of the biosensor, lactate amounts that were produced by LAB isolates were measured chronoamperometrically in triplicate. The amounts of lactic acids were calculated from the regression equation obtained from the flow-time graphs (as in Figure 3 b), y: shows the increase in flow amount, a: sensitivity, and x shows the lactic acid concentration in the solution. This equation allows us to presume the actual lactic acid concentration by measuring the lactate from the diluted solution. Results Morphological Characterization of the Bacterial Isolates According to the Gram stain assay of the bacteria isolates numbered in Table 1 , all 6 strains were found as Gram (+) and were morphologically identified as coccus. Metabolites of these 6 isolates were determined in the biosensor carried out within the scope of the study. Also, the ability of these 6 isolates to produce lactate has been confirmed by high performance liquid chromatograph analysis (data not shown) [ 35 ]. Discovering Electrochemical Characteristics of the Sensors Electrochemical characteristics of the sensors were determined by cyclic voltammetry (CV) measurement. The results were shown in Figure 1 . According to Figure 1 , it is seen that there was an increase in the oxidation peak from 0.4 V by adding 2 mM lactate into the PBS. When the lactic acid amount in PBS increased, the oxidation peak was also increased, which indicates that the enzyme layer immobilized on the electrode surface and working properly. The oxidation reaction of lactate in the presence of LOx is given in Eq. 1. When the hydrogen peroxide (H 2 O 2 ) produced from lactic acid oxidation reaches the electrode surface if there is a suitable applied potential, it gives an oxidation reaction and the electron is released as a result of this reaction. For this reason, the increase in oxidation current seen in Figure 1 was due to the oxidation of hydrogen peroxide on the sensor surface. Considering Equations 1 and 2, there is a correlation between the lactic acid present in the system and the concentration of hydrogen peroxide formed by the reaction and oxidized on the sensor surface. Thus, the amount of lactic acid in the environment can be calculated by determining the amount of hydrogen peroxide and concentration. The effect of scan rate on the electrochemical behaviour of the sensors was evaluated by conducting the CV experiments at various scan rates. These experiments were carried out in PBS solution in the presence of 2 mM lactic acid and the results are given in Figure 2 . As seen in Figure 2 a, the increase in the scan rate resulted in a significant change in the peak current, and the oxidation peaks became more pronounced. The connection of this alteration with the scan rate was shown in Figure 2 b. So, peak current demonstrated a linear change with the increasing scan rate (R 2 = 0.9998). According to these results, it was deduced that reactions that occurred on the sensor’s surface were surface-controlled. In other words, the performances of the prepared sensors show a significant dependence on electrocatalytic surface properties [ 34 , 36 ]. Activities of Biosensors to Lactic Acid Activities and analytical performances of prepared biosensors were determined by using the chronoamperometry method (CA) [ 34 , 36 ]. Lactic acid was added to PBS at certain time intervals and alterations on the current were determined and saved. In electrochemical performance experiments, the applied voltage was fixed at 0.6 V, because the oxidation potential of hydrogen peroxide at the surface of Pt was determined as 0.6 V which is shown in Figure 1 and the peak formation was around this value as seen in Figure 2 a. While no lactic acid was present in the system the current of the system seem around zero (Figure 3 ). When 0.05 mM lactic acid was added to the PBS, it was observed that the measured current increased rapidly and fixed within a short time. The addition of lactic acid was repeated periodically and after each addition, the flow was rapidly increased and fixed. It was shown that demanded sensor performance was obtained, and the prepared lactic acid biosensors are capable of determining the lactic acid healthily and efficiently. Besides, the fast increase of the current in a short period with the addition of lactic acid is an indication of giving rapid results of the prepared sensors. According to this information, the current biosensor provided rapid determination which is an important advantage for biosensors. The calibration curves were obtained according to the results in Figure 3 a by performing the same sensor three times independently under specified conditions (Figure 3 b). One of the most significant results which were deduced from the graphs (Figure 3 a and 3 b) was that the prepared sensors can be used up to 350 µM lactic acid concentration in a proper way. Thus, it was found that the sensor performances increased linearly up to the concentration of 350 µM, yet above this value, an increase in the current was tended to reach saturation and not linear to the concentration. Slopes of calibration curves give the sensitivity of biosensors. Therefore, the mean sensitivity of the sensors was found as 0.0008 µA/µM when based on the equation in Figure 3 b. Then, it was known that the diameter of the Pt electrode used in the study was 1.6 mm, and the obtained sensitivity value was 0.04 µA.µM −1 .cm −2 . The limit of detection (LOD) for the biosensors was calculated according to LOD = (s/S)x10 equation. Based on the equation, LOD of the biosensor were found as 31 µM. It was dedicated that the linear working range of the biosensor was 50-350 µM. This determination range could be considered advantageous in some aspects compared to other studies in the literature mentioned in Table 1 . Besides, the relative standard deviation (RSD%) was found as 3.9%. Detection of Lactate Producing by Foodborne Presumptive LAB with the Biosensor The amount of lactates produced by the presumptive LAB isolates were measured chronamperometrically at room temperature at 0.6 V with the amperometric lactate biosensor developed by determining the optimum working conditions and determination range given at Table 1 . The measurements were done for diluted samples and then, lactate amounts were calculated consideration dilution ratios in main samples. Therefore, the results were given as mean value and standard deviation for triplicate measurements. Table 1 The biosensor measurement results of lactic acids produced by the LAB isolates. Isolate Number Food Origin Lactic Acid Amount (µM) 11 Cheese 280±0.05 12 Cheese 270±0.03 13 Cheese 250±0.06 14 Cheese 330±0.03 23 Cheese > 24 Cheese > *>: Level of lactate amount was measured as higher than the limit of detection range of the biosensor. As seen in Table 1 , the developing biosensor was successful in measurement amounts of the lactates which were range in limit of detection of the biosensors. These were produced by numbered as 11, 12, 13 and 14 isolates that obtained cheese samples were found 280, 270, 250, 330 µM, respectively. Besides, the amount of lactic acid was found higher than the 250 µM in all samples. Discussion According to the results of the Table 1 , it is seen that the biosensor developed within the scope of this study has a very high potential in terms of use in the determination of lactate produced by food-borne LAB if the working range is improved. Also, it should be considering that lactate amounts that cannot be determined exactly in the biosensor because of the environmental factors cannot be eliminated, or secondary metabolites in the metabolites may be oxidized on the electrode surface and affect the measurement results (parasite) [ 32 , 37 – 40 ]. Nevertheless, using a biosensor for the determination of lactate is an advantageous method, and the success of applying the real samples was shown in many studies like mentioned below. Canbay and co-workes have developed a microbial biosensor (with 0.1-10 mM determination range) that determines lactic acid and pyruvic acid in their study. They checked the actual sample trials with the commercial kit (Sigma Aldrich, USA) and determined the amount of L-lactate in kefir, milk, and butter samples. Then, they dedicated that, lactate in the drinks could be determined by using the biosensor [ 37 ]. Pereira and Stradiotto (2019) were developed a biosensor that had a modified glassy carbon electrode with MIP (molecularly imprinted polymers)/AuNP(gold nanoparticles)/RGO (graphene oxide) for the determination of lactic acid was obtained by electropolymerization of the ophenylediamine monomer. They indicated that the materials used to modify the electrode allowed to provided low detection and quantification limits and also good selectivity in the presence of molecules with similar chemical groups and sizes. Besides, GCE modification and MIP construction were done in addition to the cyclic voltammetry by electrochemical impedance spectroscopy, scanning electron microscopy, and atomic force microscopy. Then, there were not observed any critical values among the results. Thus, using MIP was an original and an advantage for the study. Also, they have used the sensor for the determination of lactate on sugarcane vinasse [ 41 ]. Similarly, the study of Alizadeh and co-workers was shown the benefit of using the MIP. They have developed a novel nano-sized imprinted polymer/multi-walled carbon nanotube (MWCNTs)-based potentiometric sensor for lactic acid determination. Then, the proposed sensor was successfully applied for the determination of lactate in milk and yogurt samples [ 42 ]. Table 2 Some examples of lactate biosensors reported in the literature. The biosensor final application, sensitivity and linear range values are shown. Concept of the Biosensor Linear Range (µM) Sensitivity (µA.µM −1 .cm −2 ) Sample Type Reference CNT–LOX on a Glassy Carbon Electrode 1000–4000 - Skin and Sweat [ 33 ] CNT–LOX on a Si/ITO substrate 10000-50000 AuNNs-programmed flexible sensor 1000-25000 - Sweat [ 43 ] Carboxylated multiwalled carbon nanotubes/copper nanoparticles/polyaniline modified pencil graphite electrode 1-2500 - Human Serum Plasma, milk products and wine [ 44 ] Pt electrode was modified with a composite prepared from rGO, CNTs and AuNPs 50–100.000 35.3 Milk, Blood and Cell Culture Media [ 45 ] AuNPs-ERGO-PAH/SPE 500-3000 1.08 Yogurt and Wine [ 46 ] 4000-16000 0.28 GCE/Chit/LOx/Chit/GA (5 mM K 3 Fe(CN) 6 ) 990-5660 1.44×10 −3 (C.cm −2 .mM −1 ) Not studied. [ 47 ] GCE/GInk/LOx/PD 740-2440 4.11×10 −4 (C.cm −2 .mM −1 ) 1 mm carbon working electrode, Pt auxiliary electrode and Ag/ AgCl reference electrode 50-800 3.03 Wine [ 48 ] Pt working electrode, Ag/AgCl reference electrode and a Pt wire counter electrode ( Lactobacillus delbruecki sp. based) 100-1000 - Buttermilk, Kefir and Milk [ 37 ] MIP/AuNP/RGO/GCE 10 −4 -10 −3 1.9 × 10 5 (µA.L.mol −1 ) Sugarcane Vinasse [ 41 ] Nano-MIP/MWCNTs-based membrane electrode 1-100000 - Milk and Yogurt [ 42 ] Ag/AgCl, Pt electrode and Pt wire 50-350 0.04 Foodborne Presumptive LAB Metabolite Present Study *CNT: Carbon nanotube, MWCNTs: multi-walled carbon nanotube, Si/ITO: silicon/indium tin oxide, AuNNs: gold nanopine needles, rGO: reduced graphene oxide, AuNPs: gold nanoparticles, ERGO: electrochemically-reduced graphene oxide, PAH: poly (allylamine hydrochloride), SPE: screen-printed electrode, GCE: glassy carbon electrode, Chit: chitosan, GA: glutaraldehyde, Gink: graphite ink solution, PD: 1,10-Phenan-throline-5,6-dione, MIP: molecularly imprinted polymers According to Table 2 ., Weber and co-workers developed two different biosensors for lactate determination prepared using carbon nanotube lactate oxidase electrodes and used the dripping method that was similar to present study. Furthermore, in that study determination range of the biosensors were found between 1000–4000 and 10000-5000 µM [ 33 ]. The fact that, the LOD of the presented paper is much lower than the mentioned study. Thus, even there is very low amount of lactate is presented in a sample, it can also be detected. Similarly, Yu and co-workers developed gold nanostructure-programmed flexible electrochemical biosensor for detection of glucose and lactate in sweat, the limit of detection in sweat was determined as 54 µM which was higher than the current study even using gold nano pin needles [ 43 ]. Dagar and Pundir (2017) were constructed an amperometric L-lactate biosensor in which LOx was immobilized on carboxylated multiwalled carbon nanotubes (cMWCNT)/copper nanoparticles (CuNPs)/polyaniline (PANI) hybrid film electrodeposited modified pen graphite electrode(PGE). It was stated that this developed biosensor had a very wide determination range like 1 µM - 2500 µM and it was associated that; the pencil graphite electrode had a quite large surface area [ 44 ] however, the preparation difficulties is main disadvantage for this method. Hashemzadeh and co-workers developed a novel amperometric biosensor, which had a wide linear range 50–100.000 µM of lactate with high electrochemical sensitivity (35.3 µAmM −1 .cm −2 ) and LOD of 2.3 mM. This was associated with utilizing electrodes modified with a composite prepared from rGO, CNTs, and gold nanoparticles. According to the study, the triple composite components showed synergistic effects on the enzyme loading, electrocatalytic activity, and electron transfer between receptor and electrode surface. Thus, developed nanomaterial that precluded parasites has gained a priority to the study [ 45 ]. Similarly, Istrate et al., developed a novel biosensor for L-lactate determination in dairy products and wine samples by using the screen-printed electrode modified with a ternary composite based on gold nanoparticles, electrochemically-reduced graphene oxide, and poly (allylamine hydrochloride). Then, the LDH enzyme was immobilized on the top of the electrode by crosslinking with glutaraldehyde. Furthermore, in that study linearity and ranges of the biosensor were found between 500-3000, 4000-16000 µM, and sensitivity were found as 1.08, 0.28 µA.mM −1 .cm −2 [ 46 ]. A lactate biosensor was fabricated using a chitosan and crosslinker configuration realized solution mediation (K 3 Fe(CN) 6 ) in the study of Halpin et al. (2021). Moreover, the linear range of the biosensor was 990-5660 µM, and sensitivity was 1.44×10 −3 C.cm −2 .mM −1 . Then, they used the heterocyclic quinoid species1,10-phenanthroline-5,6-dione for acting as a proton and electron acceptor concerning FADH 2 cofactor regeneration. Besides, graphite ink was formulated and utilized as an underlying conductive layer for the enzyme immobilization and enzymatic polymerization of 1,10-phenanthroline-5,6-dioneata graphite carbon electrode. Therefore, the linear range of the biosensor had become 740-2440 µM and sensitivity was 4.11×10 −4 C.cm −2 .mM −1 . Thus, the study indicated that the novel surface-confined mediator had gained an advantage over the biosensor for lactate sensing [ 47 ]. However, with the higher sensitivity and lower limit of detection values of the biosensor, the limit of range was so high. Therefore, the biosensor developed in presented study could be more favorable for lactate determination, especially in low amounts. The determination range of biosensors varies according to electrode surface used or the application of nanomaterial membrane or interface to the electrode surface [ 44 , 48 – 50 ]. It can be concluded that the sensor, which was designed without using an interface and been surface controlled in this study, was a biosensor with a suitable determination range for use in lactate determination. Conclusion In the present study, an electrochemical enzyme-based lactate biosensor with a linear range of 50-350 µM, LOD as 31 µM and a sensitivity of 0.04 µA.µM −1 .cm −2 was constructed. The practical applicability of the sensors was evaluated by detecting lactate in metabolite produced by food-borne LAB which is the unique part of the study not to the used metabolite of LAB as substrate (in real sample) before. Apart from these, this has been demonstrated by the current study that the analysis time for the developed biosensor was measured less than 3 minutes for each sample and did not require long pre-treatment. So, the study has shown that amperometric enzyme-based biosensors are the significant alternative for the detection of lactate instead of classical methods. It is showed that improving an interface for the biosensor utilizing a modification of a composite or immobilization of a mediator or enzymes into the catalytic layer to design a interference-free measuring system can be useful by the other studies. The results shared in this study are important part of improving lactate biosensors appealing to the industry and research laboratories. Declarations Compliance with Ethical Standards Funding This study was funded by the Ankara University Coordinatorship of Scientific Research Projects (BAP) with the number 17H0415001 projects. Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Statements & Declarations This study was supported by the Ankara University Coordinatorship of Scientific Research Projects (BAP) with the number 17H0415001 projects. This study is a part of a master's degree thesis of Ozum OZOGLU submitted to the Institute of Natural and Applied Sciences of Bursa Uludağ University, Bursa-Turkey. Studies were performed at Ankara University Biotechnology Institute System Biotechnology Advanced Research Unit Laboratory and Necmettin Erbakan University Faculty of Engineering and Architecture Department of Metallurgical and Materials Engineering Laboratory. Authors’ contributions All authors contributed to the study conception. Evrim Gunes Altuntas, Mehmet Altay Unal, Mehmet Gumustas and Sibel A. Ozkan designed the research. Material preparation, data collection and analysis were performed by Ozum Ozoglu, Aytekin Uzunoglu and Mehmet Gumustas. The first draft of the manuscript was written by Ozum Ozoglu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Conflict of Interest All of the authors declare that they have no conflict of interest. References Mazzei F, Azzoni A, Cavalieri B, et al (1996) A multi-enzyme bioelectrode for the rapid determination of total lactate concentration in tomatoes, tomato juice and tomato paste. Food Chem 55:413–418. https://doi.org/10.1016/0308-8146(95)00168-9 Nikolaus N, Strehlitz B (2008) Amperometric lactate biosensors and their application in (sports) medicine, for life quality and wellbeing. Microchim Acta 160:15–55. https://doi.org/10.1007/s00604-007-0834-8 Katrlík J, Pizzariello A, Mastihuba V, et al (1999) Biosensors for L-malate and L-lactate based on solid binding matrix. 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American Society for Microbiology Altuntaş EG, Ayhan K, Okcu G, et al (2010) ÇİĞ SÜT VE PEYNİR ÖRNEKLERİNDEN İZOLE EDİLEN LAKTİK ASİT BAKTERİLERİNİN ANTİMİKROBİYEL AKTİVİTELERİ. GIDA 35:197–203 Zhao Z, Jiang H (2010) Enzyme-based Electrochemical Biosensors. In: Serra PA (ed) Biosensors. In Tech, p 302 Rocchitta G, Spanu A, Babudieri S, et al (2016) Enzyme biosensors for biomedical applications: Strategies for safeguarding analytical performances in biological fluids. Sensors (Switzerland) 16:. https://doi.org/10.3390/s16060780 Romero MR, Ahumada F, Garay F, Baruzzi AM (2010) Amperometric biosensor for direct blood lactate detection. Anal Chem 82:5568–5572. https://doi.org/10.1021/ac1004426 Weber J, Kumar A, Kumar A, Bhansali S (2006) Novel lactate and pH biosensor for skin and sweat analysis based on single walled carbon nanotubes. Sensors Actuators, B Chem 117:308–313. https://doi.org/10.1016/j.snb.2005.12.025 Uzunoglu A (2016) Nanoparticle-based electrochemical sensors for the detection of lactate and hydrogen peroxide. Purdue University Özoğlu Ö (2019) Enzim Temelli Amperometrik Laktat Biyosensörü Üretimi ve Tayin Sınırının Belirlenmesi (Production of Enzyme Based Amperometric Lactate Biosensor and Determination of Limit of Detection). Bursa Uludag Unversity Uzunoglu A, Song S, Stanciu LA (2016) A Sensitive Electrochemical H 2 O 2 Sensor Based on PdAg-Decorated Reduced Graphene Oxide Nanocomposites. J Electrochem Soc 163:B379–B384. https://doi.org/10.1149/2.1201607jes Canbay E, Habip A, Kara G, et al (2015) A microbial biosensor based on Lactobacillus delbruecki sp . bacterial cells for simultaneous determination of lactic and pyruvic acid. FOOD Chem 169:197–202. https://doi.org/10.1016/j.foodchem.2014.07.140 Cunha-Silva H, Arcos-Martinez MJ (2018) Dual range lactate oxidase-based screen printed amperometric biosensor for analysis of lactate in diversified samples. Talanta 188:779–787. https://doi.org/10.1016/j.talanta.2018.06.054 Omanovic-miklicanin E, Valzacchi S (2017) Development of new chemiluminescence biosensors for determination of biogenic amines in meat. Food Chem 235:98–103. https://doi.org/10.1016/j.foodchem.2017.05.031 Vokhmyanina D V., Andreeva KD, Komkova MA, et al (2020) ‘Artificial peroxidase’ nanozyme – enzyme based lactate biosensor. Talanta 208:1–4. https://doi.org/10.1016/j.talanta.2019.120393 Pereira TC, Stradiotto NR (2019) Electrochemical sensing of lactate by using an electrode modified with molecularly imprinted polymers, reduced graphene oxide and gold nanoparticles. Microchim Acta 186:1–10. https://doi.org/10.1007/s00604-019-3898-3 Alizadeh T, Nayeri S, Mirzaee S (2019) A high performance potentiometric sensor for lactic acid determination based on molecularly imprinted polymer/MWCNTs/PVC nanocomposite film covered carbon rod electrode. Elsevier B.V. Yu M, Li YT, Hu Y, et al (2021) Gold nanostructure-programmed flexible electrochemical biosensor for detection of glucose and lactate in sweat. J Electroanal Chem 882:115029. https://doi.org/10.1016/j.jelechem.2021.115029 Dagar K, Pundir CS (2017) Enzyme and Microbial Technology An improved amperometric L-lactate biosensor based on covalent immobilization of microbial lactate oxidase onto carboxylated multiwalled carbon nanotubes / copper nanoparticles / polyaniline modified pencil graphite electro. Enzyme Microb Technol 96:177–186. https://doi.org/10.1016/j.enzmictec.2016.10.014 Hashemzadeh S, Omidi Y, Rafii-Tabar H (2019) Amperometric lactate nanobiosensor based on reduced graphene oxide, carbon nanotube and gold nanoparticle nanocomposite. Microchim Acta 186:1–8. https://doi.org/10.1007/s00604-019-3791-0 Istrate OM, Rotariu L, Bala C (2021) Amperometric L-Lactate biosensor based upon a gold nanoparticles/reduced graphene oxide/polyallylamine hydrochloride modified screen-printed graphite electrode. Chemosensors 9:. https://doi.org/10.3390/chemosensors9040074 Halpin G, Herdman K, Dempsey E (2021) Electrochemical investigations into enzymatic polymerisation of 1,10-phenanthroline-5,6-dione as a redox mediator for lactate sensing. Sensors and Actuators Reports 3:100032. https://doi.org/10.1016/j.snr.2021.100032 Shkotova L, Bohush A, Voloshina I, et al (2019) Amperometric biosensor modified with platinum and palladium nanoparticles for detection of lactate concentrations in wine. SN Appl Sci 1:1–8. https://doi.org/10.1007/s42452-019-0315-9 Cunha-Silva H, Pires F, Dias-Cabral AC, Arcos-Martinez MJ (2019) Inhibited enzymatic reaction of crosslinked lactate oxidase through a pH-dependent mechanism. Colloids Surfaces B Biointerfaces 184:1–8. https://doi.org/10.1016/j.colsurfb.2019.110490 Kumar A, Vatsyayan P, Goswami P, Minteer SD (2009) Biosensors and Bioelectronics Recent advances in material science for developing enzyme electrodes. 24:2313–2322. https://doi.org/10.1016/j.bios.2008.09.026 Cite Share Download PDF Status: Published Journal Publication published 22 Feb, 2023 Read the published version in Journal of Bioscience and Bioengineering → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1175499","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":71986891,"identity":"0f1e9246-7360-4be5-b945-f090cbdd1be3","order_by":0,"name":"Ozum Ozoglu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYLCCBww2DHwMDAYgNmMDUVoSGNIY2BBamInScpgELfz8hw+/SGw7n9gmkbzx4xcGG9kNB/iPfcCnRbLhWJpFYtttoJa0YmkZhjTjDQeYmWfg02JwsMfMAKIlx0BaguFwIkgLXocZHOYBaTkH0mL8W4LhPxFajvEYP0hsOwDSYib5geEAYS2SPWxpDAnnko3beJ6VWTMYJBvPPMxsjFcLKMQ+fCizk+1nT95880eFnWzf8cbHeLUAAZsEjMXMA4oaImKSGR4LjD8Iqx4Fo2AUjIIRCACEFkghaMFQ3AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3600-142X","institution":"Bursa Uludag University: Bursa Uludag Universitesi","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ozum","middleName":"","lastName":"Ozoglu","suffix":""},{"id":71986892,"identity":"a3d2880e-31d0-4cae-84c3-c8d84970ded5","order_by":1,"name":"Aytekin Uzunoglu","email":"","orcid":"","institution":"Necmettin Erbakan University: Necmettin Erbakan Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aytekin","middleName":"","lastName":"Uzunoglu","suffix":""},{"id":71986893,"identity":"54110243-39ab-4518-a173-a48890fc1371","order_by":2,"name":"Mehmet Altay Unal","email":"","orcid":"","institution":"Ankara University: Ankara Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"Altay","lastName":"Unal","suffix":""},{"id":71986894,"identity":"da72bb39-edc5-49fb-960f-8f92309643db","order_by":3,"name":"Mehmet Gumustas","email":"","orcid":"","institution":"Ankara University: Ankara Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"Gumustas","suffix":""},{"id":71986895,"identity":"fa51ef81-f2c4-4fea-8b06-27bef76b2336","order_by":4,"name":"Sibel Aysıl Ozkan","email":"","orcid":"","institution":"Ankara University Faculty of Pharmacy: Ankara Universitesi Eczacilik Fakultesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sibel","middleName":"Aysıl","lastName":"Ozkan","suffix":""},{"id":71986896,"identity":"4a0886a5-a71f-4a50-9e7a-ddca4d66d4d4","order_by":5,"name":"Mihriban Korukluoglu","email":"","orcid":"","institution":"Bursa Uludag University: Bursa Uludag Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mihriban","middleName":"","lastName":"Korukluoglu","suffix":""},{"id":71986897,"identity":"e768890b-3a68-41bc-a539-212d77fedf0b","order_by":6,"name":"Evrim Gunes Altuntas","email":"","orcid":"","institution":"Ankara University: Ankara Universitesi","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Evrim","middleName":"Gunes","lastName":"Altuntas","suffix":""}],"badges":[],"createdAt":"2021-12-15 23:21:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1175499/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1175499/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.jbiosc.2022.12.014","type":"published","date":"2023-02-23T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":16836528,"identity":"d5f83a76-10f9-412d-8fd2-1fa9d70e4341","added_by":"auto","created_at":"2021-12-29 16:01:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58452,"visible":true,"origin":"","legend":"\u003cp\u003eCV curves in lactate concentrations ranging from 2 mM to 6 mM in PBS solution. A potential range of -0.6 V-0.9 V was applied to the working electrode at a scan rate of 100 mV/s.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1175499/v1/8e294487c126592b1af87452.jpg"},{"id":16835983,"identity":"9afca2ee-e32c-4942-9706-a447e26a85ee","added_by":"auto","created_at":"2021-12-29 15:58:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85785,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of scan rates changes between 10-300 mV/s on electrochemical properties of the biosensors according to CV results. \u003cstrong\u003ea:\u003c/strong\u003e Change of current according to -0.6 V-0.9 V voltage potential in CV measurements. \u003cstrong\u003eb:\u003c/strong\u003e Connection of change in current of the peak with scan rate.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1175499/v1/0d9561a2927dfd221d436cf2.jpg"},{"id":16835985,"identity":"f608510a-f082-4dea-be52-68219be830d4","added_by":"auto","created_at":"2021-12-29 15:58:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60658,"visible":true,"origin":"","legend":"\u003cp\u003eChronoamperometric response of the sensor upon successive addition of 50 µM lactic acid at 0.6 V(a), The linear calibration curves between the current and the concentration of lactic acid. (b) (n=3)\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1175499/v1/a2b31b6bd63df5c78065f74e.jpg"},{"id":33423783,"identity":"cf7d7fd4-60fc-47d4-8295-f840644284a4","added_by":"auto","created_at":"2023-02-24 19:42:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":556572,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1175499/v1/9a75bf58-1553-4243-920c-a7ab695382e5.pdf"}],"financialInterests":"","formattedTitle":"Electrochemical Detection of Lactate Produced by Foodborne Presumptive Lactic Acid Bacteria","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLactate (lactic acid) can be produced naturally by lactic acid bacteria (LAB) and have two enantiomers which are L (+) and D (-). While L lactate is an intermediate in mammalian metabolism, D lactate is produced by microorganisms, algae, and plants, and utilization of D-lactate by humans is limited [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. Besides, lactate is found in fermentative food products like pickled vegetables such as Korean Kimchi and sauerkraut, fermented dairy products like yogurt and cheese, wine, cured meat, and fish. Additionally, lactate is supplied to foods and beverages as an acidulant or food preservative or to modify the flavor (E270). Apart from these, D (-) enantiomer shows that reduced freshness in vacuum-packed meat products, a racemic mixture of bacterial contamination of tomatoes, tomato paste, and tomato juice, and spoilage of wine. In fruit juice, a higher amount of LAB is referred to as that organoleptic characteristic of the juice has damaged. Thus, detection of both D (-) and L (+) form is an important indicator for the determination of food quality and in other words, at least the total lactic acid amount should be monitored for food safety which is required in terms of sustainable agriculture [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eLactate is an important metabolite of the anaerobic metabolic pathway [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Blood lactate level is a significant parameter for diagnosis of patient conditions during intensive care and surgical operation process, multiple system organ failure and death of septic shock patients, trauma, performance level, fatigue, hydration, and sports medicine. Besides, increasing lactate levels of blood should have come from congestive heart failure, hypoxia, lactic acidosis, and diabetic ketoacidosis [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. At this point, biosensor has gained importance for measuring or observed of lactate amount by the reason of their reliability, accessibility and quickness [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eBiosensors are analytical devices that are comprised of a biological recognition element (bioreceptor) and a physical transducer. They are classified by their bioreceptors (enzyme, whole-cell, and affinity biomolecule based) and physical transducer (electrochemical, optical, piezoelectric, and thermal). Biosensors, can measure the concentration of biological materials through a signal which is produced from a transducer obtained in proportion to the specificity and the concentration of the bioreceptor [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Using electrochemical transducers especially amperometric ones on biosensors very common because, having well linear range, high sensitivity, and great accuracy with various bioreceptor [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. They are powerful alternatives instead of common analytical methods which are expensive, need a long time and trained personal like spectrophotometer, HPLC, and chromatography due to the fact that, biosensors are rapid, accurate, sensitive, easy to use, and cheap equipment [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, biosensors have been used for traceability and detection of target analytes such as microorganisms, enzymes, nucleic acids, pesticides, etc. in food, health, biotechnology, environment, and military fields [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eEspecially enzymatic amperometric types of biosensors have been used commonly for the detection of lactate in food and health sciences [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Enzyme-based amperometric biosensor for lactate determination is designed by immobilized lactate dehydrogenase (LDH) or lactate oxidase (LOx) enzymes as bioreceptors. Lactate oxidase is widely used in lactate biosensors due to the fact that, its simple reaction and easy biosensor design configuration. Thus, the detection of lactate concentration is based on the catalytic action of lactate oxidase on the oxidation of lactate to pyruvate that is shown in the following equations [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAATgAAAAkCAYAAAAdHCY+AAAIWUlEQVR4Ae2aTa9MwRaGD4mRMX/Af/AXxO9AgkiYYMJQfE3E0IwJCWLgI2YiEibEQEwIiZGQCGMRdfN07iPrltq9d59z+uxr96qkumqvWrU+3lr1nt05vVayJQKJQCIwUQTWJppXppUIJAKJQEmCyyJIBBKBySKQBDfC0V68eLGsra396R8+fOiNIuofOXKkV/9fVNi3b1+5efPmX6GDD/kPwemvzSlYaQSS4EY6/mfPnpU9e/Ys5L2LABYyEpSHEuVQvWB6U6fglAS3qZCujLEkuJGOemyC862oL33eNscmOGPNN7i+08r1GoEkuBqRLXpeBsFBRH6VjaTE174olzCUsV7LeK6/SgNN1OONsqv51oUPv3air0/yd45NGnuQ29Q3L/V8Zn/MU31s6HNovPrMcVoIJMGNdJ6bTXBeZNKJ8+hHOaNz04coIDRanNdvcJAKe2kQintmgv9+sF+i0o/PrPnVnP22SE7I6hj0qz104hz7xoKuBOc+9LviNYYcp4dAEtxIZxqJZ2gIXFAvbtce1rnUdBqX3osf90RyiHLfvNzDCNnQiFnbjq5FG/pWVscgmbnuGN/gsEGMNGP1GVmdJzL2YNs2NF71c5weAklwI51pH8FJIIySWh/BoQvhSAikFgkqphp11GM/8vrtSRLri1n72vG5JjjsQWbaVW8owbXy1Iakhs+h8bo3x+khkAQ30pm2Lp/k0hVSi+AgCfZBgr69RPLywiOj8UyPOsgjuWATgqDVBAm5uMZ6TVLIiCPKedZ/nNckF2OIa76tIZuXpz7JTyyGxDtLND8miUAS3AjHCkFw8Vqdy9lqLV1kXmT2qAMRMGekRX/Koj7rtQ77IRPJhWdISmLUl8Q1cxQ+jAE9c4KAWnaRk0e0Gf24pi/19MEoqbmmz2iHNW2EUHM6YQSS4CZ8uJlaIrDqCCTBrXoFZP6JwIQRSIKb8OFmaonAqiOQBLfqFZD5JwITRiAJbsKHm6klAquOQBLcqldA5p8ITBiBwQTnv+T5V3v8aUIfNlP517w/Vah/htCXf66vHgLWSBz9jd5U0Yg/M/LO1z/RQWdeixyjjXn6Q9YGExzGOKS+IKNTA17vb4/4DZa/Z4p26/lGimdIPoAd9YgJGfFlSwRaCPAH0fqwXmINtfZshWyZd4U8ufN1QzbvHkuE4sV+5tyxeftqP63npRIcDjfCxH3AYL8L1FayLVlf0VEQrTdWD6BlM2X/LgJv3ryZXa53796VX79+rTuRSHAY4blVR+t2sI6N1OxGYui7K113se8eE1OLePHXIsxFUt9SgiNgCI8egZbBkZsQo7omH2UcFoCqwyjbR70+MPoODVstHWPWp3H43Oc318dHAAKLnYhu3bpVtm3bVnbu3Fn27t1bjh8/Xu7du1c+ffpUfv78OTho6psapVkr1BFyaoW6ovmHkjX1qHd0HOMaclptBx3W9MncTk3qRxm+aD4bz0zY8YGPeQ0/LTvI4r3QpzKejTvaZ501YhUbn6PevPmWElwMrmsOGCYbgUEmKcZ5DWosLA7EPREEfLd6l27rYAWcWFwnlpaNb9++lefPn5cXL14s1Nnz+PHjcv/+/fLgwYPB/eHDh+X27dvlxo0bC/fr16+Xq1evlitXrizcz58/X86cOVPOnj27UGfPyZMn19UhoGPHji3cjx49Wg4ePFgOHDjwpx86dKjs37+/bN++/a/62L17dzl8+HDhzW5Iow5ijcW6gLiscWxZP+rHNXRd98LrnzVkNufos0aL83hvWIv3C333aM8aN6441rrsMb6o5zzGpi6Y6CPmrH/tMYoBei3f7qnHLSU4EzNpkiP4ePgxwHgAykmO/e5hP3o2bTvGNXXiKHBRFufsb+l4MPiPzbii7O3bt38u76lTp8rQfvr06XLu3Lly6dKlWQzEMaRfuHBhRk7Xrl0r6+mQ4927dxfujx49Kk+ePFm4P336tLx8+bK8evVq4Q6279+/X7h//PixfPnypXz9+vVP//79e7lz507ZsWPHrMZ27do1q7PLly/P/jD9+PFj8NdW6qB1aakLase6RIdn5dRtbNS79UetxXX2xjXtuJ8Y0JcQ0Lc+rV/vCaNr7q9HfdVyn+u7qJxc63vCmv7w3cLKfFt5abtv/F80e7Qj2LUqAUawXEdmgCQak0LeBQr7IzAmi594UPX+6M8Y5o19h0bOFmO0Y75RxtxiquX5/G8g8Pr163LixInZ19LPnz+X379/rytw6pwa6WqsU7ux/iSduCfeOe9AXLc2oy9rU/vWJHLvX8tXtNuax1hb6/VdVCfeY2WMxkVMxhXX8Wd+yom7Lw51GTdMcDgjsa4m4dTJK58FUTG4CURg4kHHg6rt1mAJYld8+upaN75oB5/EH4sKvSG25vnJtekgQB3W9RGzs25jzbRIh7qz9tCl7iIZsIaf6Cv6Zo/70Yl7uV+uEVucx1idx1iVxdGcoox5vMeuRVvmHWXESq7YjC3qRHnXfDDBESQO6468q8U96MRn58glDG2TMI3DQMYYddzLgQgOeh6y61E2M7iBj2gTu8aoSXwvCr57c5wWAtSHfR5pUFOxjtyD3Bbr3vvgGqPr0Y7kgD3rltpUFzlzmj6jLNofOsd+tEU89CjzfnTdlajLvG7s947Xa13Pf1vp0kx5JwIUcTycWGydm3Jh5RGYR35TBWe9d0Vy954NxScJbihSqZcIbCICvEEt+jayie5XxlQS3MocdSb6/4CAX+V4I8m2fASS4JaPcXpIBBKBkRBIghsJ+HSbCCQCy0cgCW75GKeHRCARGAmBJLiRgE+3iUAisHwEkuCWj3F6SAQSgZEQSIIbCfh0mwgkAstH4D+i6Gb/NC/7RgAAAABJRU5ErkJggg==\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;(1)\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (2)\u003c/p\u003e\n\u003cp\u003ePresent paper describes the design of an enzyme-based amperometric lactate biosensor for the detection of lactate produced by 6 different and morphologically identified presumptive lactic acid bacteria (LAB). In the first part of the study, LOx-based electrochemical lactate biosensors were constructed by drop-casting a certain amount of enzyme on a mirror-like Pt electrode surface. The electrochemical performance of the sensors was evaluated using cyclic voltammetry (CV) and chronoamperometry (CA) methods. In the second part of the study, the constructed sensors were exposed to 6 different lactic acid environments produced by presumptive LAB, and the level of lactate in those environments was determined. To the best of our knowledge, there is no other study in the literature regarding the detection of lactate production by foodborne LAB by constructing a biosensor.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003cp\u003e\u003cstrong\u003eChemicals and Reagents\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eLactate oxidase enzyme (LOx) (L0638-100UN Lactate Oxidase from \u003cem\u003ePediococus\u003c/em\u003e sp., CAS:9028-72-2), Phosphate Buffered Saline (PBS) solution (pH 7.4) and lithium lactate were purchased from Sigma Aldrich. Then, the media (M17 and MRS) used for isolation and growth of LAB were obtained from Merck. Besides, the Gram stain Kit used for characterization of LAB was acquired from Norateks Kimya.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and Morphological Characterization of LAB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLAB strains were isolated from different naturally fermented cheese samples which were provided in Ankara-Turkey. MRS (De Man Rogosa Sharpe) medium and M17 medium were used for the isolation of lactic acid bacteria from the samples. M17 medium was preferred for further analysis due to bacterial growth was slightly better than MRS. The strains were inoculated M17 media concerning pour plate technique and incubated at 30\u0026deg;C at 24-48 hours [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. To exhibit morphological properties and Gram reaction of the strains, the Gram staining method was applied to the strains by Gram stain Kit [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Then, Gram (+) ones were stored at -20\u0026deg;C in an M17 broth medium containing %15 glycerol for further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcquirement of Metabolites from LAB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphologically identified 6 presumptive LAB isolates (shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) were activated at 30\u0026deg;C for 24 hours in M17 broth. Then, supernatants of the active cultures were collected after centrifuging at 6000 rpm for 10 minutes. The supernatants were filtered through a 0.45 \u0026micro;m syringe tip filter (Sartorius, Germany) [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003cp\u003e\u003cstrong\u003eDesigning Amperometric Lactate Biosensor\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eEnzyme Immobilisation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eLactate oxidase enzyme (LOx) was used as a bioreceptor and lithium lactate was preferred as substrate. Furthermore, a thin Nafion\u0026reg; layer was created on the surface of the sensors to circumvent any possible interference effect of interfering molecules and enzyme leakage out of the sensor surface. The amount of the polymer layer drop cast on the surface was also experimented to achieve maximum efficiency [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePreparation of Biosensors\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThree-electrode-system was utilized in the electrochemical performance experiments, compromising of Ag / AgCl, Pt electrode and Pt wire as a reference, working and counter electrodes, respectively. Electrochemical sensors were prepared by a drop-casting method. Before the construction of the sensors, the surface of the Pt electrode was polished with alumina solutions (1 \u0026micro;m, 0.3 \u0026micro;M, and 0.05 \u0026micro;m). After that, it was rinsed with deionized water and ethanol mixture to remove residues from the surface. 10 \u0026micro;L PBS solution which contained 2U LOx enzyme was dropped on the surface of the dried electrode and left room temperature to dry for 2 hours. After 2 hours, 8 \u0026micro;L Nafion solution (5 wt%) was dropped on the modified enzyme electrode surface and then dried at room temperature [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePerformance Analyses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePerformance analysis of the biosensor was realized with cyclic voltammetry (CV) and chronoamperometry (CA) methods. CV and CA studies were done with an Ivium Vertex One-100 mA potentiostat and IviumSoft software package. Sensor performance assays were performed at room temperature in PBS solution (0.01 M, 8 mL, pH 7.4) which was in a glass cell with added lactic acid (Lithium lactate). The prepared sensors were kept at 4\u0026deg;C when not in use [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Lactate Produced by Foodborne Lactic acid bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter specified biochemical properties and limit of detection of the biosensor, lactate amounts that were produced by LAB isolates were measured chronoamperometrically in triplicate. The amounts of lactic acids were calculated from the regression equation obtained from the flow-time graphs (as in Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb), y: shows the increase in flow amount, a: sensitivity, and x shows the lactic acid concentration in the solution. This equation allows us to presume the actual lactic acid concentration by measuring the lactate from the diluted solution.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003cp\u003e\u003cstrong\u003eMorphological Characterization of the Bacterial Isolates\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAccording to the Gram stain assay of the bacteria isolates numbered in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, all 6 strains were found as Gram (+) and were morphologically identified as coccus. Metabolites of these 6 isolates were determined in the biosensor carried out within the scope of the study. Also, the ability of these 6 isolates to produce lactate has been confirmed by high performance liquid chromatograph analysis (data not shown) [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eDiscovering Electrochemical Characteristics of the Sensors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElectrochemical characteristics of the sensors were determined by cyclic voltammetry (CV) measurement. The results were shown in Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. According to Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, it is seen that there was an increase in the oxidation peak from 0.4 V by adding 2 mM lactate into the PBS. When the lactic acid amount in PBS increased, the oxidation peak was also increased, which indicates that the enzyme layer immobilized on the electrode surface and working properly. The oxidation reaction of lactate in the presence of LOx is given in Eq.\u0026nbsp;1. When the hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) produced from lactic acid oxidation reaches the electrode surface if there is a suitable applied potential, it gives an oxidation reaction and the electron is released as a result of this reaction. For this reason, the increase in oxidation current seen in Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e was due to the oxidation of hydrogen peroxide on the sensor surface. Considering Equations 1 and 2, there is a correlation between the lactic acid present in the system and the concentration of hydrogen peroxide formed by the reaction and oxidized on the sensor surface. Thus, the amount of lactic acid in the environment can be calculated by determining the amount of hydrogen peroxide and concentration.\u003c/p\u003e\n\u003cp\u003eThe effect of scan rate on the electrochemical behaviour of the sensors was evaluated by conducting the CV experiments at various scan rates. These experiments were carried out in PBS solution in the presence of 2 mM lactic acid and the results are given in Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. As seen in Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, the increase in the scan rate resulted in a significant change in the peak current, and the oxidation peaks became more pronounced. The connection of this alteration with the scan rate was shown in Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb. So, peak current demonstrated a linear change with the increasing scan rate (R\u003csup\u003e2\u003c/sup\u003e= 0.9998). According to these results, it was deduced that reactions that occurred on the sensor\u0026rsquo;s surface were surface-controlled. In other words, the performances of the prepared sensors show a significant dependence on electrocatalytic surface properties [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActivities of Biosensors to Lactic Acid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActivities and analytical performances of prepared biosensors were determined by using the chronoamperometry method (CA) [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Lactic acid was added to PBS at certain time intervals and alterations on the current were determined and saved. In electrochemical performance experiments, the applied voltage was fixed at 0.6 V, because the oxidation potential of hydrogen peroxide at the surface of Pt was determined as 0.6 V which is shown in Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and the peak formation was around this value as seen in Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea. While no lactic acid was present in the system the current of the system seem around zero (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). When 0.05 mM lactic acid was added to the PBS, it was observed that the measured current increased rapidly and fixed within a short time. The addition of lactic acid was repeated periodically and after each addition, the flow was rapidly increased and fixed. It was shown that demanded sensor performance was obtained, and the prepared lactic acid biosensors are capable of determining the lactic acid healthily and efficiently. Besides, the fast increase of the current in a short period with the addition of lactic acid is an indication of giving rapid results of the prepared sensors. According to this information, the current biosensor provided rapid determination which is an important advantage for biosensors. The calibration curves were obtained according to the results in Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea by performing the same sensor three times independently under specified conditions (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\n\u003cp\u003eOne of the most significant results which were deduced from the graphs (Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb) was that the prepared sensors can be used up to 350 \u0026micro;M lactic acid concentration in a proper way. Thus, it was found that the sensor performances increased linearly up to the concentration of 350 \u0026micro;M, yet above this value, an increase in the current was tended to reach saturation and not linear to the concentration.\u003c/p\u003e\n\u003cp\u003eSlopes of calibration curves give the sensitivity of biosensors. Therefore, the mean sensitivity of the sensors was found as 0.0008 \u0026micro;A/\u0026micro;M when based on the equation in Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb. Then, it was known that the diameter of the Pt electrode used in the study was 1.6 mm, and the obtained sensitivity value was 0.04 \u0026micro;A.\u0026micro;M\u003csup\u003e\u0026minus;1\u003c/sup\u003e.cm\u003csup\u003e\u0026minus;2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe limit of detection (LOD) for the biosensors was calculated according to LOD = (s/S)x10 equation. Based on the equation, LOD of the biosensor were found as 31 \u0026micro;M. It was dedicated that the linear working range of the biosensor was 50-350 \u0026micro;M. This determination range could be considered advantageous in some aspects compared to other studies in the literature mentioned in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Besides, the relative standard deviation (RSD%) was found as 3.9%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Lactate Producing by Foodborne Presumptive LAB with the Biosensor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of lactates produced by the presumptive LAB isolates were measured chronamperometrically at room temperature at 0.6 V with the amperometric lactate biosensor developed by determining the optimum working conditions and determination range given at Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The measurements were done for diluted samples and then, lactate amounts were calculated consideration dilution ratios in main samples. Therefore, the results were given as mean value and standard deviation for triplicate measurements.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe biosensor measurement results of lactic acids produced by the LAB isolates.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolate Number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFood Origin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLactic Acid Amount (\u0026micro;M)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e280\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e270\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e330\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCheese\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e*\u0026gt;: Level of lactate amount was measured as higher than the limit of detection range of the biosensor.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs seen in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the developing biosensor was successful in measurement amounts of the lactates which were range in limit of detection of the biosensors. These were produced by numbered as 11, 12, 13 and 14 isolates that obtained cheese samples were found 280, 270, 250, 330 \u0026micro;M, respectively. Besides, the amount of lactic acid was found higher than the 250 \u0026micro;M in all samples.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccording to the results of the Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, it is seen that the biosensor developed within the scope of this study has a very high potential in terms of use in the determination of lactate produced by food-borne LAB if the working range is improved. Also, it should be considering that lactate amounts that cannot be determined exactly in the biosensor because of the environmental factors cannot be eliminated, or secondary metabolites in the metabolites may be oxidized on the electrode surface and affect the measurement results (parasite) [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. Nevertheless, using a biosensor for the determination of lactate is an advantageous method, and the success of applying the real samples was shown in many studies like mentioned below.\u003c/p\u003e\n\u003cp\u003eCanbay and co-workes have developed a microbial biosensor (with 0.1-10 mM determination range) that determines lactic acid and pyruvic acid in their study. They checked the actual sample trials with the commercial kit (Sigma Aldrich, USA) and determined the amount of L-lactate in kefir, milk, and butter samples. Then, they dedicated that, lactate in the drinks could be determined by using the biosensor [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003ePereira and Stradiotto (2019) were developed a biosensor that had a modified glassy carbon electrode with MIP (molecularly imprinted polymers)/AuNP(gold nanoparticles)/RGO (graphene oxide) for the determination of lactic acid was obtained by electropolymerization of the ophenylediamine monomer. They indicated that the materials used to modify the electrode allowed to provided low detection and quantification limits and also good selectivity in the presence of molecules with similar chemical groups and sizes. Besides, GCE modification and MIP construction were done in addition to the cyclic voltammetry by electrochemical impedance spectroscopy, scanning electron microscopy, and atomic force microscopy. Then, there were not observed any critical values among the results. Thus, using MIP was an original and an advantage for the study. Also, they have used the sensor for the determination of lactate on sugarcane vinasse [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Similarly, the study of Alizadeh and co-workers was shown the benefit of using the MIP. They have developed a novel nano-sized imprinted polymer/multi-walled carbon nanotube (MWCNTs)-based potentiometric sensor for lactic acid determination. Then, the proposed sensor was successfully applied for the determination of lactate in milk and yogurt samples [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eSome examples of lactate biosensors reported in the literature. The biosensor final application, sensitivity and linear range values are shown.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eConcept of the Biosensor\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLinear Range (\u0026micro;M)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSensitivity (\u0026micro;A.\u0026micro;M\u003csup\u003e\u0026minus;1\u003c/sup\u003e.cm\u003csup\u003e\u0026minus;2\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSample Type\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eReference\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCNT\u0026ndash;LOX on a Glassy Carbon Electrode\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1000\u0026ndash;4000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eSkin and Sweat\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCNT\u0026ndash;LOX on a Si/ITO substrate\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10000-50000\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAuNNs-programmed flexible sensor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1000-25000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSweat\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCarboxylated multiwalled carbon nanotubes/copper nanoparticles/polyaniline modified pencil graphite electrode\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1-2500\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman Serum Plasma, milk products and wine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePt electrode was modified with a composite prepared from rGO, CNTs and AuNPs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e50\u0026ndash;100.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e35.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMilk, Blood and Cell Culture Media\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eAuNPs-ERGO-PAH/SPE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e500-3000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eYogurt and Wine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4000-16000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.28\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGCE/Chit/LOx/Chit/GA (5 mM K\u003csub\u003e3\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e990-5660\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.44\u0026times;10\u003csup\u003e\u0026minus;3\u003c/sup\u003e (C.cm\u003csup\u003e\u0026minus;2\u003c/sup\u003e.mM\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eNot studied.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGCE/GInk/LOx/PD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e740-2440\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.11\u0026times;10\u003csup\u003e\u0026minus;4\u003c/sup\u003e (C.cm\u003csup\u003e\u0026minus;2\u003c/sup\u003e.mM\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 mm carbon working electrode, Pt auxiliary electrode and Ag/ AgCl reference electrode\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e50-800\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePt working electrode, Ag/AgCl reference electrode and a Pt wire counter electrode (\u003cem\u003eLactobacillus delbruecki\u003c/em\u003e sp. based)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e100-1000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eButtermilk, Kefir and Milk\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMIP/AuNP/RGO/GCE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003csup\u003e\u0026minus;4\u003c/sup\u003e-10\u003csup\u003e\u0026minus;3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.9 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e (\u0026micro;A.L.mol\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSugarcane Vinasse\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNano-MIP/MWCNTs-based membrane electrode\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1-100000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMilk and Yogurt\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAg/AgCl, Pt electrode and Pt wire\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e50-350\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFoodborne Presumptive LAB Metabolite\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePresent Study\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003e*CNT: Carbon nanotube, MWCNTs: multi-walled carbon nanotube, Si/ITO: silicon/indium tin oxide, AuNNs: gold nanopine needles, rGO: reduced graphene oxide, AuNPs: gold nanoparticles, ERGO: electrochemically-reduced graphene oxide, PAH: poly (allylamine hydrochloride), SPE: screen-printed electrode, GCE: glassy carbon electrode, Chit: chitosan, GA: glutaraldehyde, Gink: graphite ink solution, PD: 1,10-Phenan-throline-5,6-dione, MIP: molecularly imprinted polymers\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e., Weber and co-workers developed two different biosensors for lactate determination prepared using carbon nanotube lactate oxidase electrodes and used the dripping method that was similar to present study. Furthermore, in that study determination range of the biosensors were found between 1000\u0026ndash;4000 and 10000-5000 \u0026micro;M [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. The fact that, the LOD of the presented paper is much lower than the mentioned study. Thus, even there is very low amount of lactate is presented in a sample, it can also be detected. Similarly, Yu and co-workers developed gold nanostructure-programmed flexible electrochemical biosensor for detection of glucose and lactate in sweat, the limit of detection in sweat was determined as 54 \u0026micro;M which was higher than the current study even using gold nano pin needles [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eDagar and Pundir (2017) were constructed an amperometric L-lactate biosensor in which LOx was immobilized on carboxylated multiwalled carbon nanotubes (cMWCNT)/copper nanoparticles (CuNPs)/polyaniline (PANI) hybrid film electrodeposited modified pen graphite electrode(PGE). It was stated that this developed biosensor had a very wide determination range like 1 \u0026micro;M - 2500 \u0026micro;M and it was associated that; the pencil graphite electrode had a quite large surface area [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e] however, the preparation difficulties is main disadvantage for this method.\u003c/p\u003e\n\u003cp\u003eHashemzadeh and co-workers developed a novel amperometric biosensor, which had a wide linear range 50\u0026ndash;100.000 \u0026micro;M of lactate with high electrochemical sensitivity (35.3 \u0026micro;AmM\u003csup\u003e\u0026minus;1\u003c/sup\u003e.cm\u003csup\u003e\u0026minus;2\u003c/sup\u003e) and LOD of 2.3 mM. This was associated with utilizing electrodes modified with a composite prepared from rGO, CNTs, and gold nanoparticles. According to the study, the triple composite components showed synergistic effects on the enzyme loading, electrocatalytic activity, and electron transfer between receptor and electrode surface. Thus, developed nanomaterial that precluded parasites has gained a priority to the study [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. Similarly, Istrate et al., developed a novel biosensor for L-lactate determination in dairy products and wine samples by using the screen-printed electrode modified with a ternary composite based on gold nanoparticles, electrochemically-reduced graphene oxide, and poly (allylamine hydrochloride). Then, the LDH enzyme was immobilized on the top of the electrode by crosslinking with glutaraldehyde. Furthermore, in that study linearity and ranges of the biosensor were found between 500-3000, 4000-16000 \u0026micro;M, and sensitivity were found as 1.08, 0.28 \u0026micro;A.mM\u003csup\u003e\u0026minus;1\u003c/sup\u003e.cm\u003csup\u003e\u0026minus;2\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eA lactate biosensor was fabricated using a chitosan and crosslinker configuration realized solution mediation (K\u003csub\u003e3\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e) in the study of Halpin et al. (2021). Moreover, the linear range of the biosensor was 990-5660 \u0026micro;M, and sensitivity was 1.44\u0026times;10\u003csup\u003e\u0026minus;3\u003c/sup\u003eC.cm\u003csup\u003e\u0026minus;2\u003c/sup\u003e.mM\u003csup\u003e\u0026minus;1\u003c/sup\u003e. Then, they used the heterocyclic quinoid species1,10-phenanthroline-5,6-dione for acting as a proton and electron acceptor concerning FADH\u003csub\u003e2\u003c/sub\u003e cofactor regeneration. Besides, graphite ink was formulated and utilized as an underlying conductive layer for the enzyme immobilization and enzymatic polymerization of 1,10-phenanthroline-5,6-dioneata graphite carbon electrode. Therefore, the linear range of the biosensor had become 740-2440 \u0026micro;M and sensitivity was 4.11\u0026times;10\u003csup\u003e\u0026minus;4\u003c/sup\u003eC.cm\u003csup\u003e\u0026minus;2\u003c/sup\u003e.mM\u003csup\u003e\u0026minus;1\u003c/sup\u003e. Thus, the study indicated that the novel surface-confined mediator had gained an advantage over the biosensor for lactate sensing [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, with the higher sensitivity and lower limit of detection values of the biosensor, the limit of range was so high. Therefore, the biosensor developed in presented study could be more favorable for lactate determination, especially in low amounts.\u003c/p\u003e\n\u003cp\u003eThe determination range of biosensors varies according to electrode surface used or the application of nanomaterial membrane or interface to the electrode surface [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. It can be concluded that the sensor, which was designed without using an interface and been surface controlled in this study, was a biosensor with a suitable determination range for use in lactate determination.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, an electrochemical enzyme-based lactate biosensor with a linear range of 50-350 \u0026micro;M, LOD as 31 \u0026micro;M and a sensitivity of 0.04 \u0026micro;A.\u0026micro;M\u003csup\u003e\u0026minus;1\u003c/sup\u003e.cm\u003csup\u003e\u0026minus;2\u003c/sup\u003e was constructed. The practical applicability of the sensors was evaluated by detecting lactate in metabolite produced by food-borne LAB which is the unique part of the study not to the used metabolite of LAB as substrate (in real sample) before. Apart from these, this has been demonstrated by the current study that the analysis time for the developed biosensor was measured less than 3 minutes for each sample and did not require long pre-treatment. So, the study has shown that amperometric enzyme-based biosensors are the significant alternative for the detection of lactate instead of classical methods. It is showed that improving an interface for the biosensor utilizing a modification of a composite or immobilization of a mediator or enzymes into the catalytic layer to design a interference-free measuring system can be useful by the other studies. The results shared in this study are important part of improving lactate biosensors appealing to the industry and research laboratories.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Ankara University Coordinatorship of Scientific Research Projects (BAP) with the number 17H0415001 projects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements \u0026amp; Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Ankara University Coordinatorship of Scientific Research Projects (BAP) with the number 17H0415001 projects. This study is a part of a master\u0026apos;s degree thesis of Ozum OZOGLU submitted to the Institute of Natural and Applied Sciences of Bursa Uludağ University, Bursa-Turkey. Studies were performed at Ankara University Biotechnology Institute System Biotechnology Advanced Research Unit Laboratory and Necmettin Erbakan University Faculty of Engineering and Architecture Department of Metallurgical and Materials Engineering Laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception. Evrim Gunes Altuntas, Mehmet Altay Unal, Mehmet Gumustas and Sibel A. Ozkan designed the research. Material preparation, data collection and analysis were performed by Ozum Ozoglu, Aytekin Uzunoglu and Mehmet Gumustas. The first draft of the manuscript was written by Ozum Ozoglu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMazzei F, Azzoni A, Cavalieri B, et al (1996) A multi-enzyme bioelectrode for the rapid determination of total lactate concentration in tomatoes, tomato juice and tomato paste. Food Chem 55:413\u0026ndash;418. https://doi.org/10.1016/0308-8146(95)00168-9\u003c/li\u003e\n \u003cli\u003eNikolaus N, Strehlitz B (2008) Amperometric lactate biosensors and their application in (sports) medicine, for life quality and wellbeing. Microchim Acta 160:15\u0026ndash;55. https://doi.org/10.1007/s00604-007-0834-8\u003c/li\u003e\n \u003cli\u003eKatrl\u0026iacute;k J, Pizzariello A, Mastihuba V, et al (1999) Biosensors for L-malate and L-lactate based on solid binding matrix. 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Enzyme Microb Technol 96:177\u0026ndash;186. https://doi.org/10.1016/j.enzmictec.2016.10.014\u003c/li\u003e\n \u003cli\u003eHashemzadeh S, Omidi Y, Rafii-Tabar H (2019) Amperometric lactate nanobiosensor based on reduced graphene oxide, carbon nanotube and gold nanoparticle nanocomposite. Microchim Acta 186:1\u0026ndash;8. https://doi.org/10.1007/s00604-019-3791-0\u003c/li\u003e\n \u003cli\u003eIstrate OM, Rotariu L, Bala C (2021) Amperometric L-Lactate biosensor based upon a gold nanoparticles/reduced graphene oxide/polyallylamine hydrochloride modified screen-printed graphite electrode. Chemosensors 9:. https://doi.org/10.3390/chemosensors9040074\u003c/li\u003e\n \u003cli\u003eHalpin G, Herdman K, Dempsey E (2021) Electrochemical investigations into enzymatic polymerisation of 1,10-phenanthroline-5,6-dione as a redox mediator for lactate sensing. Sensors and Actuators Reports 3:100032. https://doi.org/10.1016/j.snr.2021.100032\u003c/li\u003e\n \u003cli\u003eShkotova L, Bohush A, Voloshina I, et al (2019) Amperometric biosensor modified with platinum and palladium nanoparticles for detection of lactate concentrations in wine. SN Appl Sci 1:1\u0026ndash;8. https://doi.org/10.1007/s42452-019-0315-9\u003c/li\u003e\n \u003cli\u003eCunha-Silva H, Pires F, Dias-Cabral AC, Arcos-Martinez MJ (2019) Inhibited enzymatic reaction of crosslinked lactate oxidase through a pH-dependent mechanism. Colloids Surfaces B Biointerfaces 184:1\u0026ndash;8. https://doi.org/10.1016/j.colsurfb.2019.110490\u003c/li\u003e\n \u003cli\u003eKumar A, Vatsyayan P, Goswami P, Minteer SD (2009) Biosensors and Bioelectronics Recent advances in material science for developing enzyme electrodes. 24:2313\u0026ndash;2322. https://doi.org/10.1016/j.bios.2008.09.026\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lactate, amperometric biosensor, lactic acid bacteria (LAB), electrochemical detection","lastPublishedDoi":"10.21203/rs.3.rs-1175499/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1175499/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The detection of lactate is an important indicator of freshness, stability, and storage stability of products as well as degree of fermentation in food industry. In addition, it can be used as a diagnostic tool in patients’ healthcare since it is known that the lactate level in blood increases in some pathological conditions including septic shock, tissue hypoxia, and sepsis. Thus, determination of lactate level plays an important role in not only food industry but also health fields. At this point, biosensor has become important to detect lactate due to having many advantages like rapid, cheap and easy to use. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods and Results: \u003c/strong\u003eIn the current study, amperometric lactate biosensors based on lactate oxidase immobilization (with nafion 5% wt) were designed and limit of detection, linear range and sensitivity values were determined, which were found as 31 μM, 50-350 μM and 0.04 μA.μM\u003csup\u003e-1\u003c/sup\u003e.cm\u003csup\u003e-2\u003c/sup\u003e, respectively. Then, it was used for the measurement of lactic acid produced by 6 different and morphologically identified presumptive lactic acid bacteria (LAB) which were isolated from different naturally fermented cheese samples for this study. Then, the biosensors were used to perform lactate measurements successfully within 3 minutes for each sample even few of them were out of the limit of detection. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Electrochemical biosensors should be alternative and quick solutions for measurement of lactate metabolites instead of using classical methods which are required long working time. Besides, it is the first study to measure lactate produced by foodborne LAB as real sample with a biosensor.\u003c/p\u003e","manuscriptTitle":"Electrochemical Detection of Lactate Produced by Foodborne Presumptive Lactic Acid Bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-12-29 15:58:31","doi":"10.21203/rs.3.rs-1175499/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eee91fea-b0a4-4beb-81ad-9c5b12306c43","owner":[],"postedDate":"December 29th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":9363977,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2023-02-24T19:42:09+00:00","versionOfRecord":{"articleIdentity":"rs-1175499","link":"https://doi.org/10.1016/j.jbiosc.2022.12.014","journal":{"identity":"journal-of-bioscience-and-bioengineering","isVorOnly":true,"title":"Journal of Bioscience and Bioengineering"},"publishedOn":"2023-02-23 00:00:00","publishedOnDateReadable":"February 23rd, 2023"},"versionCreatedAt":"2021-12-29 15:58:31","video":"","vorDoi":"10.1016/j.jbiosc.2022.12.014","vorDoiUrl":"https://doi.org/10.1016/j.jbiosc.2022.12.014","workflowStages":[]},"version":"v1","identity":"rs-1175499","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1175499","identity":"rs-1175499","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}