New automated system based on biosensors for winemaking ... · merely on temperature control of...
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New automated system based on biosensors for winemaking
monitoring and assessment of allergen risk along the wine production
chain (SENS4WINE)
Project start date: June14, 2017
Project end date: June 13, 2019
Contract 32/14.06.2017 Contracting Authority: CNDI-UEFISCDI
Project code: ERANET-MANUNET II-SENS4WINE
Total funding awarded: 1.001.250,00 RON (222.500,00 EUR)
Co-funding : 122.600,00 RON (27.244,00 EUR)
Total costs : 1.123.850,00 RON (249.744,00 EUR)
Summary Consortium News Results 2017
Results 2018
Results 2019
PROJECT SUMMARY The continuous need for safe products of consistently high quality delivered by the food and
beverage industry can be only answered by close monitoring of the production processes, for
which reliable and low cost analytical technologies are essential. There is a rising trend in
embracing automation in wine production for obtaining wines of consistently high quality, with
important energy and cost savings. However, most of automated systems available focus
merely on temperature control of fermentation without including other quality parameters
relevant for winemakers. SENS4WINE aims to develop and manufacture a novel, cost effective
and more comprehensive automated system, based on (bio)sensors that will allow real-time
monitoring of two key processes in wine production: maceration and alcoholic fermentation.
Moreover, the system will provide low-cost analysis of lysozyme, an egg protein sometimes
used as an antimicrobial additive in wine as partial replacement of sulphites, and which poses a
potential allergen risk. The automated system includes a sampling unit, a sample preparation
unit, and a sample analysis unit (including, data acquisition& interpretation), linked to an alarm
and a modern Supervisory Control and Data Acquisition (SCADA) system. We propose a
modular sample analysis unit which, in addition to several classic sensors (e.g. temperature),
includes innovative, highly effective and robust biosensors for glucose and lysozyme, as well as
a low-cost spectro-electrochemical detector for monitoring of chromatic characteristics and
polyphenol content of wines. Alarm systems will be embedded in the automated monitoring
system, promptly signaling when critical parameters are reached (Figure 1).
Figure 1. Schematic representation of the proposed automated system for wine fermentation
monitoring and for assessment of allergen risk along the wine production chain
The main components of the prototype automated system will be designed, built and tested
first on the bench; afterwards the system will be installed on one maceration and one
fermentation tank and evaluated at winery level during the vintages of 2017 and 2018. Our goal
is to develop and manufacture prototypes (sensors, equipment, and automated system) tested
in winery conditions, ready for implementation (TRL5 level). Project activities will be carried out
by a well-balanced consortium including two SME’s and two research institutes from two
countries with complementary market share, expertise and infrastructure in development of
biosensors for wine (Partner 1), manufacturing of screen-printed electrodes and
spectroelectrochemical equipment (Partner 2), manufacturing of automation systems (Partner
3) and wine research and manufacturing (Partner 4). Through the innovative products and
processes that it will deliver, SENS4WINE will contribute to the increase in competitiveness and
visibility of SMEs and research institutions involved. Moreover it will offer to wine producers a
performant, cost-effective system for a better control of the technological process, for saving
energy, ensuring a consistently high quality of wines and facilitating innovation in the
technological process.
dr. Alina Vasilescu
International Centre of Biodynamics, Bucharest, Romania
www.biodyn.ro
Coordinator, P1
dr. Pablo Fanjul
Dropsens, Llanera ( Asturias), Spain
www.dropsens.com
Partner 2
ing. Petru Epure
Epi Sistem SRL, Brasov, Romania
www.epi.ro
Partner 3
dr.ing. Porumb Roxana
ICDVV Valea Calugareasca (Prahova), Romania
www.icdvv.ro
Partner 4
CONSORTIUM
PARTNERS DESCRIPTION (back to top)
The project consortium includes two SME’s and two research institutes from two countries
with complementary market share, expertise and infrastructure in development of biosensors
for wine (International Centre of Biodynamics, Bucharest, Partner 1), manufacturing of screen-
printed electrodes and spectroelectrochemical equipment (Dropsens, Spain, Partner 2),
manufacturing of automation systems (Epi-Sistem, Brasov, Partner 3) and wine research and
manufacturing (ICDVV Valea Calugareasca, Partner 4). SENS4WINE will deliver a product that
will address an actual need in the winemaking industry, particularly for small and medium wine
producers for better control of the technological process. to save energy, consistently produce
wines of high quality and facilitate innovation in the technological process.
The Coordinator, International Centre of Biodynamics (ICB, www.biodyn.ro) is primarly
focused on life sciences, main activity being oriented towards developing biosensors and
sensing platforms for the real time monitoring of bio-processes, with extensive documented
research activity pertaining to biomedical and food quality control fields. ICB disposes of state-
of-the-art infrastructure for sensor microfabrication, electrochemical analysis, imaging and
surface characterization. In SENS4WINE, ICB activities concern: adapting the enzymatic
biosensor for glucose developed by P2 for real-time fermentation monitoring, developing an
aptamer-based biosensor for off-line analysis of residual levels of lysozyme (an antimicrobial
additive sometimes used in wine processing, posing potential allergen risk); electrode
functionalization with nanomaterials and aptamers, defining appropriate calibration procedures
for developed sensors, including when integrated into the automated analysis system
developed by P3 and validating the new methods (with P4) in comparison with current
standard procedures.
Partner 1, DropSens, Spain (www.dropsens.com) is focused on the design, fabrication and
commercialization of electrochemical screen-printed devices that are used as (bio)sensors, as
well as on the development and manufacturing of small and portable electrochemical
instrumentation for clinical and environmental analysis. DropSens was awarded as the best
Spanish Innovative Technology-Based Firm (EIBT) by the National Association of Spanish CEEIs
(ANCES). The company has access to resources for characterization of surfaces (SEM, TEM,
AFM, XPS), lithographic and sputtering services. DropSens is an ISO9001 quality certified
company and an ISO13485 certified company for the 'Manufacturing of sensors for medical
devices'. DropSens distributes its products worldwide (in more than 60 countries: USA, UK,
China, Brazil, India…) through distributors or direct relationship with customers. In the project,
Dropsens will fabricate the sensors and biosensors in the project at factory level, optimize
manufacturing procedure of spectroelectrochemical sensors coupled to a fluidic chamber;
design and prototype a low cost system for real-time monitoring of the chromatic
characteristics and polyphenols contents of fermenting must, by combined photometrical and
electrochemical analysis; develop and manufacture new inks that include bionanocomposite
materials for screen-printing and manufacture cost-effective biosensors for wine analysis based
on new inks using rapid manufacturing techniques.
Partner 3, Epi-Sistem SRL (www.epi.ro) is focused on design and development of measurement
& control systems based mainly on National Instruments technologies, distribution of various
types of measurement equipment for research and industry, servicing various equipment and
microcontroller programming and integration for remote control and monitoring. Epi-Sistem
develops hardware and software solutions for industrial and/or research configurations (e.g. pH
monitoring, temperature monitoring and control, weight determination, and energy
consumption metering, electrocoagulation system, CO2 monitoring, solar light & solar radiation,
LORA temperature monitoring, myDAQ-EDEA integration, measurement logger). Role in the
project: development, installation (at P4) and testing of an automation system based on
(bio)sensors for monitoring wine fermentation processes. The system includes several original
hardware and software products and contains: an online liquid sampler, a low cost, robust
sample preparation unit, an analysis unit and real-time clock multichannel data logger linked to
an alarm module and SCADA. The system will be modular to have the flexibility to be installed
on either maceration or on fermentation tanks.
Partner 4, Institutul de Cercetare-Dezvoltare pentru Viticultura si Vinificatie Valea
Calugareasca (ICDVV, www.icdvv.ro) is at the forefront of the Romanian research on Viticulture
and Enology. The Institute is located in the Appellation of Controlled Origin, Dealu Mare– Valea
Calugareasca area, in southeastern Romania. The Institute manages about 100 ha of grapevine
plantations and a winery complex with a storage capacity of 25 000 hL wine. Its average annual
red, white and rosé wines production is about 4000 hL. The activity of scientific research and
technological development coordinated by ICDVV Valea Calugareasca is performed in its own
laboratories, pilot-stations and experimental fields, as well as in the 7 research stations located
in the main vineyards all over Romania. In the oenology laboratory, authorized since 2006,
physical chemical, microbiological and organoleptic analyses are conducted for commercial
wines. Role in the project: testing of the automated system at winery level (with P3) and
alerting the partners on any required improvements; performing winemaking by classic
technology and assisting with installation of automated analysis system on one 100hL rotary
maceration tank and one 500hL static fermentation tank; implementing, at micro-winemaking
level (200L vats), a new methodology involving addition of lysozyme as an antimicrobial agent
that partially replaces sulphites and performing analysis with the biosensor for lysozyme at
critical stages in winemaking; validating the new methods for chromatic characteristics,
phenols, glucose and lysozyme versus currently used standard methods (with P1).
NEWS (back to top)
January 1, 2017: project start for Spanish partner
June 14, 2017: project start for the Romanian partners
September 12, 2017: sampling and analysis system connected to 500 hL fermentation tank installed at ICDVV
September 12-30, 2017: alcoholic fermentation of white wines, samples collected 2-3 times/day
September 30-October 6, 2017: maceration-fermentation of red wines in 100 hL rotary tanks at ICDVV
October 25, 2017: all partner project meeting at ICB Bucharest
October 26, 2017: participation at Priochem conference in Bucharest: poster presented by Ana Maria Titoiu from ICB on the flow injection system method based on a biosensor for glucose monitoring during fermentation
February 26- March 6: small scale fermentation at ICDVV, using 100 kg of black Moldova grapes; samples were taken 2-3 times/day, kept at -20° C until the end of fermentation, then distributed to all partners for analysis
March, 2018: the contract of the Romanian partners with the funding authority, UEFISCDI was amended; part of budget and activities were shuffled from 2018 to 2019.
May 23-25, 2018: Ana Maria Titoiu participated at the IC-ANMBES international conference in Brasov with an oral presentation
June 30, 2018: end of project for Spanish partner
July 23, 2018: the conference “Biosensors as tools for today’s challenges” was organized at ICB, all partners participated, Alina Vasilescu from ICB presented ““Lysozyme detection: ensuring specificity in practical applications”
July30-August 1, 2018: collaborative tests Epi-Sistem/ICB at ICB to investigate problems related to the automated dilution module and to transfer to Epi-Sistem the method for phenolic compounds analysis with a biosensor based on carbon nanotubes
August 30, 2018: oral presentation by Alina Vasilescu from ICB at the 18th World Conference of analytical chemistry and mass spectrometry”
September 19-October 10, 2018: large scale fermentation at ICDVV for white wines (500 hL stationary tank) and red wines (100 hL, rotating tank) and small scale fermentation of white wines treated with lysozyme
November 6, 2018: Romanian patent application deposited at OSIM by Petru Epure
April 15-19, 2019: production of a white wine at ICDVV (micro scale, from table grapes, aprox. 25L) and monitoring the fermentation with the automated system
June 12-14, conference presentation by Epi Sistem, Exp’At 19 , Madeira, Portugal
June 13, 2019, project end. The project goals were reached.
June 26, 2019: the Partners will present the project results at theAnnual Scientific Session of ICDVV Valea Calugareasca, Romania
June 26, 2019: presentation of MSc thesis based on project results by Ana Maria Titoiu (ICB team member): Faculty of Chemistry, University of Bucharest.
RESULTS 2017 (back to top)
The first year of the project was dedicated to the development of measuring modules for
glucose, phenolic compounds and color and their inclusion into an automated system for
monitoring wine fermentation-maceration. The consortium worked on checking the possibility
to use screen printed enzyme biosensors for glucose determination in musts and wine;
development of a flow-injection system including a glucose biosensor with adequate analytical
characteristics and stability and the corresponding protocol to measure glucose in white and
red musts and wine; developing (preliminary) of a automated system with sampling from the
fermentation tank, automated dilution and inclusion of the measuring modules for glucose and
phenolic compounds; performing fermentation of white wines in 500 hL tank in the period
September 10-October 02, 2017 and analyzing the evolution of glucose during the fermentation
with monitoring also of the temperature; performing the maceration-fermentation of red wines
in 100 hL rotary tank in the period September 29–October 06, 2017 and analyzing the evolution
of glucose and phenolic compounds during the maceration-fermentation; evaluating a low cost
colorimeter for determining the chromatic characteristics, to be included in the automated
system; evaluated optical and electrochemical methods for the detection of phenolic
compounds in red musts/wine. During the all partner meeting at ICB on October 25, 2017, the
partners presented their progress in implementing the project activities. Dropsens presented
their activities related to the detection of phenolic compounds and sugar. The sensors
developed by Dropsens and ICB have good performances. However there were large
differences in absolute values between the results obtained by the electrochemical method and
the classic Folin Ciocalteu spectrophotometric test for the total phenolic compounds. ICB will
continue investigating the differences and propose a suitable solution. The cost related to the
fast optical detection of phenolic compounds by UV via measuring the phenolic index at 280 nm
was deemed too expensive and qualitative. Therefore, the electrochemical method was chosen
for the determination of phenolic compounds, to be integrated in the automated system. The
partners agreed on a small scale fermentation to be conducted at ICDVV in February 2018,
where samples taken periodically should be distributed to be analyzed by all partners.
Measurement of the chromatic characteristics of wine were also discussed. The partners
agreed that the low cost colorimeter evaluated in 2017 is not sensitive enough for monitoring
the fermentation, a better spectrophotometer will be evaluated in 2018.
RESULTS 2018 (back to top)
The year 2018 marked the end of the project for Dropsens (in June), while Romanian
partners will continue until June 2019. The activities of the consortium were centered on
further developing and optimizing the automated system for monitoring wine fermentation,
as well as the development of the lysozyme analysis module.
Dropsens developed and further evaluated sensors for sugar analysis based on screen-
printed electrodes. Romanian partners were involved in six scientific and one dissemination
activities. ICDVV produced 2 wines at large scale fermentation level. The white wine
fermentation took place in stationary, 500 hL fermentation tank at 15° C, while the maceration-
fermentation of red wine (blend of Burgund, Merlot and Feteasca Neagra grape varieties) took
place in 100 hL rotary tank at 25 C°. Additionally, two small scale fermentation processes were
conducted : one from Moldova black grapes, in February when 100 L of wine was produced for
the purpose to check the sensors, equipment and methods developped by all partners and to
prepare for the main fermentation in autumn. A white wine (50 L) was also produced in
September where sulphur dioxide was partially replaced with lysozyme as antimicrobial agent.
Five experimental variants were produced and this experiment was aimed at verifying the
lyzoyme biosensor and the corresponding analysis module. Besides producing wines, hosting
and assisting with the installment of the automated system during testing sessions, ICDVV
contributed with the standard laboratory testing for all the wines produced. ICB has optimized
the methods based on (bio)sensors for the determination of phenolic compounds and
lysozyme, has developed the method for measuring the chromatic characteristics of wine, has
transferred all the developed methods to Epi-Sistem for integration in the automated system,
has coordonated the project meetings, has organized a common testing session with Epi-Sistem
for the faster implementation of electrochemical methods and has organized a conference for
disseminating the project results. Epi-sistem’s activity was centered on integrating the various
novel analysis methods for phenolic compounds, chromatic characteristics and lysozyme within
the automated system, solving technical issues, validating the automated dilution module,
validating the remote access of the automated system for real- time vizualising of information
pertaining to the progress of fermentation, has optimized the various components (such as the
electrochemical cell, the fluidics part, the graphical interface etc) and optimising the sample
prelevation module for both stationary and rotary fermentation tanks. While previous results
obtained with finished wines and fruit juices have validated the accuracy of the sensors
developed by Dropsens for sugar analysis, the results on must samples were different from
those obtained by the standard method at ICDVV due to must evolution (fermentation) during
transport from Romania to Spain. The differences in the project start date and project duration
between the Spanish and Romanian partners have represented a significant disadvantage for
the project. The Romanian partners will continue in 2019 and have agreed on a last small scale
fermentation to be conducted in Spring 2019, for a final evaluation of the automated system.
DISSEMINATION (back to top)
- article published: Vasilescu A, Hayat A, Gaspar S, Marty J-L, “Advantages of Carbon
Nanomaterials in Electrochemical Aptasensors for Food Analysis”, Electroanalysis (2018), 30, 2-
19,doi: 10.1002/elan.201700578
-book chapter published: Vasilescu A., Gáspár S., David S., Gheorghiu M., Boukherroub R.,
Szunerits S., “Lysozyme detection with graphene oxide–coated plasmonic interfaces: specificity
brought by aptamer and cells for biorecognition”, in the Series of Micro and Nanoengineering,
vol 26, « Nanotechnologies and nanomaterials for various applications”, editors Maria
Zaharescu, Marius Enachescu, Dan Dascalu, Publishing House of the Romanian Academy, 2018,
ISBN: 978-973-27-2954-8, p 88-105.
-national patent application submitted: Epure P, Porumb R, Vasilescu A, Gaspar S, Titoiu AM,
“Automated system based on biosensor for monitoring some parameters of the wine-making
process”, registration number nr A100872/6.11.2018
-conference presentations:
Oral presentations
1. Titoiu AM, “Flow injection analyis system integrating electrochemical sensors for monitoring the alcoholic fermentation of wines”, IC-ANMBES, Brasov, Romania, May 23-25, 2018
2. Vasilescu A, “Biosensing approaches for lysozyme detection with graphene oxide-coated plasmonic interfaces”, 18th World Conference of analytical chemistry and mass spectrometry”, Toronto, Canada, August 29-30, 2018
3. Vasilescu A, “Lysozyme detection: ensuring specificity in practical applications”, conferinta ”Biosensors as tools for today’s challenges”, International Centre of Biodynamics, Bucharest, Romania, July 23, 2018
4. Vasilescu A, Gaspar S, David S, Titoiu AM, Purcarea C, Zamfir M, Development of a SPR
aptasensor: towards a robust tool for detecting traces of lysozyme dimer in oligomeric
and aggregated mixtures, NanoBioMed, Barcelona, Spain, November 22-24, 2017
Poster Titoiu A.M, Munteanu R., Epure P, Porumb R, Brinduse E, Gáspár S,
Vasilescu A, Flow injection system integrating a glucose biosensor for
monitoring the alcoholic fermentation of wines, Priochem, Bucharest,
October 25-27, 2017.
First international conference presentation, congratulations Ana Maria!
Conference organized in the frame of the project: “Biosensors as tools for today’s challenges”, CIB, Bucharest, July 23, 2018 with participants from France, USA, Canada and various institutes from Romania, from Bucharest, Cluj-Napoca and Arad. (figure 2). The conference was centered on discussing some problems preventing the practical implementation of biosensors in various fields.
Figure 2. Images from the conference presentations (prof. Michael Thompson, University of Toronto, Canada, lecturer dr. Madalina Tudorache, Universitaty of Bucharest, prof. Silvana Andreescu, Clarkson University, USA) and some of the participants.
RESULTS 2019 (back to top)
The last part of the project was dedicated to the fine tuning and final check of the automated monitoring system for wine fermentation, as well as to the validation of the lysozyme analysis module. For this purpose, ICDVV has produced a white wine at micro winemaking level ( about 25 L of wine were produced). Table grapes bought from the supermarket were used to make the wine as these were the only available at the time of experiments (April 15-19, 2019). The grapes had a low amount of sugar and the fermentation was fast: after only 4 days, a wine with 9.2% alcohol was obtained. ICDVV performed physic-chemical analysis of the wines produced in the project and assisted Epi Sistem with the installation of the automated monitoring system at ICDVV in April 2019 (Figure 3). Epi—Sistem optimized and validated the different modules of the automated wine monitoring system (sample collection, dilution, analysis). Among others, it brought several improvements to allow the extensive use of the amperometric detector for periods of up to 10 days, enhancing the reliability of the automated dilution module, optimizing the fluidics for various configurations and optimizing the automated sample collection.
Figure 3 Production of a white wine
at ICDVV ( at micro winemaking level) and the monitoring the alcoholic fermentation with the automated monitoring system installed in April 2019 at ICDVV.
ICB has optimized and validated the biosensor based method for the detection of lysozyme and has applied the method to measure the content of lysozyme in the experimental wine samples produced by ICDVV in 2018. For these experimental wines, a classic
winemaking technological process was followed with the exception of using lysozyme in different proportions as a partial replacement for sulphur dioxide. In March 2019 the wines were conditioned by bentonite treatment and filtration at ICDVV. Samples were collected at critical stages during the wine production, in 2018 and 2019 and were kept at -20°C until the analysis with the biosensor in April. The biosensor was compared with a standard chromatographic method showing good correlation, moreover it was demonstrated that the biosensor can be used successfully to measure accurately the concentration of lysozyme in wine during wine production, from levels as high as 375 mg/L (added initially in the must) to around 1 mg/L residual levels after wine conditioning. ICDVV has analyzed the physico-chemical
and organoleptic characteristics of the conditioned wines. Three of the wines, treated with different quantities of lysozyme between 190 and 375 mg/L by a single added dose in the must had organoleptic characteristics that were no different from the wine obtained in the classic way, using exclusively sulphur dioxide. By contrast, the sample V1, where lysozyme was added at 375 mg/L immediately after the alcoholic fermentation, was slightly opalescent, slightly amber, with a slight acetaldehyde odor (Figure 3). This sample had around 200 mg/L after conditioning and filtration. The study shows the importance of the moment chosen for lysozyme addition in relation to wine characteristics; it moreover stressed the importance of having a fast, sensitive, simple and affordable analytical tool like the biosensor developed in the project for assisting to optimize the technological process of wine production. Epi Sistem developed the lysozyme analysis module (including the software interface), that integrates the biosensors and allows users to register voltamograms for the standards and samples, calculate and store data, determine the calibration curve and display the concentration of lysozyme in a sample, as is calculated automatically by interpolation on the calibration curve.
Figure 4. A. Wines treates with SO2, with and without lysozyme, immediately aftre the addition of lysozyme in the must in September 2018 (left) and after conditioning, in March 2019 (right). B. Schematic representation of the analysis of lysozyme treated wines with the electrochemical aptasensor for lysozyme.
At the end of these activities, the main goal of the project, the development of an automated system for monitoring wine fermentation based on (bio) sensors was reached. The automated system is modular, having flexibility for future development and applications and the cost is estimated at 12400 Euro, including the software.
DISSEMINATION
Articles:
1. Titoiu A-M, Porumb R, Fanjul-Bollado P,- Epure P, Zamfir M,- Vasilescu A Detection of allergenic lysozyme during winemaking with an electrochemical aptasensor,Electroanalysis in evaluare
2. Vasilescu A, Fanjul-Bollado P, Titoiu AM, Porumb R, Epure P, Progress in electrochemical biosensors for monitoring wine production, Chemosensors, in pregatire
Conference participation
1. Epure P, Ursutiu D, Samoila C, Remote Monitoring and Control of Winemaking Parameters, EXP.AT’19, Madeira, Portugalia, 12-14 iunie 2019, oral presentation.
2. Epure P, Porumb R, Vasilescu A, Automated system for determining the content of glucose and phenolic compounds during the fermentation process, Annual Scientific Session, ICDVV Valea Calugareasca, June 26, 2019, oral presentation
3. Titoiu A.M, Porumb R, Epure P, Vasilescu A, Monitoring lysozyme in white wine by using an electrochemical biosensor, , Annual Scientific Session, ICDVV Valea Calugareasca, June 26, 2019, poster
Other:
Products
Epure P, Maciuca B, Perju A, Automated system for wine fermentation monitoring
Software
Epure P, Perju A, Offline lysozyme analysis module(software)
Equipment
Epure P, Maciuca B, Perju A, Sample collection module
MSc Thesis
MSc thesis work entitled « Development of an electrochemical aptasensor for lysozyme :, student : Ana Maria Titoiu (ICB team member), based on the results obtained in the frame of the SENS4WINE project, to be presented on June 26, 2019 at the Faculty of Chemistry, University of Bucharest.
Project application The project partners have applied in the 2019 EraNet Manunet competition ; the proposal passed in the second stage of the competition.