1. Biogenic amines in Hardanger ciders : the effect of native cider yeasts on biogenic amine productionUrban Česnik, Mitja Martelanc, Branka Mozetič Vodopivec, Ingunn Ovsthus, Lorena Butinar, 2023, objavljeni povzetek znanstvenega prispevka na konferenci Opis: The presence of bioactive compounds in food and beverages of plant origin is mainly connected with higher nutritional value and better sensory properties. However, some of them can pose a threat to food product quality and human health. For example, excess biogenic amines (BAs) intake can cause different allergenic responses in individuals who have such intolerances.
BAs have been intensively studied in recent years, especially in fermented foods like wine, meat, fish, and cheese. Among fermented beverages is apple cider still rather unexplored from this perspective. Especially since no such data exist for the Norwegian cider. Norwegian cider is becoming more and more popular in Norway in recent years among producers and consumers. Hardanger cider from Western Norway is very different from French, English, or Spanish ciders in terms of sensory characteristics, apple cultivars, and in the fermentation process. In Hardanger, the traditional cider is still produced by spontaneous fermentation of apple juice with naturally occurring yeasts that originate from the fruit or processing equipment surfaces.
Lactic acid bacteria are known to be associated with BA formation. However, several studies reported about the BA-producing yeasts in winemaking. Due to the important role of natural yeasts in the production of Hardanger cider, we focused on the ability of BA formation by native yeasts. Thus, in our study, we followed the amounts of BAs in the Hardanger ciders during the fermentation process and characterize isolated yeasts if they have the ability to produce BAs under cidermaking conditions by performing a micro-fermentation experiment.
From must/cider samples, taken during the fermentation process at 13 producers in the Hardanger region, we isolated 530 yeast isolates. Based on the sequencing of the D1/D2 domain of the 26S rDNA we identified 25 different yeast species. As expected, yeast diversity was higher at the beginning compared to the middle fermentation stage, when mostly different non-Saccharomyces yeast species prevailed, while at the end of fermentation mainly Saccharomyces species with high ethanol tolerance were present.
BAs were analyzed with the HPLC-UV method. In all apple juice/cider samples 4 different BAs (putrescine, cadaverine, histamine, and tyramine) were detected and quantified with external calibration. On average in all cider samples from the producers total BA (summation of all BAs) concentration reached 9,45 mg/L, however in one case even 25 mg/L. Tyramine was the most abundant BA in all fermentation stages. 40 isolated yeast strains were further tested for BA formation in a small-scale experiment by fermenting apple juice. Results show that non-Saccharomyces yeasts mainly form histamine (1,68 mg/L) and tyramine (1,30 mg/L), while Saccharomyces yeasts putrescine (0,48 mg/L) and tyramine (3,53 mg/L). As a general conclusion, the occurrence and distribution of BA concentrations in the small-scale fermentation were lower (2,96 mg/L and 4,01 mg/L) and less variable than in the real ciders (average in final ciders 9,45 mg/L) and with tyramine being the most abundant BA in all samples analyzed. Ključne besede: cider, yeast, biogenic amines, Hardanger, biodiversity Objavljeno v RUNG: 23.06.2023; Ogledov: 1761; Prenosov: 7 Povezava na datoteko Gradivo ima več datotek! Več... |
2. Novel analytical approaches in quality and safety control in production of fermented beverages : dissertationJelena Topić, 2022, doktorska disertacija Opis: The exploitation of microorganisms for fermentation goes back centuries. Two types of fermentation are usually used in the winemaking process – alcoholic fermentation and malolactic fermentation. Nowadays, inoculated fermentations with the use of starter cultures are commonly used in order to produce wine with more consistent quality. However, wines can lack in flavour complexity, so scientists and the industry are constantly looking for new and improved starters that can be adapted to different types of wine.
In this work we focused on the development and implementation of novel analytical methods for wine quality control. In the course of method development native yeasts and lactic acid bacteria isolates were characterized for wine starter properties. We focused on the determination of biologically active compounds that determine wine quality and safety. Yeasts can influence wine colour through their adsorption capacity and synthesis of stable colour pigments pyranoanthocyanins and lactic acid bacteria can produce biogenic amines which can have adverse detrimental health effects on sensitive consumers when they are present in wines. Ključne besede: Saccharomyces yeasts, non-Saccharomyces yeasts, pyranoanthocyanins, thermal-lens spectrometry, lactic acid bacteria, biogenic amines, thin layer chromatography, dissertations Objavljeno v RUNG: 18.02.2022; Ogledov: 3741; Prenosov: 152 Povezava na celotno besedilo Gradivo ima več datotek! Več... |
3. Determination of biogenic amines formation by autochthonous lactic acid bacteria from ‘Refošk’ grapes using different analytical methodsJelena Topić, Lorena Butinar, Martina Bergant Marušič, Dorota Korte, Branka Mozetič Vodopivec, 2022, izvirni znanstveni članek Opis: Different analytical methods were tested and optimized for the determination of four biogenic amines (BA) histamine, putrescine, cadaverine and tyramine produced by grape-associated lactic acid bacteria (LAB). Autochthonous LAB were isolated from ‘Refošk’ grapes belonging to Slovenian-Italian Karst region as they represent a potential pool of beneficial LAB starter bacteria for improving the typicality and quality of wine. Six isolated strains were screened by multiplex PCR, of which four strains were positive for BA-forming genes (MKBT-49, MKBT-282, MKBT-568, MKBT-570). The production of BA was evaluated by thin-layer chromatography (TLC), high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) and enzymatic method. The HPLC-DAD results showed that strain MKBT-49 (tyrdc+) had high tyramine production (386.6 ± 0.14 mg/L), which was confirmed by TLC. The ability to produce putrescine was confirmed in strain MKBT-282 by PCR, HPLC-DAD (16.4 ± 1.72 mg/L), and TLC. Histamine-producing ability was detected in strain MKBT-570, with a concentration below the limit of detection of the HPLC-DAD (<0.2 mg/L), while the other two methods were not sensitive enough for confirmation. This study shows that the production of BA can detected in native LAB and that relatively simple method such as TLC can be used effectively for the initial screening. Ključne besede: grapes, wine-like matrices, biogenic amines, autochthonous lactic acid bacteria, analytical methods Objavljeno v RUNG: 03.01.2022; Ogledov: 2545; Prenosov: 10 Povezava na celotno besedilo Gradivo ima več datotek! Več... |
4. Determination of biogenic amine-forming potential of naturally occuring lactic acid bacteria (LAB) strains isolated from the grapes of Vitis vinifera cv. Refošk grape varietyJelena Topić, Lorena Butinar, Martina Bergant Marušič, Dorota Korte, Branka Mozetič Vodopivec, 2020, objavljeni povzetek znanstvenega prispevka na konferenci Opis: Biogenic amines are naturally occurring compounds that have been reported in variety of food, such as fish, meat, cheese, vegetables, and wines. When biogenic amines are formed by microorganisms such as lactic acid bacteria (LAB) they may have adverse effect on human health. The most common biogenic amines found in foodstuff are histamine, tyramine, putrescine and cadaverine. LAB are used as starters in the production of several foods and beverages. In wines they are used in malolactic fermentation, where the conversion of malic acid to lactic acid occurs. The aim of this work was to determine biogenic amine-forming capacity of indigenous LAB strains isolated from grapes which were PCR positive and screening medium positive for biogenic amines. Three methods for the confirmation of LAB biogenic amine-forming potential were developed and compared: HPLC-DAD and TLC methods both using derivatization agent dansyl chloride and enzymatic method using horseradish peroxidase (HRP) and diamine oxidase (DAO) enzymes. The applicability of methods was tested on the three known biogenic amine-producing LAB strains: 0006, 9809, 9906 (IOEB, France). The limit of detection (LOD) of the methods was <1 mg/L for HPLC-DAD, 5 mg/L for TLC and 25 mg/L for enzymatic method. Although the LODs between the proposed methods differed, all of the three methods confirmed BA-forming potential of the three strains. Furthermore, the proposed methods were applied for the analysis of three indigenous strains: MKBT-282 (BA+), MKBT-307 (BA-) and MKBT-325 (BA+). The positive or negative BA-forming potential was confirmed with all of the three proposed methods. Ključne besede: lactic acid bacteria, biogenic amines, histamine, grape Objavljeno v RUNG: 10.07.2020; Ogledov: 3790; Prenosov: 0 Gradivo ima več datotek! Več... |
5. Occurence of biogenic amine - producing lactic acid bacteria in Refošk grape and wineJelena Topić Božič, Jan Reščič, Martina Bergant Marušič, Darja Lisjak, Branka Mozetič Vodopivec, Lorena Butinar, 2019, objavljeni povzetek znanstvenega prispevka na konferenci Opis: Lactic acid bacteria (LAB) are naturally present in grapes, musts and wines. During malolactic fermentation, besides the beneficial conversion of L-malic to L-lactic acid, LAB can form also other compounds like biogenic amines (BAs). BAs are formed through decarboxylation of corresponding amino acids, through the action of enzymes. Consumption of food containing BAs can lead to food poisoning such as histamine poisoning. Histamine, the most studied BA, is known to cause headaches, oedema, vomiting.
In the last years, BAs associated pathways have been described as strain dependent and not as species dependent. Because of the high variability of microorganisms to decarboxylase amino acids, the detection of bacteria that have the possibility to transform precursor amino acid into BAs is very important in order to estimate the risk of BAs accumulation in wines. Since in previous years during project AGROTUR I we detected the higher BA content in some wines, we decided to focus on the presence of BA-producing LAB in Refošk grapes and wines. Therefore, during AGROTUR II project LAB collection of autochthonous lactic acid bacteria was set-up by isolating LAB from Refošk grapes and wines, originated from the grape growing cross-border region Karst (Slovenia). Over 600 isolates were obtained using MRS medium with added cycloheximide and 2% tomato juice. In parallel, also selective enrichment was performed using MRS medium described above with additionally added 5% ethanol. BA-producing LAB were primarily detected in microtiter-plate format using the decarboxylase screening medium with added amino acids (histidine, lysine, ornithine or tyrosine), which enable us to identify histamine-, cadaverine-, ornithine and tyrosine-producing LAB. Selected representatives from each BA-producing LAB groups were further on confirmed with chromatographic analysis (HPLC) and molecular methods (multiplex PCR method). BA-producing LAB were in majority represented by thyramin-producers, thereafter followed cadaverine-producers, with putrescine and histamine producers being the least presented. This screening of the BA-producing LAB is also incorporated in our on-going accurate selection of LAB starters for potential production of Refošk wines. Ključne besede: biogenic amines, lactic acid bacteria, refošk, grape, wine Objavljeno v RUNG: 22.01.2020; Ogledov: 4114; Prenosov: 0 Gradivo ima več datotek! Več... |
6. Characterization of lactic bacteria for biogenic amine formationJelena Topic, Lorena Butinar, Martina Bergant Marušič, Dorota Korte, Branka Mozetič Vodopivec, 2018, samostojni znanstveni sestavek ali poglavje v monografski publikaciji Opis: Biogenic amines are compounds present in many different foods and beverages (wine, beer, dairy products, fermented vegetables and soy products, fish, etc.). Their presence in foodstuff is a result of a microbial action during storage and ageing. The most important are histamine, tryptamine, β-phenylethylamine and tryptamine, which can induce undesirable physiological effects in humans. They are formed through decarboxylation of corresponding amino acids, through the action of enzymes. Consumption of food containing biogenic amines can lead to food poisoning such as histamine poisoning. Histamine, the most studied biogenic amine, is known to cause headaches, oedema, vomiting, etc. [1]–[4]. Monitoring of the content of biogenic amines in foods is of concern for public health in their relation to the food safety, food spoilage and food intolerance. Because microorganisms are used in food productions as starters and biopreservers, characterization of microorganisms for their ability to produce biogenic amines is equally important. Lactic acid bacteria are often used as biopreservers as they can produce antimicrobial metabolites and antifungal peptides. Some strains can also produce undesirable biogenic amines [5]. In order to use lactic acid bacteria as starters or biopreservers, the selection of strains that would not produce biogenic amines is necessary.
When considering studies of biogenic amines in foods, focus should be on developing new or improving analysis methods for biogenic amines detection. Secondly, the connections between microorganisms capable of producing biogenic amines and the content of biogenic amines in foods should be investigated [3]. The most widely technique used for quantification of biogenic amines in foodstuff is liquid chromatography, Alternatively to chromatographic techniques, other techniques such as enzymatic biosensors, ELISA and flow-injection analysis have also been employed. Sensors are interesting due to the fact that they do not require special instrumentations, and there is no need for sample clean-up and derivatization, which are the main drawback of chromatographic methods [4]. Detection of biogenic amines producing lactic bacteria is important due to the concerns for public health and there is a need for the early and rapid detection of such microorganisms. Most of the methods that are used for screening involved the measurement of amino acid-decarboxylase activity, although there were been some methods reported that used differential media and pH indicators. Nowadays, molecular methods are replacing culture methods. Molecular approaches are used to determine the presence or absence of genes responsible for biogenic amines formation. The main advantages of DNA hybridization and PCR methods are speed, simplicity, sensitivity and specificity as they allow detection of targeted genes. Culture independent methods which are based on PCR techniques are now regarded as most suitable methods for screening isolates [5].
[1] A. R. Shalaby, “Significance of biogenic amines to food safety and human health,” Food Res. Int., vol. 29, no. 7, pp. 675–690, Oct. 1996.
[2] J. M. Landete, S. Ferrer, and I. Pardo, “Biogenic amine production by lactic acid bacteria, acetic bacteria and yeast isolated from wine,” Food Control, vol. 18, pp. 1569–1574, 2007.
[3] F. B. Erim, “Recent analytical approaches to the analysis of biogenic amines in food samples,” TrAC - Trends in Analytical Chemistry, vol. 52. pp. 239–247, 2013.
[4] J. L. Ordóñez, A. M. Troncoso, M. D. C. García-Parrilla, and R. M. Callejón, “Recent trends in the determination of biogenic amines in fermented beverages – A review,” Analytica Chimica Acta, vol. 939. pp. 10–25, 2016.
[5] R. M. Elsanhoty and M. F. Ramadan, “Genetic screening of biogenic amines production capacity from some lactic acid bacteria strains,” Food Control, vol. 68, pp. 220–228, Oct. 2016. Ključne besede: lactic bacteria, biogenic amines Objavljeno v RUNG: 13.12.2018; Ogledov: 4419; Prenosov: 0 Gradivo ima več datotek! Več... |
7. The detection and study of biologically active compounds in environmental processes and samplesMojca Žorž Furlan, doktorska disertacija Opis: Environmental pollution in the 21th century still represents a global problem for human and animal health. Despite general awareness about released substances and their degradation products their fate and possibilities of removal are not well investigated. Even though the chemicals released are dispersed and diluted in water cycles, their poor biodegradability and/or strong accumulation can result in the intoxication of exposed organisms. Similarly, as a part of the environment, food can get contaminated by bioactive substances during different steps of preparation. Not only artificial compounds such as pesticides or pharmaceuticals, but also natural toxins enter the food chain and impact negatively on humans' and animals' health. In addition, the activity of some bacteria can influence the production of amines from amino acids after fermentation, to which the human body responds with several symptoms of intoxication.
Several analytical methods for the determination of trace levels of broad range contaminants have been developed. Due to the largely robust, selective and sensitive features of the conventional (rearguard) techniques, they represent the first choice for analysing multiple organic compounds in frequently very complex matrices. However, screening (vanguard) methods are paving the way in the chemical analytics as a solution that provides simplicity and rapid analytical responses with binary (yes/no) answers. They require little or no sample treatment as well as more economically-efficient instrumentation. The combination of vanguard-rearguard analytical strategies hence offers a compromise between classical analytical figures of merit and productivity-related characteristics.
In the first part of our research feasibility studies for the application of TLS and/or TLM in novel analytical methods for the determination of lipid-lowering drug atorvastatin and a mycotoxin ochratoxin A . The survey on atorvastatin performed spectrophotometrically has shown a decrease of ATV-sulpho-vanillin product at the wavelength of its maximum absorbance after dilution by organic solvent, which was investigated due to the possible increasing of the method sensitivity. As the predicted LODs that could be obtained by TLM (0.3 mg/L) could not reach the concentration of ATV usually present in the environment (ng/L-g/L) further experiments on this subject were therefore not justified. On the other hand, the ELISA assay for the determination of ochratoxin A was performed. In case of μFIA-TLM, the measurements were influenced by high background signal resulting in high LODs of TLM (470 pg/mL), which is known as a background limited technique. It was estimated that the LODs of standard ELISA assay could not be significantly improved, therefore no further research was conducted in this direction.
In the second part of the dissertation, a sensitive rearguard system by coupling HPLC and TLS for the determination of biogenic amines in wine samples was developed. Putrescine, cadaverine, histamine and tyramine were separated and detected on a HPLC-TLS system after derivatization by dabsyl chloride. The method was optimized in terms of chromatographic conditions and in terms of TLS parameters. Also, the sensitivity of the newly developed method was evaluated by comparing the TLS detection with DAD detection in terms of LOD values, where TLS showed 3.6-fold improvement compared to DAD. Afterwards, the standard addition calibration was performed and evaluated for its recoveries (86−117%) in the determination of the four BAs. The applicability of the novel method was tested by the analysis of real white and red wine samples and by comparing the results to the standard HPLC-FL method and concentrations of BAs in wine samples were in good accordance. In addition, the dabsylated BAs showed better stability compared to the OPA derivatives as they have not lost the peak intensity after 17h of storage.
In the third part, a vanguard system for detection of the overall biogenic amines concentration was developed by employing μFIA-TLM. Initially, NH4Cl standard solutions were applied in the indophenol reaction for batch mode, off-line μFIA-TLM and in an on-line indophenol formation for μFIA-TLM detection. By adding 50 % of EtOH to indophenol we obtained 9-fold improvement. In addition, indophenol showed good stability under TLM conditions. We optimized the microfluidic and TLM parameters in the off-line and on-line indophenol reaction. The addition of 5% ethanol to the reagent in the on-line reaction resulted in the 3-fold improvement of the signal-to-noise ratio. Further on, the overall reaction, including the enzymatic and the following indophenol reaction, was optimized by choosing the optimal buffer (pH=7, 0.5 M) and alkaline conditions (2M NaOH). The influence of interferences from amino compounds was also evaluated and discussed. The off-line and on-line μFIA-TLM were evaluated by their performance characteristics. The LOD for ammonia detection reached 2.3 μM and the applicability in ammonia detection in water samples was discussed. Similar LOD of 3.2 μM was obtained for the overall concentration of BAs and LOD of 3.8 μM for histamine, which is more than 4-folds lower value as the lowest suggested limits of intake for histamine in wine samples (2 mg/L; 18 μM). Finally, an immobilization procedure on magnetic nanoparticles was developed for the possible implementation of the selected enzyme in a miniaturized biosensor. Ključne besede: thermal lens spectrometry, thermal lens microscopy, high performance liquid chromatography, microfluidics, biogenic amines, microbial transglutaminase, indophenol (Berthelot) reaction Objavljeno v RUNG: 04.06.2018; Ogledov: 6735; Prenosov: 251 Celotno besedilo (2,90 MB) Gradivo ima več datotek! Več... |
8. NOVEL APPROACHES FOR THE DETERMINATION OF BIOGENIC AMINES IN FOOD SAMPLESMladen Franko, Mojca Žorž, Mitja Martelanc, Sara Budal, 2017, izvirni znanstveni članek Opis: In this work, new analytical approaches for determination of biogenic amines in wines were developed.
For the first time, we studied the derivatization of BAs in wines with naphthalene- 2,3-dicarboxaldehyde (NDA) and with dabsyl chloride (DBS) and analysis of derivatized BAs by HPLC coupled to fluorescence (HPLC-NDA-FL) and thermal lens spectrometry (HPLC-DBS-TLS) detectors. The sensitivity of the two methods (LODs HPLC-NDA-FL in the range 27-73 μg/L; LODs HPLC-DBS-TLS in the range 3.4-11 μg/L) was higher than that of the official method for biogenic amines in wines, OIV-MA-AS315-18 (60-77 μg/L). For its best performances, the HPLC-DBS-TLS technique was applied to the analysis of putrescine, cadaverine, histamine and tyramine in two white wine samples. Additionally, exploiting the Berthelot reaction, the TLS fast screening of biogenic amines in wines, following the release of ammonia by transglutaminase, was also proposed. This approach allowed us to determine total biogenic amount content in concentrations below 0.1 mg/L, expressed as equivalents of histamine. Ključne besede: biogenic amines, NDA, liquid chromatography, TLS, fluorescence, wine Objavljeno v RUNG: 02.11.2017; Ogledov: 5237; Prenosov: 278 Celotno besedilo (441,06 KB) |
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10. Determination of biogenic amines by thermal lens microscopic detection of enzymatically released ammoniumMojca Žorž, Mladen Franko, 2016, objavljeni povzetek znanstvenega prispevka na konferenci Opis: Biogenic amines (BAs) are organic amines present in meat, fish, dairy produce and wine due to the breakdown of amino acids, catalysed by microbial decarboxylases. BAs determination in food is important not only because of possible toxicological effects such as nausea, sweating and headache but also due to their possible role as indicators of food spoilage. Chromatographic methods are traditionally applied for determination of BAs in food [1], which usually require preliminary operations for sample pre-treatment that are laborious and difficult to automate. On the other hand, screening analytical systems provide simple, low cost and rapid analysis with the possibility of subjecting high number of samples to the screening system in a short time [2]. In this work we present a novel method for screening determination of BAs using a microfluidic system with the detection by highly sensitive thermal lens microscope (μFIA-TLM).
Four biogenic amines (putrescine, cadaverine, histamine and tyramine) were subjected to enzymatic catalysis by transglutaminase, where ammonia was released as a product of acyl transfer reaction between the peptide bound glutamine (Gln) and the amino group of BAs. Ammonia was further transformed into indophenol blue by the Berthelot reaction. The coloured product was detected in batch (static) mode in a 100 μm sample cell or in μFIA (flowing) mode in a microchip with the same optical path length. The detection was performed on a TLM system applying a solid-state diode as an excitation source (660 nm). Organic solvents were tested for signal enhancement.
For evaluation of the sensitivity and determination of LOD values (S/N = 3 basis), the NH4Cl standard solution was applied in Berthelot reaction with further detection on TLM system. The LODs for NH4+ in batch mode and in μFIA were 24 μg/L and 109 μg/L, respectively. Both LOD values are lower than the LOD achieved with conventional spectrophotometry (180 μg/L). When mixtures of the indophenol standard solutions and EtOH in the ratio of 1:1 were prepared the LOD in batch mode was improved to 3 μg/L, achieving 60-times improvement compared to spectrophotometry. Ključne besede: Biogenic amines, Thermal lens microscopy, microfluidics, translgutaminase Objavljeno v RUNG: 05.07.2016; Ogledov: 6036; Prenosov: 0 Gradivo ima več datotek! Več... |