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Abstract: 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.
Keywords: Cider, yeast, biogenic amines, Hardanger, biodiversity
Published in RUNG: 23.06.2023; Views: 362; Downloads: 4
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Keywords: Cider, vino, jabolčni sok, HPLC-UV, Amino kisline, biogeni amini.
Published in RUNG: 20.02.2023; Views: 519; Downloads: 0
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Abstract: In the Hardanger area in Western Norway, the production of cider has a long tradition that goes back to the 12th century, when monks introduced apple growing in this area. Nowadays, this is also the main area of fruit production in Norway. Despite the strict regulation of the alcoholic beverage production in Norway, traditional cider is still produced on some farms in this area. Therefore, our aim was to study the ecology and biodiversity of the yeasts associated with the cider production in the Hardanger area during fermentation process; especially of traditional cider, which is produced by a spontaneous fermentation of apple juice, performed by naturally occurring indigenous yeasts that originate from the fruit or the surfaces of the processing equipment. In our study, samples of fermenting juice/cider were taken during fermentation process from 12 producers, located in 12 different locations in Hardanger region. Classical cultivation methods using WL (Wallerstein Laboratories) agar medium with added chloramphenicol enable us to isolate a total of 530 yeast isolates that were stored in in-house yeast collection at the NIBIO and included also at the Wine Research Centre collection. Based on the sequencing of the D1/D2 domain of the 26S rDNA we managed to identify 357 isolates and distinguished 27 different yeast species as follows: Aureobasidium pullulans, Candida californica, C. oleophila, C, sake, Hanseniaspora meyeri, H. uvarum, H. valbyensis. Kregervanrija fluxuum, Kregervanrija sp., Metschnikowia andauensis, M. chrysoperlae, M. fructicola, M. pulcherrima, Metschnikowia sp, Pichia fermentans, P. kluyveri, P. membranifaciens, P. nakasei, Piskurozyma capsuligena, Rhodotorula nothofagi, Saccharomyces bayanus, S. cerevisiae, S. paradoxus, S. pastorianus, Saccharomyces sp., S. uvarum and Torulaspora delbrueckii. Even though we were not able to obtain samples in three different fermentation stages (beginning, middle and at the end of fermentation) from all producers, we could observe yeast succession during fermentation progress. Yeast diversity was higher at the beginning comparing to the middle of fermentation, when mostly different non-Saccharomyces yeast species prevailed, while in the middle of fermentation 11 species were detected (Candida californica, H. uvarum, H. valbyensis, Kregervanrija sp., K. fluxuum, Pichia membranifaciens, Metschnikowia pulcherrima, Saccharomyces sp, S. bayanus, S. uvarum and S. cerevisie). On the other hand, at the end of fermentation mainly Saccharomyces species with high ethanol tolerance were present (Saccharomyces sp., S. cerevisiae, bayanus, S. uvarum and P. fermentans). In samples that were collected from three producers in all three fermentation stages also quality parameters were determined (ethanol, organic acids, sugars, biogenic amines) with in-house developed methods using HPLC-UV/RID. The most important sugars in ciders were fructose and glucose, as expected. Two producers added sugar to increase the level of ethanol in the middle of fermentation, which is a common procedure in the Hardanger area. Ethanol and organic acid analysis indicated that fermentations went in the right direction, since all parameters were within normal limits. Including the acetic acid level, an indicator of low cider quality, was very low (average around 0,06 g/L). The alcohol incised from the beginning to end fermentation in all samples analysed and minimum concentration was 2,71 g/L. In ciders we detected four biogenic amines (putrescin, cadaverine, histamine and tyramine). The average amount was 32 mg/L and the most abundant was tyramine.
Keywords: indigenous yeasts, biodiversity, spontaneous fermentation, cider-making
Published in RUNG: 18.10.2022; Views: 609; Downloads: 0
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Abstract: Botrytis cinerea Pers., the fungal plant pathogen and the causal agent of gray mould diseases in grapevine, is vastly responsible for substantial economic losses in table and wine grapes production worldwide by negatively affecting plant growth and causing the reduction of grape and wine quality. The conventional approach for pathogen control has been up to date based on synthetic fungicides with good effectiveness against pathogens but a negative impact on the environment. The growing level of harmful residues in the environment and some also detected in wines have led the European Union and many winemakers to limit the application of synthetic fungicides to earlier season. However, with a high risk of disease also late in the season, the need for other solutions is clear. Consequently, more and more research is focused on finding potential alternatives in the form of effective biological control agents. Although there are several reports of yeast’s biocontrol activity, they are up to date still poorly commercialized for such purposes. As the yeasts represent an important part of the grape microflora, competing with other microorganisms (including pathogens) for nutrients and space, we decided to examine the potential of autochthonous yeasts as "green" alternatives in fighting against phytopathogens such as B. cinerea in viticulture. With this aim, we tested the biocontrol activity of 119 different indigenous yeasts belonging to 30 different species of 17 genera against filamentous fungus B. cinerea, the causal agent of grey mould or botrytis bunch rot in grape. The yeasts were screened for putative multidimensional modes of action such as antifungal volatiles (VOC), in vitro inhibition of fungal mycelial growth, competition for nutrients, hydrolytic enzyme activities, and yeast tolerance to fungicides like copper, iprodione and cyprodinil/fludioxonil combination. With a qualitative detection of the hydrolysis activity by using screening methods based on solid medium with chitin or βD-glucosides as substrates, we found that many tested yeasts were capable of producing lytic enzymes with the ability to degrade the cell wall of phytopathogenic fungi and are potentially also able to produce VOCs via hydrolysis of grape glycosides as a result of β-glucosidase presence. Furthermore, we observed the capability of tested yeast to inhibit fungal mycelia growth on plate and assimilation of a wide variety of carbon sources; however, no siderophore producers were detected. In general, the yeasts under observation were tolerant to the tested fungicides. Their fungicide resistance can indeed be regarded as a beneficial trait for potential biofungicide agent (PBA) candidates due to open possibilities of applications and combinations within low input pest management strategies in the vineyard. Finally, a field experiment in Pinot noir and Pinot gris vineyards was designed to study different combinations of optimized canopy microclimate manipulation (CMM) techniques and potential biocontrol agent (PBA) application. In experimental conditions, the ability of PBA’s to maintain appropriate population density for disease prevention was observed. In addition, the grape and wine quality parameters were analysed to observe the possible impact of implemented biocontrol yeast on final products. The biocontrol yeast Pichia guilliermondii ZIM 624 was selected and applied in experimental vineyards based on yeast testing results. We were able to detect and confirm PBA yeast’s suitable density on grapes until harvest. In the case of early defoliation for both varieties lower grape compactness was observed together with lower yield/ plant, regardless of PBA yeast/ no yeast application. Among grape basic quality parameters, the optimized techniques showed a positive effect on sugar content. Still, unexpectedly, in the treatments with biocontrol yeasts some trends toward higher acidity were noticed in Pinot gris.
Keywords: sustainable viticulture, Botrytis cinerea, gray mould, yeasts, biocontrol, canopy microclimate manipulation, grapevine metabolite, dissertations
Published in RUNG: 07.07.2022; Views: 1145; Downloads: 113
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Abstract: 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.
Keywords: grapes, wine-like matrices, biogenic amines, autochthonous lactic acid bacteria, analytical methods
Published in RUNG: 03.01.2022; Views: 1144; Downloads: 6
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