Evaluation of physicochemical, sensory, and antimicrobial properties of small-scale produced fruit vinegars


 Hungarian fruit vinegars were characterised in terms of physicochemical attributes (total polyphenol content, antioxidant characteristics/FRAP, CUPRAC, ABTS/, ascorbic acid content, pH, total soluble solids), sensory profiles, and antimicrobial properties.
 Both compositional and sensory profiles showed distinct patterns depending on the type of vinegar (Tokaj wine, balsamic or apple) and the additional fruit used. Balsamic vinegars maturated on rosehip, sea buckthorn, and raspberry showed outstanding antioxidant performances. Rosehip, raspberry, and quince vinegars, as well as vinegars produced from Tokaji aszú and balsamic apple obtained high scores for fruity and sweet notes.
 Antimicrobial activities were tested on Gram-negative and Gram-positive organisms, including probiotic bacteria. Generally, only weak activities were obtained, which was attributed to the natural sugar content of the samples, depending on the type of the vinegar and the fruit. Similar results, but more pronounced bacterial growth inhibitions were obtained for probiotic strains, however, some probiotic strains were resistant to at least two of the vinegars. Based on these, balsamic apple, raspberry, rosehip, quince, and sea buckthorn may qualify as potential functional components of probiotic preparations containing some of the strains tested.


INTRODUCTION
Being more than a versatile food ingredient, its bioactive components maintain vinegar in the focus of current research. Recently, the demand for fruit vinegars has increased due to their reputation as health-promoting food products, fitting into the global trends demanding enrichment of food with natural antioxidants (Dziki et al., 2014). Within fruit vinegars, small-scale produced vinegars represent a valuable quality category, enabling the selection of specific raw material and adequate technology appropriate to local conditions and requirements . Fruits are used not only for flavour, aroma, and colour addition, but also as precious sources of phytochemicals, including phenolic compounds. In addition to products fermented directly from fruits as carbohydrate sources, vinegar-derived condiments obtained by maceration with fruits are gaining an increasing interest due to the various polyphenols, micronutrients, and other bioactive compounds present in these vinegars (Zhang et al., 2020). Phenolics transferred from fruits into vinegars considerably enhance the antioxidant potential of the product (Liu et al., 2019;Stagos, 2020). Fruit polyphenols may also exert their specific antimicrobial effects in vinegars, proven on various microorganisms, including food-borne pathogens like Escherichia coli and Salmonella Typhi (Priyadarshini et al., 2014;Soorya et al., 2019).
Next to their health-promoting properties, fruit-derived compounds also define the main sensory attributes of vinegars, thus playing a key role in formation of taste, aroma, and colour (Cejudo-Bastante et al., 2016;Boonsupa, 2019). Nevertheless, only limited information is available on the comparative sensory evaluation of vinegars maturated on different fruit beds.
In the present research, a range of selected small-scale domestic vinegar products were studied in terms of their physicochemical, antioxidative, sensory, and antimicrobial properties.

Samples
A total of 8 wine-or cider-based small-scale vinegars from the Hungarian Tokaj region were analysed: a group of vinegars maturated on fruit bed (quince -TQ, rosehip -TRH, raspberry -TRB, sea buckthorn -TST) and a group of naturally fermented vinegars (cider -TC) including balsamic ones (balsamic apple -TAB, Tokaj aszú -TA) and a thyme-infused product (TT). These were compared to commercial samples (plain vinegar -V, white balsamic vinegar -WB, cider vinegar -OC, red wine vinegar -RW) (Supplementary Table 1S). Wine-or cider-based Tokaj vinegars were naturally fermented and maturated for a minimum of 6 months in oak barrels. In the case of balsamic vinegars, concentrated grape or apple musts were added to the base vinegars upon maturation. For maceration on fruit beds, maturated vinegars were subsequently transferred to stainless steel reservoirs, where fresh fruits were added in amounts according to the individual recipes of the manufacturer, then maturation on fruit beds (or thyme) was accomplished for 1-2 months. Results are reported for 1-1 bottle per sample, however, antioxidant measurements were performed on more bottles for most of Tokaj vinegars (Supplementary Table S2). Vinegars from Tokaj were kindly donated by Borecet Művek Ltd., Bodrogkeresztúr.

Analytical methods
Analytical measurements were performed in five replicates (except for vitamin C contenttwo replicates). For spectrophotometry, a Thermo Helios Alpha UV-VIS instrument (±0.001 units of absorbance, 1 cm light path) was used. Proper dilutions (10-or 100-fold) were applied for antioxidant assays.
2.3.1. Total polyphenol content, ferric reducing antioxidant power (FRAP), cupric reduction antioxidant capacity (CUPRAC), and ABTS assays. Total polyphenol content was evaluated following a method adapted from Singleton and Rossi (1965). In FRAP experiments, the procedure described by Benzie and Strain (1996) was adapted. ABTS antioxidant capacity was performed according to Re et al. (1999). CUPRAC assay was accomplished according to Apak et al. (2007).
2.3.3. Acetic acid content. Acetic acid content for small-scale vinegars was determined by titration of acidity with 0.1 N NaOH against phenolphthalein, reporting the results as m/V % acetic acid.

Sensory analysis
Tokaj vinegar samples (50 mL of each) were presented to panel members undiluted, in plastic cups, with 3-digit random codes. Two groups of samples were set up based on their production technology: vinegars maturated on fruit beds (4 samples) and other vinegars (4 samples). Twelve trained panellists were given a complete list of the pre-defined taste and aroma descriptors (see Figs 1 and 2) and were asked to score them on scales from 0 to 100. Measurements were performed in two replicates. Neutral mineral water was used as taste neutraliser. Sensory tests were carried out meeting all criteria of ISO 13299 (ISO, 2016). For determination of sensory Acta Alimentaria profiles, the intensity values for the reference samples (sea buckthorn and cider vinegars) were pre-defined in both groups for each attribute on the evaluation scales.

Microbiological analysis
Antimicrobial activity of vinegars was tested on both Gram-positive (Staphylococcus aureus ATCC 6538, Enterococcus faecalis T1, and Listeria monocytogenes CCM 4699) and Gram- negative (E. coli T1, Proteus mirabilis T1) spoilage and pathogenic bacteria. The following probiotic strains were also tested: Lactobacillus acidophilus LA-5, Lactobacillus casei 01, Lactobacillus acidophilus 150 Exqium, and Lactobacillus plantarum 299V. Details regarding the origin of the strains used are presented in Supplementary material Table S3. Agar well diffusion tests were performed on CASO agar plates for non-probiotic strains and MRS agar plates (20 mL/plate) for probiotic strains. Plates were inoculated with a 10 6 CFU mL À1 initial count inoculum of the test microorganism by spread plating. The volume of inoculum was 0.1 mL. The wells were prepared by punching the inoculated plates with a 5 mm diameter standard sterile stainless steel cork borer. The wells were filled up with 50 mL of each vinegar sample, and the plates were incubated at 37 8C. After 24 h of incubation, the diameter of the zone of inhibition was measured using a ruler, then results were quoted by subtracting the diameter of the well and dividing the results by two. Interpretation of inhibition zones of test culture was adopted from Priyadarshini et al. (2014)

Statistical methods
Pearson correlation (P 5 0.05) was used for verifying correlations between antioxidant properties, significant differences were checked by one-way analysis of variance (ANOVA) completed by Tukey's post-hoc test (https://www.statskingdom.com/180Anova1way.html). Sensory tests were evaluated by ProfiSens v. 2012 software (Kókai et al., 2002). Profile analysis was evaluated by one-way ANOVA (P 5 0.05) followed by LSD (least significant differences) post-hoc tests. Principal Component Analysis (PCA) on sensory data was performed with the Past statistical package (Hammer et al., 2001).

Polyphenol content and antioxidant capacity
More in vitro assays (Folin-Ciocalteu TPC, FRAP, CUPRAC, and ABTS) were used for a complex characterisation of the antioxidant properties of the vinegars (Table 1)

Ascorbic acid content
The highest ascorbic acid contents were measured in rosehip followed by sea buckthorn and raspberry, showing the valuable contribution of fruit extracts, partly still preserved in the final products. Vitamin C in vinegars originates exclusively from fresh fruits, it has not been detected in any of the other Tokaj vinegars (Table 1). Ascorbic acid content is also strongly correlated with antioxidant capacity (correlation coefficients: 0.82, 0.89, and 0.90 for FRAP, CUPRAC, and ABTS, respectively, P 5 0.05). Our results also correlate with those obtained for a papaya-based fermented product (2.32 mg/100 mL) (Kong et al., 2018).

Sensory analysis
Sensory characteristics of vinegars result from the complex effect of raw materials, technology and various biochemical processes, as parts of a dynamic equilibrium (Cejudo-Bastante et al., 2016). Sensory profile of vinegars maturated on fruit bed (see Supplementary material, Fig. 1S) showed that vinegar-like aroma and flavour, as well as sour flavour were the most pronounced for sea buckthorn vinegar (TST; even though its acidity was not higher than of the other samples), their values having been significantly higher (P 5 0.05) than those obtained for quince (TQ), and for raspberry (TRB) (vinegar-like flavour) or rosehip (TRH) (vinegar-like aroma) samples or both of these (sour taste). These findings are also supported by the PCA biplot (Fig. 1), where these attribute vectors point towards the quadrant where the TST sample is. Fruity flavour, fruity aroma, and sweet taste were the most intensive for TQ. Both TRB and TRH showed significantly higher scores (P 5 0.01) than TST, however, TRH was less fruity than TQ (P 5 0.05). The same applies for fruity aroma, where values were significantly higher for TQ and TRB compared to TST (P 5 0.01 and P 5 0.05, respectively). Mild sweet-caramellic aroma and taste were the strongest for TRB and TRH, both preceding TST (P 5 0.01 and P 5 0.05, respectively). TQ was found to be the sweetest (significantly sweeter than TST and TRH, P 5 0.05) and the least sour. A significant dominance of caramellic flavour was noticed for TRB and TRH over TST (P 5 0.01 and P 5 0.05, respectively). Sensory profile of vinegars not maturated on fruit bed (see Supplementary material, Fig. 2S) showed much higher differences, reflecting the different character of the samples; i.e. cider (TC), balsamic apple (TAB), Tokaj aszú (TA), and thyme-flavoured (TT) wine vinegars. Although TT had the highest acidity, vinegar-like notes and sour taste were the most pronounced for TC (P 5 0.01, Fig. 2). The same applies for wine-like aroma (P 5 0.01). On the other hand, sweet-caramellic aroma and taste and sweet taste were the most pronounced for TA, significantly more intense than for TT and TC (P 5 0.01). Fruity notes were the strongest for TAB, both aroma and taste were significantly more intensive than in the case of TC and TT (P 5 0.01).

Microbiological analysis
3.4.1. Antimicrobial activity of vinegars against selected spoilage organisms and pathogens. Antimicrobial activity of vinegars was tested on Gram-positive and Gram-negative spoilage and pathogenic bacteria. The results (Table 2) showed that all vinegars possessed antimicrobial activity against all bacteria, however, only a few microorganisms were highly sensitive to some of the vinegars tested. Gram-positive bacteria, especially L. monocytogenes and E. faecalis, were the most susceptible, while both Gram-negative species were less sensitive. Although vinegar is reported to be generally efficient against many microorganisms (e.g. L. monocytogenes, S. aureus, and E. coli (Medina et al., 2007)), these effects were not proven for the fruit vinegars tested, even though many of the polyphenols transferred from fruits have their own antimicrobial effects as well, although this effect is strain-dependent (Bouarab-Chibane et al., 2019). The relatively weak antimicrobial activity of these products is attributed to the antagonistic effect induced by the fairly high sugar content (from both maceration on fruits and addition of concentrated musts). This sugar content is reflected in the Brix values, which correlate with the antimicrobial properties, i.e. the highest inhibition zones were obtained for cider and thyme-flavoured vinegars, these having the lowest Brix values and containing neither fruit extracts nor fruit must. The stronger antimicrobial effect of thyme-flavoured vinegar can also be explained by its active compounds (carvacrol and thymol) (Kiskó and Roller, 2005).

Antimicrobial activity of vinegars against selected probiotics.
Based on the weak antimicrobial effects observed above, fruit vinegars were tested on probiotic strains as well (Table 3). Our main goal was to select probiotic bacteria not susceptible to the action of these products and thus, to be able to utilise the potential of fruit vinegars as functional ingredients in probiotic food preparations. In the case of Lactobacillus strains the effect of sugar content was also observed, similarly to spoilage and pathogenic strains, while cider and thyme-flavoured vinegars had the most inhibitory effects against probiotic strains. Due to the known higher sensitivity of probiotics, the inhibition zones were generally larger, especially for L. casei 01, where thyme vinegar reached a moderate antimicrobial effect. However, most of the vinegars exerted only a weak antibacterial effect on most Lactobacillus strains. The most promising results were obtained for L. acidophilus 150 and L. plantarum 299V, which were practically resistant to at least two of the vinegars used (raspberry and sea buckthorn; balsamic apple, quince and sea buckthorn, respectively). Quince vinegar showed no growth inhibition on L. acidophilus LA-5 either. Thus, some of these probiotic strains can be used successfully in functional foods enriched with selected fruit vinegars as valuable sources of phytochemicals and minerals, while also delivering additional flavour and aroma.

CONCLUSIONS
High polyphenol contents and antioxidant capacities were detected in vinegars maturated on fruits with exceptionally high values obtained for rosehip balsamic vinegar followed by raspberry and sea buckthorn products. Sensory properties were determined by the type of the fruit macerated: sea buckthorn delivered vinegar-like notes, sourness; raspberry and rosehip provided sweet-caramellic taste and aroma, whereas fruity notes dominated in quince-based vinegar. For vinegars not maturated on fruit bed, the highest vinegar-like scores were obtained for cider vinegar. Vinegar enriched with Tokaj aszú showed a pronounced sweet-caramellic flavour and aroma, while balsamic apple vinegar was dominated by fruity notes. Microbiological analysis revealed that fruit-containing vinegars exert only weak antimicrobial actions against the selected food spoilage and foodborne pathogens, this diminished effect being attributed to their sugar content (from either fruit or must concentrate). Their antimicrobial effects were generally more pronounced on probiotic strains, though some of these showed promising resistance in the presence of vinegars maturated on fruits.
In addition to their versatile sensory features, the remarkable nutritional assets of small-scale vinegars highlighted here (especially those maturated on fruit beds and, to some extent, balsamic vinegars), i.e. high polyphenol content and antioxidant capacity or even ascorbic acid content for the fruit-bed ones, qualify these products as valuable food ingredients, contributing to dietary antioxidant intake. Moreover, some of the vinegars maturated on fruit beds may have a potential as functional additives in probiotic food preparations.