Construction of IsoVoc Database for the Authentication of Natural Flavours.

Flavour is an important quality trait of food and beverages. As the demand for natural aromas increases and the cost of raw materials go up, so does the potential for economically motivated adulteration. In this study, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis of volatile fruit compounds, sampled using headspace-solid phase microextraction (HS-SPME), is used as a tool to differentiate between synthetic and naturally produced volatile aroma compounds (VOCs). The result is an extensive stable isotope database (IsoVoc-Isotope Volatile organic compounds) consisting of 39 authentic flavour compounds with well-defined origin: apple (148), strawberry (33), raspberry (12), pear (9), blueberry (7), and sour cherry (4) samples. Synthetically derived VOCs (48) were also characterised. Comparing isotope ratios of volatile compounds between distillates and fresh apples and strawberries proved the suitability of using fresh samples to create a database covering the natural variability in δ13C values and range of VOCs. In total, 25 aroma compounds were identified and used to test 33 flavoured commercial products to evaluate the usefulness of the IsoVoc database for fruit flavour authenticity studies. The results revealed the possible falsification for several fruit aroma compounds.

Fruit Quality Characteristics and Biochemical Composition of Fully Ripe Blackberries Harvested at Different Times.

We investigated how the quality of blackberry fruit changes during the ripening period. Since it is difficult to predict picking dates for blackberries, we were interested in how the quality of fully ripe fruit changed depending on the sampling date (from 28 July to 1 September). Blackberries (at full ripeness) were sampled at six weekly intervals and the contents of sugars, vitamin C, organic acids and phenolic components were analysed by high performance liquid chromatography combined with mass spectrometry. The colour parameters, total soluble solids and weight of the fruits were also measured. The results showed that the fruits at the last sampling had a significantly lower fruit weight and higher soluble solids. 'Cacanska Bestrna' had the highest fruit weight and vitamin C content (11.43 mg/100 g). The main sugars in blackberries were fructose (13.8-33.4 g/kg FW) and glucose (13.0-33.2 g/kg FW). 'Loch Ness' and 'Navaho' had a sweeter taste since they had the highest ratio of sugars and acids (S/A approx. 5.8) and 'Smoothstem' and 'Thornfree' had the sourest taste, with a ratio of S/A 2.5. Blackberries harvested at the first two samplings had lower anthocyanin contents than fruits from later sampling times. There were no significant differences in the content of flavonols, ellagitannins, flavanols or hydroxycinnamic acids during the ripening period. The content of vitamin C in the fruits did not change among samplings, but the fruits had a higher content of organic acids at the first two or three samplings. The results may be useful for both the processing industry and growers to set quality standards for each variety and to determine the optimal harvest time.

The impact of food processing on the phenolic content in products made from juneberry (Amelanchier lamarckii) fruits.

Sugars, organic acids, and phenolic compounds were analyzed in the fruits of juneberry (Amelanchier lamarckii). Different food products were made from fresh juneberry fruits: jam, liqueur, juice, and tea. A detailed analysis of the phenolic component was performed using high-pressure liquid chromatography coupled with mass spectrometry, and the content of phenolics in different products was compared with the control treatment (fresh fruits extracted with 70% methanol: 27% water: 3% formic acid; v/v/v). Four sugars and six organic acids were determined in the fruits. The main sugars were glucose (61 g/kg fresh weight [FW]) and fructose (64 g/kg FW) and the acids: malic (5.85 g/kg FW) and citric acid (2.6 g/kg) were abundant. Hydroxycinnamic acids, anthocyanins, and flavonol glycosides were the major phenolic groups in juneberry fruits. Fruit processing significantly affected the content of phenolic compounds in the different fruit products. Liqueur had 17% higher phenolic acid contents than the control treatment (fresh fruits extracted with a solution of 70% MeOH/27% H2 O/3% formic acid) and jam had 14% higher content than the control, calculated on the dry mass of fruit. Juneberry juice had the highest content of total analyzed phenolics (298 mg/100 mL), followed by liqueur (108 mg/100 mL) and tea (8 mg/100 mL). Fruits of juneberry are rich in bioactive compounds and a useful source for the food industry for making various health snacks, jellies, marmalades, alcoholic drinks, juices, and so on. PRACTICAL APPLICATION: Juneberry is becoming a desirable organically grown fruit species with many views as new functional food. The content of phenolic compounds has been measured in different food products made from juneberries. The results could be useful for food and nutraceutical industry.

A wild 'albino' bilberry (Vaccinium myrtillus L.) from Slovenia shows three bottlenecks in the anthocyanin pathway and significant differences in the expression of several regulatory genes compared to the common blue berry type.

Relative expressions of structural genes and a number of transcription factors of the anthocyanin pathway relevant in Vaccinium species, and related key enzyme activities were compared with the composition and content of metabolites in skins of ripe fruits of wild albino and blue bilberry (Vaccinium myrtillus) found in Slovenia. Compared to the common blue type, the albino variant had a 151-fold lower total anthocyanin and a 7-fold lower total phenolic content in their berry skin, which correlated with lower gene expression of flavonoid 3-O-glycosyltransferase (FGT; 33-fold), flavanone 3-hydroxylase (FHT; 18-fold), anthocyanidin synthase (ANS; 11-fold), chalcone synthase (CHS, 7.6-fold) and MYBPA1 transcription factor (22-fold). The expression of chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductase (LAR), anthocyanidin reductase (ANR) and MYBC2 transcription factor was reduced only by a factor of 1.5-2 in the albino berry skins, while MYBR3 and flavonoid 3',5'-hydroxylase (F3'5'H) were increased to a similar extent. Expression of the SQUAMOSA class transcription factor TDR4, in contrast, was independent of the color type and does therefore not seem to be correlated with anthocyanin formation in this variant. At the level of enzymes, significantly lower FHT and DFR activities, but not of phenylalanine ammonia-lyase (PAL) and CHS/CHI, were observed in the fruit skins of albino bilberries. A strong increase in relative hydroxycinnamic acid derivative concentrations indicates the presence of an additional bottleneck in the general phenylpropanoid pathway at a so far unknown step between PAL and CHS.

Polyphenol metabolism in differently colored cultivars of red currant (Ribes rubrum L.) through fruit ripening.

MAIN CONCLUSION: Rare red currants colors caused by low anthocyanin content in the pink and a lack of anthocyanins in the white cultivar correlated with low ANS gene expression, enzyme activity, and increased sugar/acid ratios. Changes in the contents of polyphenols, sugars, and organic acids in berries of the three differently colored Ribes rubrum L. cultivars ('Jonkheer van Tets', 'Pink Champagne', and 'Zitavia') were determined by LC-MS and HPLC at 4 sampling times during the last month of fruit ripening. The activities of the main flavonoid enzymes, chalcone synthase/chalcone isomerase (CHS/CHI), flavanone 3-hydroxylase (FHT), and dihydroflavonol 4-reductase (DFR), and the expression of anthocyanidin synthase (ANS) were additionally measured. Despite many attempts, activities of flavonol synthase and glycosyltransferase did not show reliable results, the reason of which they could not be demonstrated in this study. The pink fruited cultivar 'Pink Champagne' showed generally lower enzyme activity than the red cultivar 'Jonkheer van Tets'. The white cultivar 'Zitavia' showed very low CHS/CHI activity and ANS expression and no FHT and DFR activities were detected. The DFR of R. rubrum L. clearly preferred dihydromyricetin as substrate although no 3',4',5'-hydroxylated anthocyanins were present. The anthocyanin content of the red cultivar slightly increased during the last three weeks of ripening and reached a maximum of 890 mg kg-1 FW. Contrary to this, the pink cultivar showed low accumulation of anthocyanins; however, in the last three weeks of ripening, their content increased from 14 to 105 mg kg-1 FW. Simultaneously, the content of polyphenols slightly decreased in all 3 cultivars, while the sugar/acid ratio increased. The white cultivar had no anthocyanins, but the sugar/acid ratios were the highest. In the white and pink cultivars, reduction/lack of anthocyanins was mainly compensated by increased relative concentrations of hydroxycinnamic acids and flavonols.

Do optimally ripe blackberries contain the highest levels of metabolites?

Five blackberry cultivars were selected for the study ('Chester Thornless', 'Cacanska Bestrna', 'Loch Ness', 'Smoothstem' and 'Thornfree') and harvested at three different maturity stages (under-, optimal- and over-ripe). Optimally ripe and over-ripe blackberries contained significantly higher levels of total sugars compared to under-ripe fruit. 'Loch Ness' cultivar was characterized by 2.2-2.6-fold higher levels of total sugars than other cultivars and consequently, the highest sugar/acids ratio. 'Chester Thornless' stands out as the cultivar with the highest level of vitamin C in under-ripe (125.87mgkg(-1)) and optimally mature fruit (127.66mgkg(-1)). Maturity stage significantly affected the accumulation of phenolic compounds. The content of total anthocyanins increased for 43% at optimal maturity stage and cinnamic acid derivatives for 57% compared to under-ripe fruit. Over-ripe blackberries were distinguished by the highest content of total phenolics (1251-2115mg GAE kg(-1) FW) and greatest FRAP values (25.9-43.2mM TE kg(-1) FW).

White versus blue: Does the wild 'albino' bilberry (Vaccinium myrtillus L.) differ in fruit quality compared to the blue one?

Wild albino and blue bilberry fruit were analyzed to compare different fruit characteristics linked to the composition of primary and secondary metabolites. Compounds were identified using HPLC-MS and standard quality parameters were determined. Albino berries were significantly smaller, accumulated less water and were characterized by 23% higher dry weight. Soluble solids content and pH value were higher in albino bilberry and their surface was lighter and characterized by a yellow hue. Both bilberry forms accumulated identical individual sugars and organic acids; however, the albino form had 33% higher content of total sugars and 9% higher content of total organic acids compared to the blue type. Fifteen anthocyanins were identified in both forms, but in albino bilberries, individual anthocyanins were only detected in traces. Blue bilberry contained 1.6-fold higher levels of flavanols, 2.1-fold higher levels of flavonols, 2.5-fold higher levels of hydroxycinnamic acid derivatives and consequently, 4.6-fold higher total phenolic content.

Changes in fruit quality parameters of four Ribes species during ripening.

There are no defined maturity parameters for berry fruit which often results in harvesting and marketing of inedible, immature or over ripe berries. In the present study the changes in quality characteristics of different white-, red-, and black-currant cultivars as well as gooseberry and jostaberry fruit were investigated at three maturity stages. Colour parameters, berry juice pH level and the content of sugars, organic acids and phenolic compounds have been evaluated. A total of 65 different phenolic compounds were detected by HPLC-PDA/MS. The results indicate that, during the last 2-4 weeks of fruit ripening, significant changes occur in colour parameters, primary and secondary metabolites and radical scavenging activity. The results suggest that the average content of specific phenolic groups and sugars/acids ratio can be used as a simple and reliable maturity index for optimisation of harvest time.

Changes in the contents of anthocyanins and other compounds in blackberry fruits due to freezing and long-term frozen storage.

The aim of this study was to evaluate the effect of fast and slow freezing and frozen storage on the metabolite content of six blackberry cultivars. The content of metabolites determined with HPLC RI/PDA-MS in stored blackberries was compared with the initial content of the fruit. During frozen storage of fruits a loss of vitamin C up to 80% has been recorded along with changes of color values, which shifted to blue and yellow hues. The color changes were accompanied with increased pH levels and content of anthocyanins. Most of the phenolic groups, sugars, and organic acids showed a better extraction after storage, especially in the slow freezing treatment due to a higher degree of tissue damage by freezing. The 'Thornless Evergreen' cultivar was especially rich in sugars, vitamin C, and phenolic compounds, but the highest levels of anthocyanins were determined in 'Loch Ness' cultivar.

Alteration of the content of primary and secondary metabolites in strawberry fruit by Colletotrichum nymphaeae infection.

The effects of infection with Colletotrichum nymphaeae , the causative agent of strawberry black spot, were studied on two strawberry cultivars: susceptible 'Elsanta' and tolerant 'Honeoye' cultivars. Four treatments were established: (1) artificial inoculation; (2) spray application of pyraclostrobin + boscalid (Signum); (3) foliar spraying with calcium (Stopit); and (4) control (spraying with water). Primary metabolites (sugars and organic acids) and secondary metabolites (phenolic compounds) were determined in strawberry fruit with the use of HPLC-MS(n). Infected fruit accumulated large amounts of total sugars and low levels of organic acids. The sugar/acid ratio was higher in the infected and in Ca-treated strawberries. The contents of ellagic acid derivatives, flavonols, oligomeric procyanidins, flavan-3-ols, and total phenolics were highest in inoculated strawberry fruit. Results indicated that fungicide and calcium sprayings did not alter polyphenolic levels in plant tissue.

Phenolics in Slovenian bilberries ( Vaccinium myrtillus L.) and blueberries ( Vaccinium corymbosum L.).

Phenolics from bilberries ( Vaccinium myrtillus L.) sampled from seven different locations and highbush blueberries ( Vaccinium corymbosum L.) from one location in Slovenia were analyzed. In samples of both species 15 anthocyanins were identified by LC-MS/MS. Their contents were expressed as cyanidin 3-glucoside equivalents (C3GE); bilberries contained 1210.3 ± 111.5 mg C3GE/100 g fw and blueberries 212.4 ± 14.1 mg C3GE/100 g fw. Glycosides of delphinidin and cyanidin were predominant (488.5 vs 363.6 mg C3GE/100 g fw) in the bilberries and glycosides of malvidin (108.0 vs 100.8 mg C3GE/100 g fw) in the blueberries, whereas the contents of peonidin were lowest (74.5 vs 4.8 mg C3GE/100 g fw) in both berries. The contents of flavanols, flavonols, phenolic acids, and stilbenes were determined by LC-MS. For the first time, rutin was identified (bilberries, 0.2 ± 0.0 mg/100 g fw; blueberries, 3.1 ± 0.1 mg/100 g fw). Chlorogenic acid (as 3-caffeoylquinic acid) was the most abundant among the phenolic acids (23.1 ± 1.0 mg/100 g fw in bilberries and 70.0 ± 3.4 mg/100 g fw in blueberries). Statistical analysis shows that the content of 27 individual flavonoids, phenolic acids, and stilbenes can be used to identify the picking region of these Slovenian bilberries.