Apples are grown in temperate zones and are a common fruit all over the world. Apples are a rich source of phytochemicals, and epidemiological studies have linked consumption of apples to have positive effects on many lifestyle diseases [1
Apples can be specially grown for fresh consumption or for production of beverages like juice, wine or cider. Cultivar, horticultural practice and year of cropping are important factors determining the chemical composition of apples. The chemical compounds might also respond differently to thermal, mechanical and biochemical processes. Polyphenols are important for flavor and aroma in apples, as well as in processed products. Apple content of phytochemicals, including flavonols (mainly quercetin glycosides), flavan-3-ols (catechins and procyanidins), dihydrochalcones (essentially phloretin glycosides) and hydroxycinnamic acids (mainly chlorogenic acids), depends on cultivar and climatic conditions, especially during maturation of the fruits [2
]. Guyot et al. [3
] found that procyanidins were the most abundant group of polyphenols in apples, both in the flesh and in the peel, while flavonols were mostly found in the apple peel.
The interest in craft cider has grown steadily in the last several years. Knowledge of the fruit quality and variation between cultivars is, therefore, important for choosing the proper raw material. Content of various polyphenols, combined with acidity, defines important quality parameters for apples. These also define the potential use of the various apple cultivars for cider production. The total polyphenol content associated with the total acidity of the must is used to classify French and English cider apple cultivars in different taste categories [4
]. Most of the polyphenols contribute to the final colour of the cider, since they are directly involved in the enzymatic oxidation phenomenon that leads to the formation of coloured oxidation products during apple crushing and pressing. More specifically, tannins (procyanidin oligomers and polymers) are responsible for bitterness and astringency that are important for taste and mouthfeel in those products [3
Content of polyphenols varies between cultivars, year of harvest and postharvest storage conditions [5
]. The most common polyphenols in apples are chlorogenic acid, caffeic acid, procyanidin B2, (−)-epicatechin and quercetin. Apples also exhibit antioxidant activity [6
]. However, Valavanidis et al. did not find any significant difference between antioxidant activity or phenolic compounds in apples grown in organic or conventional agriculture [7
]. Tsao et al. concluded that antioxidant activity was positively correlated with total phenolic concentration in apples, and that the flavan-3-ols/procyanidins were the most important contributors to the in vitro antioxidant capacity of apples. Among them, procyanidin B2 and epicatechin were the most important individual contributors [8
It is often believed that older varieties have higher nutrient content than newer varieties. Jakobek et al. characterised old apple varieties from the Balkans and found relatively high contents of polyphenols, while Wojdylo et al. found no difference between old and new varieties in their study [9
Nitrogen content in apples varies between cultivars, age of the apple tree and, mainly, horticultural practices with respect to soil, climate and fertilisation. Kahle et al. found that local growing conditions were more important than cultivar for nitrogen content in the apples [11
]. Uptake of nitrogen in apples and leaves depends on soil quality, what type of fertilizer that is used and also in which period of the growing season the fertilization is done [12
]. In a study by Karl et al. [13
], they found that fertilizer treatment of the soil had no effect on acidity and total polyphenol concentration in the apple juice while concentration of yeast assimilable nitrogen (YAN) increased. This, in turn, might affect the fermentation rate during cider processing. YAN includes free amino nitrogen (FAN), ammonia and ammonium [14
]. FAN is a measure of amino acids and small peptides that can be utilized by the yeast in the fermentation process. In a study by Boudreau et al. [15
], they found a correlation between content of FAN and YAN. On average FAN comprised 85% total YAN. If the YAN concentration is low, it is possible that fermentation rate will be low, which can result in development of unwanted sulphur compounds. In a different study, they found that addition of methionine decreased H2
S production when fermenting with some yeast strains, especially if YAN was low, but also an increase at specific YAN concentrations [16
]. Thus, the YAN concentration in the fermenting liquid seems to be important for flavour development, positively or negatively.
Di Maro et al. found that the main amino acids in apples were asparagine, aspartic acid and glutamic acid, and that these amino acids accumulate when nitrogen is available, depending on horticultural conditions [17
], while Wu et al. [18
] reported that asparagine was the most important and serine the second most important amino acid. Ma et al. [14
] found that, on average, asparagine was the most abundant amino acid in their study of 13 different cultivars of apples, and ranged phenylalanine as the second most important, while Zuo et al. and Ye et al. [19
] did not report on any asparagine content in apple juice for cider production.
Alcoholic and malolactic fermentation reduce the content of most amino acids, the main nitrogenous nutrients for yeast. When initial nitrogen content of juice is low enough the cider can be quite stabile against microbial growth after fermentation (French craft cider making method) [21
Sugar and nitrogen content in apples are often negatively correlated, and late maturating apples are often higher in sugar content due to starch hydrolysis and, for some cultivars, water loss during maturation.
The apple tree originates from the Caucasus and has been cultivated for thousands of years. It is now grown on almost all continents, mostly in temperate and cooler climates. The apple was known in Scandinavia 1000 years ago. Wild apples are found all along the coastline nearly up to the Arctic Circle. There is a long tradition for using apples in various dishes and beverages. In Norway, especially in Hardanger region, commercial production of cider has been known since the end of 1700. Today, there is increased interest in renewing the old traditions of using apples as raw materials for juice, cider and apple spirit. The producers frequently use a large variation of cultivars available on the market. Of particular interest are the cultivars Gravenstein and Aroma, the most important apple cultivars for cider production in Norway [22
]. Most of the cider production is based on dessert apples commonly grown in Norway, but import and cultivation of cider apple trees to Norway to obtain apple cultivars of specific quality for cider production is at the experimental stage. Norwegian apples are, in general, rather acidic and low in bitterness. If sugar content is low, external sugar can be added. Mixing of apples to balance bitterness, acidity and sweetness is common. In France and England cider apples are cultivated for the purpose of cider production, while the use of dessert apples is more common in, for example, North America, China and some European countries [23
The aim of this study was to characterize the biochemical composition of some apple cultivars grown in Norway for potential use in cider production. It was also of interest to investigate whether there were differences between apple varieties that could be characterized as new or old cultivars, and whether place of growth could have an impact on quality. This study adds useful information on apples grown in the Nordic/arctic climate.
2. Materials and Methods
Seventeen samples of apples were grown in two experimental orchards in Norway A: Akershus/south-east (10°77′ E. 59°67′ N at the NMBU/Norwegian University of Life Sciences) and H: Hardanger/west (6°66′ E. 60°32′ N at the Nibio Ullensvang Norwegian Institute of Bioeconomy Research) were harvested in September and October 2013 and stored at 2 °C until pressing at full maturity in December. Hardanger is an important region for cultivation of fruits and berries. The climate in Hardanger is characterized by mild winters and cool summers, and the region is known for the narrow fiords and steep growing locations. The annual rainfall is approximately 3000 mm. In the experimental field in Akershus, the winters are colder and the summers warmer, and the area for cultivation is more open farmland. The annual rainfall is approximately 750 mm. The orchards were all fertilized using complete fertilizer “12-4-18” (chlorine-poor, high in phosphorus, potassium, calcium, magnesium, and sulphur), 250 g per tree.
Approximately 12 kg of each cultivar were harvested from two or three trees per cultivar. The cultivars were: Aroma Amorosa (A), Aroma (A + H), Bramley Seedling (H), Delcorf (A), Elstar (A), Filippa (A), Gravenstein (A + H), James Grieve (H), Jonagold (A), Mutsu (A), Rubin (A), Summerred (A), Sunrise (A), Torstein red (A) and Torstein (H).
More detailed description of all apple varieties is shown in Table 1
2.2. Sample Preparation
Apple Juice Extraction and Sampling
Maturity of the apples was analyzed by dipping one slice from each cultivar of apples into an iodine solution and comparing the color changes to a CTIFL color chart. All samples showed complete starch regression at the time of pressing. Three replicates of 2 to 2.5 kg of apple for each variety were pressed to produce juice for chemical analyses. The apples were mashed and pressed using a small laboratory press (model HP5, 5 L, Hafico, Fischer & Co. Dusseldorf, Germany). The hydraulic pressure rose to 400 bars giving a maximum pressure of 24 bars in the press cake. The pressing time was 15 min. The juice was then sampled for the different analysis and kept frozen. NaF (sodium fluoride; 1 mg/mL juice) was added to samples for HPLC analyses of polyphenols. NaF was used to stop enzymatic oxidation immediately after pressing by inhibition of polyphenoloxidase [25
]. Samples were either freeze-dried or thawed before analyses.
2.3. Chemical Analyses
2.3.1. UV-MS Reversed Phase HPLC of Polyphenols Directly or after Phloroglucinolysis Reaction
Direct analysis of simple polyphenols in apple juice: freeze-dried juice samples (0.5 mL) were dispersed by sonication for 20 min in 1.2 mL MeOH-Ac (1% v/v).
Samples were filtered (syringe filter; 0.45 µm PTFE) before HPLC analyses. The HPLC procedure was carried out using reversed-phase analyses using a RP18 column and UV-visible and mass spectrometry (MS) detection. Phenolic compounds were identified based on their retention time, UV-visible spectrum, full MS characteristics and MS fragmentation pattern in comparison with available standards.
Phloroglucinolysis: acidolysis in the presence of phloroglucinol allows the complete depolymerisation of flavanol oligomers and polymers (i.e., procyanidins and condensed tannins), giving both qualitative and quantitative information related to those major polyphenolic compounds in complex matrixes [26
]. A freeze-dried juice sample (0.5 mL) was dispersed in 0.8 mL PLG (phloroglucinol) and 0.4 mL MeOH/HCl (0.3 M) by agitation in a vortex mixer then the tubes were set in a water bath at 50 °C for 30 min and the reaction was immediately stopped by cooling the tubes on ice and adding 1.2 mL sodium acetate. The reaction mixture was filtered on a PTFE 0.45 µm filter before HPLC analysis. Total flavanols (including catechins and procyanidins) were quantified as previously described by Malec et al. [26
2.3.2. Total Phenols (Folin Ciocalteu Assay)
Total phenols were analysed by the Folin Ciocalteu method (ISO14502-1). The reaction was carried out by mixing 0.5 mL of apple juice (diluted 1:10) and 2.5 mL 10% Folin Ciocalteu reagent (diluted in water) and 2.0 mL buffer (7.5% Na2CO3). Samples were incubated at ambient temperature for 60 min. The absorbance was read at 765 nm. Samples were analysed in triplicate. Standard solutions (epicatechin—1 g/L in water) for the standard curve were constructed. Quantifications were obtained using a calibration curve of epicatechin (EPI) and expressed as mg/L EPI equiv.
2.3.3. Density (d20/20)
Density measurements were used to estimate the sugar concentration in the juice. The relative density was determined using a Density Meter, DMA 58 (Anton Paar). Results were expressed as g/L.
pH was measured using a pH-meter (Radiometer PHM 92. Copenhagen).
Total nitrogen in the apple juice was analysed by the Kjeldahl method, according to IDF 2001.
2.3.6. Free Amino Acids (FAA)
Free amino acids were analysed using HPLC with o-phthaldialdehyde (OPA) and fluorenyl methyl chloroformate (FMOC) derivatisation according to a method described by Moe et al. [28
]. The amino acids were identified and quantified by comparing standard solutions of the following amino acids: aspartic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, citrulline, arginine, alanine, GABA, tyrosine, valine, methionine, isoleucine, phenylalanine, tryptophan, leucine, ornithine and lysine. Norvaline was used as an internal standard. Sum of amino acids are reported as mg/kg. Percentage share of only the most important amino acids is reported.
2.4. Statistical Analyses
The results are expressed as mean ± standard deviation (STD) from two to five independent measurements. A correlation test, one-way analysis of variance (ANOVA) and principal component analysis (PCA) were performed to find differences between varieties, groups of apple varieties (old and new, sharp and dessert apples) and growing sites in terms of chemical composition in the juice of the various apples. Statistical analyses were performed with Minitab statistical software version 17 (Minitab Ltd., State College, PA, USA).