Impact of steam pasteurization on the sensory profile and phenolic composition of rooibos (Aspalathus linearis) herbal tea infusions

Food Research International 53 (2013) 704–712

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Food Research International journal homepage: www.elsevier.com/locate/foodres

Impact of steam pasteurization on the sensory profile and phenolic composition of rooibos (Aspalathus linearis) herbal tea infusions I.S. Koch a, N. Muller a, D. de Beer b, T. Næs c, E. Joubert a, b,⁎ a b c

Department of Food Science, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch) 7602, South Africa Post-Harvest & Wine Technology Division, ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa NOFIMA, Osloveien 1, 1430 Ås, Norway

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Article history: Received 22 August 2012 Received in revised form 2 October 2012 Accepted 10 October 2012 Keywords: Rooibos Aspalathus linearis Herbal tea Steam pasteurization Sensory profile Aspalathin

a b s t r a c t The effect of steam pasteurization of fermented rooibos leaves and stems on the sensory characteristics and phenolic composition of infusions was determined. The extent to which this processing step changes the sensory profile and whether compositional changes influence taste and astringency of the beverage was determined. These were achieved by examining the changes in the concentrations of soluble solids (SS), total polyphenols (TP) and 14 individual non-volatile monomeric phenolic compounds, as well as the changes in 17 aroma, flavor, taste and mouthfeel attributes of rooibos infusions. Steam pasteurization significantly reduced the SS, TP and aspalathin contents, as well as the “total color” (area under the curve: 380 to 520 nm). Neither the intensities of the taste attributes, sweetness and bitterness, nor the levels of individual phenolic compounds changed significantly, except that of aspalathin which were significantly reduced. A small but significant decrease in the astringency of rooibos infusions was observed. The intensities of most of the aroma and flavor attributes decreased significantly as a result of steam pasteurization. “Green” and “caramel” notes exhibited the largest reductions in attribute intensity. The prominent “green” flavor of unpasteurized rooibos was frequently changed to a “hay-like” flavor after steam pasteurization. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Leaves and fine stems of the endemic South African fynbos plant, Aspalathus linearis, are processed to produce rooibos, a herbal tea of increasing global popularity. It is mostly consumed in the “fermented” (oxidized) form (Joubert & De Beer, 2011) which has a characteristic rooibos flavor that can be described as a combination of honey, woody and herbal-floral notes with a slightly sweet taste and subtle astringency (Koch, Muller, Joubert, Van der Rijst, & Næs, 2012). It contains no caffeine (Joubert & De Beer, 2011), which could contribute to a bitter taste. Flavor plays a predominant role in the grading of rooibos. The characteristic sensory attributes and absence of negative attributes are associated with high quality tea. Attributes such as “green grass” and “hay-like” aroma notes which are undesirable in some products (Hongsoongnern & Chambers, 2008) could have a negative impact on the quality and grade of rooibos as these would signify underfermentation. Commercial processing of rooibos involves shredding of the shoots followed by wetting with water and bruising to initiate enzymatic and chemical oxidation before overnight fermentation at ambient ⁎ Corresponding author at: Post-Harvest & Wine Technology Division, ARC InfruitecNietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa. Tel.: +27 21 8093444; fax: +27 21 8093400. E-mail address: [email protected] (E. Joubert). 0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2012.10.017

temperature and sun-drying the next day (Joubert & Schulz, 2006). Before packaging the dried and sieved product is steam pasteurized at 96 °C for 60 s to ensure that the final product is microbiologically safe. After the introduction of steam pasteurization in the 1980s, consumers noted a softening of the flavor and the prominent “medicinal aroma” of rooibos. This led to a more acceptable product for some consumers while others preferred the flavor of unpasteurized rooibos (A. Redelinghuys, Rooibos Ltd., Clanwilliam, pers. comm.). These changes in the sensory quality of rooibos infusions due to steam pasteurization have not yet been scientifically substantiated, nor have such changes been accurately described or quantified. Recently a rooibos sensory wheel was developed (Koch et al., 2012) summarizing the variation in sensory attributes of rooibos infusions. The development of the sensory wheel has provided a basis for subsequent studies on the sensory quality of rooibos, because it has supplied the necessary terminology with which changes in the sensory profile of rooibos could be described and quantified. The objective of this study was thus to determine the effect of steam pasteurization of fermented rooibos leaves and fine stems on the sensory characteristics of the infusion and to profile the changes in aroma, flavor, taste and mouthfeel (astringency) attributes. Furthermore, the link between the changes in the sensory profile and the phenolic composition of rooibos infusions was examined to determine whether compositional changes in non-volatile compounds could be associated with changes in taste and mouthfeel attributes.

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2. Materials and methods 2.1. Chemicals Chemicals required for HPLC analysis were 99.8% acetic acid (Fluka, Sigma-Aldrich, Steinheim, Germany), acetonitrile (LiChrosolv, gradient grade for liquid chromatography, Merck, Darmstadt, Germany) and ascorbic acid (Sigma-Aldrich). Enolic phenylpyruvic acid-2-O-glucoside (PPAG), isolated from green rooibos (purity> 95% by HPLC and LC–MS), was supplied by the Post-Harvest & Wine Technology Division of the Agricultural Research Council of South Africa (ARC InfruitecNietvoorbij, Stellenbosch, South Africa). Aspalathin and nothofagin (purity > 95% by HPLC and LC–MS) were supplied by the PROMEC Unit of the Medical Research Council of South Africa (Cape Town, South Africa). Iso-orientin, iso-vitexin, luteolin, chrysoeriol and hyperoside were obtained from Extrasynthese (Genay, France). Sigma-Aldrich provided quercetin and rutin, while Roth (Karlsruhe, Germany) supplied orientin, vitexin, quercetin-3-O-glucoside (isoquercitrin) and luteolin7-O-glucoside. Laboratory grade deionized water was purified using a Milli-Q 185 Academic Plus water purifier (Merck Millipore, Billerica, MA, USA) to obtain HPLC grade water. The reagents required for the quantification of the total polyphenol content were Folin–Ciocalteu's phenol reagent (Merck), anhydrous sodium carbonate (Saarchem, South Africa) and gallic acid (Sigma Aldrich). 2.2. Rooibos samples Prior to pasteurization, 69 samples, representing different production batches of fermented rooibos of the 2009 harvest season, were randomly collected and graded by an expert industry grading panel (Koch et al., 2012). The samples comprised nine Grade A samples and 20 samples each of Grades B, C and D, with Grades A and D representing the highest and the lowest quality, respectively. The sample codes used in this study indicate the quality grade and sample number assigned to each batch (e.g. B13 = Grade B, Sample 13). A reference sample composed of six different Grade B rooibos samples was prepared. Grade B samples were chosen as this grade present good quality tea produced in the highest quantities. The sensory attributes of the reference sample were considered to be representative of the profile typically associated with rooibos, i.e. having a mixture of honey, woody and herbal-floral notes with a slight sweet taste and subtle astringency (Koch et al., 2012). It thus served as a “fixed” point during descriptive sensory analysis (DA), allowing panelists to calibrate their sensory perception at the start of each session.

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(Koch et al., 2012). Infusions were strained through fine-mesh stainless steel tea strainers into preheated stainless steel thermos flasks. Panelists were served ca. 100 mL of the infusions “as-is” in preheated porcelain mugs with plastic lids. The mugs were kept in water-baths at 65 °C throughout the sensory analysis session to keep the temperature of the infusion as constant as possible. 2.5. Descriptive analysis (DA) 2.5.1. Sensory panel Nine female judges, who previously received extensive training in DA, as well as the sensory analysis of rooibos infusions (Koch et al., 2012; Lawless & Heymann, 2010), participated in the study. During 22 1-hour sessions the panel generated the aroma, flavor, taste and mouthfeel descriptors that were used to assemble the rooibos sensory wheel and lexicon as described by Koch et al. (2012). The current study is therefore based on the sensory terminology that was developed in the previous study. 2.5.2. Intensity rating The panel was requested to rate the intensities of 17 aroma, flavor, taste and mouthfeel attributes for each of the samples as described by Koch et al. (2012). In each session the UPAS sample and its PAS counterpart were presented together with the reference standard so that the panelists could not only directly compare the two samples with one another, but also with the reference standard. The panelists were informed that they were to receive a number of sample pairs each consisting of an UPAS sample with its corresponding PAS sample. Samples were labeled with three-digit codes, while the reference sample was labeled as such so that it could be identified by the panelists. The presentation order of the different sample pairs, as well as the order of the UPAS and the PAS samples within each pair, was randomized. All 69 UPAS and PAS samples were analyzed in duplicate. The samples were tested during 40 sessions over a period of 8 weeks, with 2 sessions conducted per day during which 6 and 8 samples were analyzed, respectively. 2.6. Compositional analysis 2.6.1. Sample preparation An aliquot (200 mL) of each infusion of UPAS and PAS rooibos prepared for sensory analysis was filtered through Whatman No. 4 filter paper, allowed to cool and the soluble solids (SS) content determined. The remaining part of the filtrate was transferred into 2 mL microfuge tubes which were stored in a freezer at − 18 °C until required for further analyses.

2.3. Steam pasteurization of rooibos samples A sub-sample of each unpasteurized rooibos sample (UPAS) was steam pasteurized for direct comparison. The finely cut, dried leaves and stems (50 g) were spread in a thin layer on stainless steel, 30-mesh trays which were placed in a pre-heated steam cabinet at ± 96 °C for 60 s to simulate the industrial process. The steam pressure, generated with a THE 400 NM Electropac electrode boiler (John Thompson Boilers, Cape Town), was maintained at 2.76 N/m 2 at the inlet of the cabinet. In order to remove superficial moisture and reduce the moisture content of the pasteurized rooibos (PAS) below 10%, the trays were placed in a cross-flow dehydrator set to 40 °C for 10 min. The dried rooibos was then stored in air-tight, re-sealable plastic bags. 2.4. Preparation of tea infusions Infusions of unpasteurized and pasteurized samples were prepared by adding freshly boiled distilled water (900 g) to 17.4 g rooibos and stirring for about 5 s, followed by a 5 min infusion period

2.6.2. Determination of soluble solids (SS) content The SS contents of the infusions were determined gravimetrically by evaporating 20 mL aliquots of the filtrate to dryness on a steam bath in triplicate, followed by oven drying at 100 °C for 1.5 h. The moisture dishes were allowed to cool in a desiccator before re-weighing. 2.6.3. Determination of total polyphenol (TP) content The TP contents of the rooibos infusions were determined according to the method developed by Singleton and Rossi (1965), scaled-down to 96-well microplate format as described by Arthur, Joubert, De Beer, Malherbe, and Witthuhn (2011). Gallic acid was used as calibration standard and results expressed as mg gallic acid equivalents (GAE)/L infusion. 2.6.4. Quantification of monomeric phenolic compounds with HPLC Quantification of 14 monomeric phenolic compounds was achieved by high-performance liquid chromatography with diode-array detection (HPLC–DAD) using an Agilent 1200 system (Agilent, Santa Clara, CA, USA) comprising a quaternary pump, autosampler, in-line degasser,

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2.6.5. Color measurements Spectrophotometric measurements of 100 μL of the filtered infusion mixed with 100 μL of water were carried out in triplicate using a Biotek Synergy HT microplate reader (Biotek Instruments, Winooski, Vermont, USA). Absorbance was measured at 10 nm intervals ranging from 380 nm to 520 nm. Using Gen5 Secure software, values for the integral of the absorbance spectrum were obtained (Area under the curve — AUC), reflecting the “total color” of the diluted sample across the wavelength range. 2.7. Statistical analysis The data were subjected to analysis of variance (ANOVA) using SAS® version 9.2 (SAS Institute, Cary, NC, USA). The Shapiro–Wilk test was used to test for non-normality of the residuals (Shapiro & Wilk, 1965). In the event of significant non-normality (p b 0.05) outliers were identified and residuals larger than 3 were removed. Student's t-test's least significant difference (LSD) was calculated at the 5% level to facilitate comparison between UPAS and PAS samples. Principal component analysis (PCA), based on the correlation matrix, was conducted using XLStat (Version 7.5.2, Addinsoft, New York, USA) to determine correlations between attributes and to visualize and elucidate the relationships between the samples and their attributes (Næs, Brockhoff, & Tomic, 2010). 3. Results and discussion

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column thermostat and diode-array detector. A Zorbax SB-C18 column (Rapid Resolution HT, 4.6× 100 mm, 1.8 μm particle size, Agilent) with an in-line filter frit (2.1 mm, 0.2 μm pore size, Waters) and protected by an Acquity UPLC® BEH C18 pre-column (Van Guard, 2.1× 5 mm, 1.7 μm particle size, Waters, Milford, USA) was used for separation of the compounds at 36 °C with acetonitrile and 2% acetic acid as solvents. The flow rate was set to 1 mL/min and the gradient profile in terms of acetonitrile was as follows: 0–2 min, 10%; 2–14 min, 10–14.8%; 14–38 min, 14.8–50%; 38–40 min, 50%; 40–41 min, 50–10%; and 41–50 min, 10%. Stock solutions of standards were prepared with dimethyl sulfoxide and aliquots frozen until analysis. Different injection volumes (1, 5, 10, 20, 30 and 40 μL) of a standard mixture were used to generate a standard curve for each compound. Samples were prepared by mixing 1000 μL of each sample with 100 μL ascorbic acid solution (10% m/v) to prevent oxidation of the phenolic compounds (Beelders, Sigge, Joubert, De Beer, & De Villiers, 2012) and filtering the mixture through 0.45 μm Millex-HV hydrophilic PVDF syringe filters (Merck Millipore, Billerica, MA, USA). For each sample an injection volume of 30 μL was injected in duplicate. Retention times and spectral characteristics were used for peak identification. The peak areas of aspalathin, nothofagin and PPAG were determined at 288 nm, while all other compounds were quantified at 350 nm.

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F1 (51.91 %) Fig 1. PCA loading (a) and score (b) plots showing the positioning of rooibos attributes and Grade × Treatment averages and the changes in sensory characteristics due to steam pasteurization. Except for astringency, the letters “A”, “F” and “T” in front of an attribute refer to aroma, flavor and taste attributes, respectively. The terms UPAS and PAS refer to unpasteurized and pasteurized samples, respectively. Letters A to D on the score plot (b) refer to grade.

3.1. Changes in sensory attributes The effect of steam pasteurization on the sensory characteristics of rooibos infusions can be displayed by means of PCA loading and score plots (Fig. 1a) which reflect, respectively, the positioning of the sensory attributes with respect to each other, and the translation of the score vectors across the quadrants. The loading plot shows that the “positive” attributes, i.e. sensory attributes typically associated with good quality rooibos, are separated from the “negative” attributes, i.e. attributes associated with poor quality along PC1 from right to left. This corresponds to the separation of the score vectors from high quality Grade A rooibos on the right side of the score plot to low quality Grade D rooibos on the left (Fig. 1b). Most of the attributes, except “dusty” and “caramel” aroma, and “sweet” taste, are distributed across the upper two quadrants of the loading plot. On the

score plot all four grade averages of the UPAS samples are positioned in the upper quadrants while the grade averages for the PAS samples are positioned in the bottom quadrants. This movement from the UPAS sample averages to its PAS counterparts indicates that the score vectors moved away from the attributes, revealing a decrease in the attribute intensity as a result of steam pasteurization. The average intensities of the attributes for UPAS and PAS samples are displayed in Fig. 2. Steam pasteurization had a significant effect on the intensities of most of the 17 sensory attributes. The average intensities of the PAS samples were significantly lower for all aroma attributes except for “hay” and “dusty” aromas (Fig. 2a). The average intensities of the palate attributes, i.e. “woody”, “floral” and “green”

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Fig. 2. Average intensity values for rooibos aroma attributes (a) and flavor, taste and mouthfeel attributes (b) of unpasteurized (UPAS) and pasteurized (PAS) samples. Bars for the same attribute (same color) but different letters differ significantly from each other (p b 0.05).

flavors, “sour” taste, and astringency were also slightly, but significantly lower in the PAS samples (Fig. 2b). The percentage change in the average attribute intensity between the UPAS and PAS samples was calculated to determine which attributes decreased the most in intensity (Fig. 3). The attributes exhibiting by far the greatest changes in intensity were “green” aroma and flavor which decreased by 26% and 32%, respectively. The other attributes did not decrease as substantially as a result of steam pasteurization. The relative changes due to pasteurization in selected attribute intensities were also examined for each quality grade separately (Fig. 4), because certain aroma attributes associate more closely with certain quality grades (Fig. 1). The “green” aroma of Grades B, C and D decreased considerably, especially that of Grade C samples,

whereas the largest reduction in “caramel” aroma was observed for Grade A samples. The “woody” aroma decreased more or less to the same extent for all grades, whereas “hay” aroma showed an increase for Grades B, C and D, but a decrease for Grade A samples. These results indicate that steam pasteurization of rooibos does not have the same effect on the aroma characteristics of all the quality grades. The initial sensory profile of a sample, before steam pasteurization, determines the effect of the heat treatment on its sensory characteristics. Unpasteurized Grade C samples, which most strongly associated with “green” flavor and aroma (data not shown), thus showed the largest decrease in “green” flavor due to pasteurization (Fig. 4). Similarly, “caramel” aroma and flavor were most closely associated with Grade A samples (data not shown) and, accordingly, these samples decreased to the largest extent in “caramel” aroma (Fig. 4).

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-35 Fig. 3. The percentage change in attribute intensity as a result of steam pasteurization. Except for astringency, the letters “A”, “F” and “T” in front of an attribute refer to aroma, flavor and taste attributes, respectively. The terms UPAS and PAS refer to unpasteurized and pasteurized samples, respectively, while ns indicates attributes that did not change significantly.

The comparison between the PCA loading plots for UPAS and PAS samples is depicted in Fig. 5. Other than a slight anti-clockwise rotation, the relative attribute positioning with respect to each other remained similar. However, while the “green” and “hay” attributes are separate and distinct on the UPAS PCA plot, these attributes are closely associated for the PAS samples. Furthermore, “hay” aroma was the only attribute that, on average, increased slightly after steam pasteurization, even though this increase was not significant (Fig. 3). The change in the “green” and “hay” character of rooibos infusions was also revealed when comparing the sensory profiles of two

individual UPAS and PAS sample pairs with strong “green” notes (Fig. 6). Assessors who were not reliable and consistent in rating “green” flavor and aroma were removed before generating the spider plots. The spider plot of sample C7 (Fig. 6a) showed a reduction in the intensity of “green” aroma and flavor as a result of steam pasteurization. No hay-like notes were perceived after steam pasteurization. In the case of sample C20 (Fig. 6b), however, the “green” aroma and flavor was completely lost during steam pasteurization and replaced by a hay-like aroma and flavor. 3.2. Changes in the composition of rooibos infusions

Grade A Grade B Grade C Grade D

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The effect of steam pasteurization on the chemical/instrumental parameters is summarized in Table 1. The mean values for the contents of SS, TP and monomeric phenolic compounds, and for the “total color” (AUC) measurements for UPAS and PAS samples are given. Even though a spectrum of absorbance values was obtained at 15 different wavelengths, all of these values were highly correlated, and, therefore, the integral of the absorbance spectrum (AUC) was chosen to represent the “total color”. The SS, TP, and aspalathin contents, as well as the “total color” were significantly (p b 0.05) higher in UPAS samples than in PAS samples (Table 1). The content of all other individual phenolic compounds did not change significantly as a result of steam pasteurization, although all the means, except for quercetin, luteolin and chrysoeriol, were slightly lower for the PAS samples. The SS content and TP content are strongly correlated (r = 0.991); therefore, it can be concluded that the decrease in soluble solids as a result of steam pasteurization was largely due to the decrease in the level of soluble polyphenols. However, except for aspalathin, none of the individual phenolic components that were quantified in this study were significantly lower after steam pasteurization. Polymeric compounds may therefore have been affected. A decrease in the content of polyphenolic compounds as a result of heat treatments is a

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Fig. 5. Change in attribute distribution for unpasteurized (UPAS) (a) and pasteurized (PAS) (b) samples indicating a change in the “green” attribute. Except for astringency, the letters “A”, “F” and “T” in front of an attribute refer to aroma, flavor and taste attributes, respectively.

Fig. 6. Spider plots reflecting the differences in the sensory profile between unpasteurized (UPAS) and pasteurized (PAS) samples with a strong “green” flavor. Except for astringency, the letters “A”, “F” and “T” in front of an attribute refer to aroma, flavor and taste attributes, respectively.

well-established phenomenon that has been demonstrated in a number of studies involving other teas or food products (e.g. Devic, Guyot, Daudin, & Bonazzi, 2010; Joubert, Manley, Maicu, & De Beer, 2010; Katsube, Tsurunaga, Sugiyama, Furuno, & Yamasaki, 2009; Larrauri, Ruperez, & Saura-Calixto, 1997; Sanderson et al., 1976; Wang, Kim, & Lee, 2000). Heat-induced losses in aspalathin and its flavone glycosides, orientin and iso-orientin, have been demonstrated for ready-to-drink rooibos iced tea (Joubert, Viljoen, De Beer, & Manley, 2009). It has been shown that oxidation reactions result in aspalathin being converted to iso-orientin and orientin, as well as dimers and other oxidation products (Heinrich, Willenberg, & Glomb, 2012; Krafczyk & Glomb, 2008). However, even though the aspalathin level is significantly lower in PAS samples (Table 1) the reduction in aspalathin is most likely not due to its conversion to iso-orientin

and orientin, since the levels of the two flavones did not increase due to steam pasteurization. The formation of other compounds from aspalathin, as demonstrated by Heinrich et al. (2012), may be predominant. The dimers that form upon oxidation of aspalathin are colorless, while the dibenzofurans are key chromophores in the color of rooibos infusions. Breakdown of the dibenzofurans gives rise to high molecular weight tannin-like structures during the late stages of aspalathin oxidation and a change in color from yellow to dark orange. Pasteurization resulted in a significant change in the “total color” value. Relatively weak, but significant correlations existed between the “total color” and the TP content (r = 0.628), as well as with the SS content (r = 0.551). The absorbance values (A380 to A520) were normalized in terms of the SS content to determine whether

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differences in absorbance measurements resulted from compositional differences between the infusions. The spectra of the actual absorbance values and the values normalized in terms of the SS content for UPAS and PAS samples are shown in Fig. 7. The mean absorbance values of PAS samples were consistently lower than those of the UPAS samples, even when normalized. The decrease in the normalized absorbance due to steam pasteurization confirms that this processing step causes changes in the phenolic constituents of rooibos leaves and stems which affect the absorbance characteristics of the infusion. The overall reduction in the sum total content of individual phenolic compounds may be one of the contributing factors. The largest differences in normalized absorbance values of UPAS and PAS samples were observed between A440 and A460 (Fig. 7b) suggesting that changes must be taking place in compounds with absorbance maxima at around 450 nm. Krafczyk, Heinrich, Porzel, and Glomb (2009) demonstrated that oxidation of aspalathin resulted in the formation of higher molecular weight browning products which was accompanied by an increase in A450. The decrease in aspalathin without an accompanying increase in A450 could be the effect of one or more occurrences, e.g. brown oxidation products may not have formed during pasteurization or their solubility may have decreased during steam pasteurization, or thermal degradation of these compounds may have resulted in a reduction in their absorbance. Further investigation of this aspect is required to provide insight into these changes in color taking place during steam pasteurization.

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SS = soluble solids, TP = total polyphenols, AUC = area under curve, PPAG = phenylpyruvic acid-2-O-glucoside, glc = glucoside. Values in the same row with different letters are significantly different. (P b 0.05) after significantly different. a Parameters given in mg/L.

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2197.3 b 613.11 b 123.61 b 15.83 b 27.06 a 20.59 a 12.37 a 4.99 a 7.39 a 1.78 a 7.30 a 2.43 a 1.00 a 0.43 a 0.32 a 11.12 a 1.22 a

390

2240.0 a 624.98 a 134.34 a 16.66 a 27.28 a 20.62 a 12.49 a 5.01 a 7.47 a 1.81 a 7.38 a 2.45 a 1.00 a 0.43 a 0.32 a 11.15 a 1.25 a

400

SS (mg/L) TP (mg GAE/L) AUC Aspalathina Iso-orientina Orientina Vitexina Hyperosidea Rutina Quercetin-3-O-glc Iso-vitexin a Luteolin-7-O-glc a Quercetina Luteolin a Chrysoeriola PPAG a Nothofagina

3.0

380

PAS

390

UPAS

380

Parameter

(a)

Absorbance

Table 1 Mean values of chemical/instrumental data for unpasteurized (UPAS) and pasteurized samples (PAS).

Absorbance

710

Wavelengths (nm) Fig. 7. Differences between the absorbance spectra of unpasteurized (UPAS) and pasteurized (PAS) samples. Actual absorbance values (a) and absorbance values normalized according to the average soluble solids content (b) are shown.

3.3. Compositional changes in relation to changes in sensory attributes

coefficient between “woody” flavor and total polyphenol content was considerably higher compared to that of other flavor attributes (data not shown), “woody” flavor was therefore also included in the table. The correlation coefficients were extremely low, primarily as a result of the large variation found in the samples which is typical of a processed plant material, in particular a product such as rooibos that is fermented under uncontrolled conditions and sun-dried (Joubert & De Beer, 2011). Even though correlation coefficients were low (r b 0.5), significant correlations (p b 0.05) between certain parameters did exist.

Polyphenolic compounds are commonly responsible for sweet or bitter taste or astringent mouthfeel characteristics of certain fruit and beverage products (e.g. grapes, apples, wine, beer and tea) depending on their solubility and structure (Cabarello, Trugo, & Finglas, 2003; El Gharra, 2009). For the purpose of this study monomeric phenolic constituents of rooibos were quantified and the changes in these compounds related to changes in basic taste and mouthfeel characteristics of the infusions. Insight into the association between sensory and chemical/instrumental variables can be obtained by determining their correlation coefficients. Since non-volatile phenolic compounds are linked to the taste and not to the aroma of a product, only the correlation coefficients between the compositional parameters and sweet taste, bitter taste, sour taste and astringency are shown (Table 2). The correlation

3.3.1. Bitterness and sweetness The TP content of UPAS samples was weakly, but significantly correlated to sweetness (r = 0.246) and bitterness (r = 0.256) (Table 2). Several phenolic compounds were also associated with these sensory attributes, e.g. PPAG, quercetin-3-O-glucoside and iso-orientin correlated with sweet taste, and luteolin, quercetin and aspalathin with bitterness (Table 2). Interestingly, PPAG was demonstrated by our group to be bitter tasting (unpublished results), yet it correlated with the sweet taste of rooibos infusion, indicating that its taste modality is modulated when in the presence of other compounds. Since more than one phenolic compound correlated significantly with sweetness and bitterness, it is likely that the sum of sweet- or bitter-tasting components, rather than one specific sweet or bitter compound, influences the taste characteristics of the infusion. No

I.S. Koch et al. / Food Research International 53 (2013) 704–712

711

Table 2 Correlation coefficients between compositional parameters and selected sensory attributes. Variables

TSweet UPAS

SS TP AUC Aspalathin Iso-orientin Orientin Vitexin Hyperoside Rutin Quercetin-3-O-glc Iso-vitexin Luteolin-7-O-glc Quercetin Luteoli Chrysoeriol PPAG Nothofagin AGreen FGreen FHay FWoody

TBitter PAS

0.318 0.246⁎ 0.244⁎ 0.131 0.334⁎ 0.325⁎

0.215 0.160 0.183 0.013 0.266⁎ 0.287⁎

0.201 0.268⁎ 0.195 0.344⁎ 0.286⁎ 0.294⁎

0.161 0.229 0.191 0.219 0.248⁎ 0.254⁎

0.058 −0.099 0.002 0.356⁎ 0.098

0.038 −0.014 −0.009 0.284⁎ −0.044

UPAS 0.199 0.256⁎ 0.023 0.283⁎ 0.116 0.128 0.176 0.144 0.185 0.042 0.114 −0.002 0.289⁎ 0.300⁎ 0.238⁎ −0.023 0.240⁎

TSour PAS 0.074 0.113 −0.096 0.220 0.078 0.044 0.167 0.051 0.134 −0.083 0.053 −0.057 0.178 0.191 0.246⁎ 0.033 0.142

UPAS 0.012 0.037 −0.057 −0.186 0.079 −0.016 −0.058 0.050 −0.015 0.082 0.054 −0.005 0.150 −0.110 −0.195 −0.188 −0.032 0.248 0.342

FWoody

Astringency

PAS

UPAS

0.186 0.085 0.008 −0.199 0.165 0.023 0.010 −0.006 0.083 0.038 −0.017 0.024 0.102 −0.068 −0.073 −0.110 0.123 0.147 0.162

0.309⁎ 0.405⁎ 0.264⁎

0.313⁎ 0.359⁎ 0.309⁎

0.234 0.251⁎ 0.277⁎ 0.367⁎

0.216 0.303⁎ 0.325⁎ 0.395⁎

0.225 0.369⁎ 0.180 0.273⁎ 0.210 0.421⁎ 0.323⁎ 0.252⁎

0.216 0.345⁎ 0.161 0.266⁎ 0.161 0.393⁎

0.071 0.077

PAS

0.236 0.158 0.026 −0.035

UPAS 0.101 0.159 −0.027 0.231 0.017 0.018 0.208 −0.003 0.238⁎ −0.070 0.045 0.139 0.236 0.223 −0.014 −0.118 0.036 0.435⁎ 0.175 0.517⁎

PAS 0.279⁎ 0.327⁎ 0.158 0.286⁎ 0.143 0.140 0.248⁎ 0.085 0.242⁎ −0.031 0.119 0.114 0.334⁎ 0.205 0.078 0.023 0.109 0.152 0.329⁎ 0.679⁎

SS = soluble solids, TP = Total polyphenols, AUC = area under curve, PPAG = phenylpyruvic acid-2-O-glucoside, glc = glucoside. Except for astringency, the letters “A”, “F” and “T” in front of an attribute refer to aroma, flavor and taste attributes, respectively. ⁎ Values in bold and marked with an asterisk are significantly different from 0 with a significance level p b 0.05.

significant changes in bitterness and sweetness were observed between UPAS and PAS samples (Fig. 2) and since none of these flavonoids were significantly reduced by pasteurization (Table 1), it is to be expected that sweet and bitter taste characteristics would not decrease significantly as a result of steam pasteurization. 3.3.2. Sourness No significant correlations were observed between the chemical/instrumental parameters and sour taste (Table 2). Sourness in food or beverage products is caused by small, soluble inorganic cations and not by polyphenolic compounds (Jackson, 2009), although it has been reported that certain simple phenolic acids, e.g. p-hydroxybenzoic acid and ferulic acid, have acidic or sour taste characteristics at fairly high detection thresholds (Huang & Zayas, 1991; Peleg & Noble, 1995). Steam pasteurization resulted in a small but significant decrease in sour taste (Figs. 2 and 3). Since sourness was significantly correlated to “green” flavor and aroma of UPAS, but not of PAS samples (Table 2), it is possible that sourness is related to the compounds responsible for the “green” flavor of certain UPAS samples. The fact that “green” flavor and aroma decreased most significantly as a result of steam pasteurization (Fig. 3) also suggests that a reduction in the sour taste quality of certain rooibos infusions is due to the loss of “green” volatiles rather than the loss of sour-tasting phenolic acids. 3.3.3. Astringency Only rutin (quercetin-3-O-rutinoside) was significantly correlated with the astringency of UPAS samples, but for the PAS samples, both the SS and TP contents, as well as quercetin, aspalathin, vitexin and rutin contents correlated significantly with astringency (Table 2). Scharbert, Holzmann, and Hofmann (2004) found that rutin induces a “silky, mouth-drying, and mouth-coating sensation”, and that the oral threshold for this compound is extraordinarily low at 0.001 mg/L compared to the 3-O-glucoside (0.65 mg/L) and -galactoside (0.43 mg/L) derivatives of quercetin. Whether aspalathin is associated with astringency has not yet been established, whereas the slightly astringent character of quercetin and luteolin has been mentioned in literature (Ley, 2008; Stark, Bareuther, & Hofmann, 2005). In other studies it was shown that vitexin (apigenin-8-C-glucoside), quercetin-3-O-glucoside and luteolin-7-O-glucoside have astringent mouthfeel characteristics

at different threshold concentrations (Scharbert et al., 2004; Stark, Bareuther, & Hofmann, 2006). Even though no significant correlations were seen between these flavonoids and astringency, it is likely that they contribute to some extent to the mouthfeel sensation of rooibos tea. Astringency was found to be significantly lower in PAS samples (Fig. 2). The content of rutin, vitexin and quercetin did not differ significantly between UPAS and PAS samples (Table 1). Therefore, the decrease in astringency cannot be attributed to changes in these flavonoids alone. With aspalathin being the only flavonoid that was significantly reduced by pasteurization, it is possible that the reduction in astringency is associated with the decrease in aspalathin. Furthermore, it is possible that the cumulative decrease in components exhibiting astringent characteristics may have brought about the slight reduction in the perceived astringency. The correlation coefficients between astringency and “green”, “hay” and “woody” flavors indicate that astringency was significantly correlated with the “green” flavor of UPAS samples, but not of PAS samples, and vice versa for “hay” flavor (Table 2), while a “woody” flavor was significantly correlated with astringency of both UPAS (r = 0.517) and PAS (r = 0.679) samples. These differences in the strength of the correlations between astringency and “green”, “hay” and “woody” attributes for UPAS and PAS samples suggest that there was not only a change in the intensity, but also a change in the character of astringency as a result of steam pasteurization. While astringency was associated with “green” flavor in UPAS samples, it correlated with “hay” and “woody” flavor in PAS samples. Heat-induced changes in the character of astringency of tea were also observed by Sanderson et al. (1976) who described the astringency of unfired black tea (i.e. which has not been dried at high temperatures) as “strong non-tangy”, while fired tea was described as having “pleasant tangy astringency”. Furthermore, terms and definitions for a variety of astringent sensations have been developed, e.g. “resinous” (astringency perceived as if chewing on a piece of raw wood), “sappy” (astringency with high acid and slightly bitter; reminiscent of the astringency elicited by chewing on a green grape stalk) and “unripe” (a negative hedonic grouping consisting of an astringent feel associated with excessive acidity and associated green flavor notes) (Gawel, Iland, & Francis, 2001). With regard to these

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definitions it is evident that different types of astringent characteristics may occur, each of which is associated with other sensory properties. Similar descriptions and definitions for astringency may be relevant in the context of the current study. The astringent character of UPAS samples, for example, could be described as “green” astringency which becomes more subtle and “woody” as a result of steam pasteurization. Also, it has been found that structural differences between tannin-like compounds can affect the intensity of the individual astringency attributes, such as “drying”, “chalkiness” and “coarseness” (Vidal et al., 2003). This indicates that changes in the astringency of rooibos infusions may not only depend on the concentration of astringent components, but also on alterations in their structural properties as a result of steam pasteurization. 4. Conclusions Steam pasteurization of rooibos resulted in significant changes in the SS, TP and aspalathin contents and the absorbance and “total color” (AUC) values of the infusions. These changes may, to some extent, be attributed to the instability of aspalathin during heat treatment. The anecdotal evidence that steam pasteurization of rooibos leads to subtle, but notable changes in the sensory characteristics of rooibos infusions was confirmed. The decrease in “green” flavor and aroma could improve the sensory quality of low-quality batches of rooibos, therefore steam pasteurization may be beneficial to quality in such instances. Future research should validate this finding, especially if fermentation time could be shortened in combination with steam pasteurization conditions conducive to reducing “green” notes. This could potentially negate the negative attributes associated with under-fermentation while gaining processing capacity. Acknowledgments Financial support for the study was supplied by the South African Rooibos Council (SARC) and THRIP (Grant UID 72065 to E.J.), an initiative of the Department of Trade and Industry, South Africa. The authors thank Johann Basson of Rooibos Ltd. (Clanwilliam, South Africa) for supplying the unpasteurized samples. SARC and THRIP played no other role than providing funding and a platform to report data to the industry. References Arthur, H., Joubert, E., De Beer, D., Malherbe, C. J., & Witthuhn, R. C. (2011). Phenylethanoid glycosides as major antioxidants in Lippia multiflora herbal infusion and their stability during steam pasteurisation of plant material. Food Chemistry, 127, 581–588. Beelders, T., Sigge, G. O., Joubert, E., De Beer, D., & De Villiers, A. (2012). Kinetic optimisation of the reversed phase liquid chromatographic separation of rooibos tea (Aspalathus linearis) phenolics on conventional high performance liquid chromatographic instrumentation. Journal of Chromatography. A, 1219, 128–139. Cabarello, B., Trugo, L. C., & Finglas, P. M. (2003). Tea. Encyclopedia of food sciences and nutrition (pp. 5373–5761). (2nd ed.). Oxford, UK: Elsevier Science Ltd. Devic, E., Guyot, S., Daudin, J. -D., & Bonazzi, C. (2010). Effect of temperature and cultivar on polyphenol retention and mass transfer during osmotic dehydration of apples. Journal of Agricultural and Food Chemistry, 58, 606–614. El Gharra, H. (2009). Polyphenols: Food sources, properties and applications — A review. International Journal of Food Science and Technology, 44, 2512–2518.

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