Phenolic content and antioxidant activity of rooibos food ingredient extracts

Journal of Food Composition and Analysis 27 (2012) 45–51

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Original Research Article

Phenolic content and antioxidant activity of rooibos food ingredient extracts Elizabeth Joubert a,b,*, Dalene de Beer a a b

Post-Harvest & Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7599, South Africa Department of Food Science, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 October 2011 Received in revised form 2 March 2012 Accepted 25 March 2012

Hot water extracts of the herbal tea, rooibos, are increasingly used as an ingredient in ready-to-drink beverages and a variety of food products. The quantity of extract solids used in the product is occasionally related to the equivalent number of one-cup-servings, yet to date no comprehensive data have been available to serve as guideline. The extent of variation in total polyphenol, aspalathin, orientin and isoorientin contents, as well as total antioxidant capacity (TAC) of hot water extract of fermented rooibos was determined and compared with that of the hot water soluble solids of infusions, prepared similar to a cup of tea. Extract preparation from a large number of individual rooibos production batches (n = 74) partly simulated industrial processing, while infusions were prepared from a sub-set of samples (n = 20). Based on the total polyphenol and aspalathin contents, rooibos extract and infusion were equivalent when compared on a soluble solids basis. The isoorientin and orientin contents of the soluble solids of the infusion were slightly higher than those of the extract. The TAC of the soluble solids of the infusion, measured with the oxygen radical absorbance (ORAC) assay, was slightly higher than that of the extract, while the opposite was observed for the TAC, measured with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. ß 2012 Elsevier Inc. All rights reserved.

Keywords: Rooibos Dihydrochalcones Flavones Antioxidant activity Beverage Food analysis Food composition Food ingredient

1. Introduction Tea, especially green tea, and herbal teas have received much attention in recent years due to their health-promoting properties, in particular their antioxidant properties. The focus fell largely on phenolic compounds as the bioactive phytochemicals responsible for the antioxidant capacity of these beverages (Arthur et al., 2011; Krafczyk et al., 2009; Lambert and Elias, 2010). Consumption of rooibos, a South African herbal tea produced from Aspalathus linearis, has grown substantially since its first introduction to the domestic market in 1904. In 2010 rooibos represented approximately 23% of the South African tea market with sales reaching more than 5000 tons. It is enjoying popularity among an estimated 10.9 million households. Germany, the Netherlands, the United Kingdom, Japan and the United States of America represent 86% of the export market of 6000 tons in 2010 (Joubert and De Beer, 2011). One of the contributing factors to the growing popularity of rooibos is its antioxidant activity and associated health-promoting properties (Joubert et al., 2008). It also led to the commercial production of hot water extracts for use as a food ingredient in a variety of food and beverage products, among

* Corresponding author at: Post-Harvest & Wine Technology Division, Agricultural Research Council (ARC) Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7599, South Africa. Tel.: +27 21 809 3444; fax: +27 21 809 3430. E-mail address: [email protected] (E. Joubert). 0889-1575/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jfca.2012.03.011

others ready-to-drink iced teas, yogurt, drinking yogurt, jam and ‘instant cappuccino’ (Joubert and De Beer, 2011), thereby increasing dietary exposure to the secondary metabolites of A. linearis. Recent research, demonstrating that daily consumption of six cups of fermented rooibos infusion over a 6-week period improved the lipid profile and redox status in adults at risk of developing cardiovascular disease (Marnewick et al., 2011), supports its ˜ o et al. (2010) potential as a health-promoting beverage. Villan demonstrated that the plasma antioxidant status of the volunteers peaked 1 h after consuming 500 mL of unfermented and fermented RTD rooibos beverages, confirming rooibos tea and its products are a source of dietary antioxidants in humans. Other studies focussed specifically on the bioavailability of the rooibos dihydrochalcone C-glucoside, aspalathin (Breiter et al., 2011; Courts and Williamson, 2009; Stalmach et al., 2009), as rooibos is the only source to date of this bioactive flavonoid. The bioactivity of aspalathin relates to its antioxidant activity (Joubert et al., 2005; Krafczyk et al., 2009; Snijman et al., 2009; Von Gadow et al., 1997), antimutagenicity (Snijman et al., 2007) and glucoselowering effect (Kawano et al., 2009; Mose Larsen et al., 2008). The presence of aspalathin and its metabolites in the plasma of volunteers after consumption of rooibos (Breiter et al., 2011; Stalmach et al., 2009) underscores its relevance as a rooibos bioactive compound. Apart from aspalathin, rooibos also contributes nothofagin, another rare C–C linked dihydrochalcone glucoside and antioxidant to the diet. The major rooibos flavones, orientin and isoorientin, are oxidation products of aspalathin.

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The aspalathin content of rooibos extracts depend on its concentration in the plant (Schulz et al., 2003), extent of fermentation of the plant material (Joubert, 1996) and processing conditions. Analysis of commercial RTD rooibos iced teas, containing hot water extracts of fermented rooibos, showed that some of the products contained no aspalathin or its oxidation products, orientin and isoorientin, suggesting either that very poor quality rooibos extract, or no rooibos extract, was used in the formulation of these products (Joubert et al., 2009). Recently, a food product claiming to provide the equivalent of ‘six cups of rooibos tea’ in a single portion was introduced to the South African market. This raises questions of batch-to-batch variation of the extract and the basis used to ensure that the required number of cups of rooibos tea is achieved in the product. Presently, no standardisation of fermented rooibos extract in terms of aspalathin is done, but a standardised extract, based on isoorientin and orientin content (>0.5% total), is available from an international ingredient company. Normally, South African extract producers use total polyphenol content and total antioxidant capacity, measured using the Folin-Ciocalteu and DPPH radical scavenging assays, respectively, as quality indicators. Several antioxidant assays, each with their own advantages and disadvantages, are available to assess plant extracts (Karadag et al., 2009; Niki and Noguchi, 2000; Prior et al., 2005). The choice of assay for quality control depends to some extent on the intended market of products. The DPPH radical scavenging assay was selected for quality control purposes of rooibos extract as it employs a stable radical and is easy to execute (Prior et al., 2005). The ORAC assay, despite its many drawbacks, is often used to evaluate total antioxidant capacity of food products and ingredients destined for the American market (Bell and Ou, 2007). Ninfali et al. (2009), analysing different commercial herbal extracts, including rooibos extract, recommended the use of ORAC values, as well as a marker compound or group of similar compounds, for improving standardisation of herbal extracts. Rutin was chosen as the marker compound for rooibos, but they concluded that it is not representative of the antioxidant activity of the extract (Ninfali et al., 2009). The aim of this study was to determine the extent of variation in total polyphenol, aspalathin, nothofagin, orientin and isoorientin contents, as well as total antioxidant capacity of simulated industrial hot water extracts of fermented rooibos, prepared from a large number of individual rooibos production batches. The same parameters were used to compare the hot water extract and infusion (‘cup of tea’) to determine whether the extract, when used at the same soluble solids content present in the infusion, would deliver the same phenolic content and antioxidant activity. 2. Materials and methods 2.1. Chemicals Chemicals and reagents were purchased from Sigma–Aldrich (Steinheim, Germany; 2,20 -azo-bis-(2-methylpropionamidine) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid (97%), ascorbic acid), Merck (Darmstadt, Germany; HPLC gradient grade acetonitrile, Folin-Ciocalteu reagent), Fluka (Buchs, Switzerland; glacial acetic acid), Riedel de Hae¨n (Seelze, Germany; sodium fluorescein), Extrasynthese (Genay, France; isoorientin (99%)) and Carl Roth (Karlsruhe, Germany; orientin (99%)). Aspalathin (97%) and nothofagin (97%) were obtained from the PROMEC unit of the Medical Research Council (Parow, South Africa). General analytical grade laboratory reagents were purchased from Sigma–Aldrich or Merck. A Modulab purifier (Continental Water Systems Corp., San Antonio, USA) was used to

prepare laboratory grade deionised water, which was further purified to HPLC grade using a Milli-Q academic water purifier (Millipore, Bedford, USA). 2.2. Sourcing of rooibos samples Fermented rooibos samples (n = 74; 500 g each), representing unrefined plant material (i.e. comminuted, but not sieved) of different production batches, were collected randomly over a sixmonth-period during the 2009 harvest year from the major rooibos processing and marketing company (Rooibos Ltd., Clanwilliam, South Africa). The samples originated from more than 55 producers spread over the production area and represented the most common quality grades, B and C, as determined by Rooibos Ltd. Grading of samples affects remuneration of producers and is based on leaf colour, infusion colour, infusion flavour and the percentage yield of refined rooibos. 2.3. Determination of yield of refined rooibos The yield was determined according to industry practice by sieving 400 g of unrefined fermented plant material through 10 and 40 mesh sieves for 1.5 min at 190 rpm using a SMC Mini-sifter (JM Quality Services, Cape Town, South Africa). The fractions <40 mesh and >10 mesh represent dust and coarse tea, respectively, while the fraction >40 mesh and <10 mesh represents refined rooibos. The percentage yield of each fraction was determined gravimetrically. 2.4. Preparation of simulated industrial extracts Duplicate extracts from each production batch were prepared by adding boiling water (100 mL) to unrefined rooibos (10 g) in a screw-cap glass extraction vessel, which was placed in a water bath at 93 8C for 30 min. The mixture was stirred every 5 min. The resulting extract was filtered through Whatman #4 filter paper while warm, followed by cooling to room temperature in a water bath. Extracts were frozen and freeze-dried using a VirTis Advantage Plus freeze-drier (SP Scientific, Warminster, PA, USA). 2.5. Preparation of ‘cup of tea’ infusions Ten samples were randomly selected from each quality grade for preparation of ‘cup of tea’ infusions (n = 20). Refined rooibos (250 g), spread in a thin layer on 40 mesh stainless steel trays, was subjected to steam pasteurisation for 2 min at >96 8C. The steam pressure, generated with a THE 400 NS Electropac electrode boiler (John Thompson Boilers, Cape Town, South Africa) was maintained at 2.76 N/m2 at the inlet to the cabinet. On removal from the cabinet, the trays were placed in a forced circulation drying tunnel for 20 min at 40 8C to remove superficial moisture and decrease the moisture content to <10%. Preparation of a ‘cup of tea’ infusion entailed adding 200 mL boiling deionised water to 2.5 g refined, pasteurised rooibos and infusing for 5 min before filtering through a tea strainer followed by filtration through Whatman #4 filter paper. Aliquots (1 mL) of each infusion, prepared in duplicate, were frozen at ca. 20 8C until total polyphenol content, flavonoid content and total antioxidant capacity analyses. 2.6. Determination of soluble solids content Soluble solids content of simulated industrial extracts before freeze-drying, and of ‘cup of tea’ infusions were determined gravimetrically by evaporating duplicate aliquots of 5 mL and 20 mL, respectively, on a steam bath followed by drying in a forced air oven at 100 8C for 60 min.

2.7. Determination of total polyphenol content and total antioxidant capacity (TAC) The total polyphenol content of the freeze-dried simulated industrial extracts reconstituted in deionised water and ‘cup of tea’ infusions was determined in triplicate using the Folin-Ciocalteu method as described by Arthur et al. (2011). The TAC of the reconstituted extracts and ‘cup of tea’ infusions was determined in triplicate using the DPPH scavenging (Arthur et al., 2011) and the oxygen radical absorbance capacity (ORAC) (Huang et al., 2002) assays. Modification of the ORAC protocol described by Huang et al. (2002) consisted of adding 300 mL of deionised water to the outside wells of each plate as a thermal barrier. A BioTek Synergy HT microplate reader with Gen5 software for data acquisition (Vermont, USA) was employed for absorbance and fluorescence readings.

(a)

19

Soluble solids extracted (g/100 g)

E. Joubert, D. de Beer / Journal of Food Composition and Analysis 27 (2012) 45–51

18

47

17

16

15

14

13

12

2.8. Quantification of individual flavonoids

0

2.9. Statistical analysis Descriptive statistical analysis and Pearson correlation analysis were performed using XLSTAT software (Version 7.5.2, Addinsoft, New York, USA). The data were subjected to analysis of variance using SAS version 9.3 (SAS Institute, Cary, NC) and analysed for normality using the Shapiro–Wilk test. The Student t-test (P < 0.05) was used to ascertain whether there were significant differences between treatments.

10

15

20

25

% Coarse fraction

(b)

1.6 1.4

Aspalathin content (g/100 g)

Quantification of aspalathin, nothofagin, isoorientin and orientin was performed in duplicate by HPLC with diode-array detection as previously described by Joubert et al. (2009) on an Agilent 1200 system (Agilent, Santa Clara, CA, USA). Calibration curves were prepared using five concentrations of aspalathin, nothofagin, isoorientin and orientin from 0.01 to 2 mg per injection. Peaks were identified based on comparison of their retention time and spectral characteristics with those of authentic reference standards.

5

1.2 1 0.8 0.6 0.4 0.2 0 0

3. Results

0.05

0.1

0.15

0.2

Nothofagin content (g/100 g)

3.1. Simulated industrial extract

(c)

1.1

Isoorientin content (g/100 g)

The soluble solids content of unrefined rooibos and its characterisation in terms of particle size fractions, i.e. coarse material (>10 mesh), refined tea (<10 mesh; >40 mesh) and dust (<40 mesh), are summarised in Table 1. Extraction of unrefined rooibos gave extract concentrations ranging from 12.46 to 18.31% soluble solids. Unrefined rooibos consisted of approximately 13% coarse plant material and 4% ‘dust’ or very fine plant material. A weak (r = 0.300), but significant (P = 0.009), negative correlation was obtained between the coarse fraction and soluble solids content of the unrefined rooibos (Fig. 1a). The phenolic composition and total antioxidant capacity of the simulated industrial extracts of the unrefined rooibos are presented as box plots in Fig. 2a and b, respectively. Total

1.2

1 0.9 0.8 0.7 0.6 0.5

Table 1 Soluble solids extracted from unrefined rooibos tea (g/100 g unrefined tea) and percentage yield of refined tea, coarse tea and dust (n = 74). Parameter

Value

Soluble solids Refined tea Coarse tea Dust

15.24  1.31 82.85  3.70 12.97  3.85 3.91  1.78

(12.46–18.31) (74.46–93.55) (4.45–23.82) (1.32–8.60)

0.4 0.4

0.5

0.6

0.7

0.8

0.9

Orientin content (g/100 g) Fig. 1. Correlation of (a) soluble solids extracted from unrefined tea and percentage coarse fraction in unrefined tea, (b) aspalathin and nothofagin contents, and (c) isoorientin and orientin contents.

E. Joubert, D. de Beer / Journal of Food Composition and Analysis 27 (2012) 45–51

Content (g/100 g)

(a)

35

(b)

30

TAC ( mol/g extract)

48

25 20

24.10 - 30.69 (27.09)

15 10

Table 2 Correlations (r) of phenolic compound and total polyphenol contents with total antioxidant capacity (TAC) of rooibos simulated extracts (values in bold indicates P < 0.01).

12000 10000 8000 6000

1808 - 2518 (2180)

4000 2000

5

DPPH

Content (g/100 g extract)

TP

1.8 1.6

TACDPPH

TACORAC

0.224 0.407 0.486 0.179 0.170 0.178 0.420 0.388

0.003 (P = 0.978) 0.065 (P = 0.583) 0.067 (P = 0.570) 0.256 (P = 0.028) 0.220 (P = 0.060) 0.243 (P = 0.037) 0.053 (P = 0.654) 0.140 (P = 0.235)

(P = 0.055) (P < 0.001) (P < 0.001) (P = 0.127) (P = 0.147) (P = 0.130) (P < 0.001) (P = 0.001)

0

0

(c)

7021 - 9968 (8448)

Parameter Total polyphenols Aspalathin Nothofagin Isoorientin Orientin Sum of flavones Sum of dihydrochalcones Sum of quantified compounds

ORAC

0.156 - 1.524 (0.581)

1.4

0.473 - 1.026 0.443 - 0.895 (0.834) (0.789)

1.2 1.0 0.8 0.6

3.2. ‘Cup of tea’ infusion

0.029 - 0.175 (0.069)

0.4 0.2 0.0 Aspalathin

Nothofagin

significantly negative correlation with TACORAC. Although not significant (P = 0.060), the same trend was observed for orientin. 3D-plots of the values of the current quality parameters, total polyphenol content and TACDPPH used by industry, combined with a single phenolic marker, did not show clear grouping according to the two quality grades, B and C (data not shown). Changing TACDPPH for TACORAC, however, almost separated the two grades (Fig. 3a–c).

Isoorientin

Orientin

Fig. 2. Boxplots for (a) total polyphenol (TP) content, (b) total antioxidant activity (TAC) and (c) phenolic composition of rooibos simulated industrial extracts (range and mean shown above or below each box).

antioxidant capacity is represented by DPPH scavenging ability and ORAC, both expressed in Trolox equivalents. The variation in the content of the dihydrochalcones, aspalathin and nothofagin, and the flavones, orientin and isoorientin, is depicted in Fig. 2c. Hot water extract of unrefined fermented rooibos contained on average 27.09% of total polyphenols, expressed as gallic acid equivalents. The values varied between 24.10 and 30.69%. The four flavonoids comprised 2.22% of the extract, with nothofagin present at less than 0.1%. Isoorientin and orientin combined comprised 1.57% of the extract. The greatest variation in individual flavonoid content was observed for aspalathin with values ranging from 0.156 to 1.524% (maximum/minimum ratio = 9.8). Nothofagin was present in substantially lower quantities (range 0.029–0.175%; maximum/ minimum ratio = 6.1). A moderate correlation (r = 0.844) was observed between the aspalathin and nothofagin contents of the extracts (Fig. 1b). Less variation was observed between the maximum and minimum values for the aspalathin oxidation products, isoorientin and orientin. Their contents varied, respectively, between 0.473 and 1.026% (maximum/minimum ratio = 2.17) and between 0.443 and 0.895% (maximum/minimum ratio = 2.0). A good correlation (r = 0.946) existed between the isoorientin and orientin contents of the extracts (Fig. 1c). The total antioxidant capacity obtained with the ORAC assay (TACORAC) was substantially higher than for the DPPH scavenging assay (TACDPPH) (ca. 3.9 times higher) (Fig. 2b). No correlation between the total polyphenol content and either of the TAC values was observed (Table 2). The ORAC assay gave larger variation in the total antioxidant capacity of the extracts than the DPPH scavenging assay. Poor, but significant correlations (P < 0.001) between a single compound and the antioxidant assays were demonstrated for nothofagin and aspalathin with TACDPPH (Table 2). The isoorientin content of the extracts showed a poor, but

Refined rooibos, prepared from a sub-set of randomly selected samples, comprised the fraction (<10 mesh; >40 mesh) that is used in pasteurised form by the retail sector for loose tea and tea bags. Infusions were prepared from the sub-set of refined rooibos samples under conditions simulating the preparation of a cup of rooibos. The hot water soluble solids content of a cup of rooibos infusion, equalling 200 mL, ranged from 176 to 268 mg with an average of 227 mg (Table 3). Under these condition 9.08% of the refined rooibos was solubilised. The total polyphenol content of the hot water soluble solids, released during the preparation of the infusions, varied between 22.98 and 27.95% (Table 3), and were slightly lower than that of the simulated industrial extract (Fig. 1a). Similarly, lower minimum and maximum TACDPPH values were obtained for the infusions than for the extracts, but the reverse was true for TACORAC. Considering the individual compounds, the average aspalathin and nothofagin contents of the soluble solids in the infusions were within the range observed for the simulated industrial extracts. Higher values were, however, observed for the isoorientin and orientin contents of the infusions (Table 3). 3.3. Simulated industrial extract equivalent to a ‘six-cup-serving’ Since the equivalent of six cups of rooibos recently became a ‘gold standard’ by industry as it relates to the quantity perceived as eliciting a measurable beneficial health effect, the phenolic composition and total antioxidant capacity of the simulated industrial extract was re-evaluated, using the average soluble solids content of a cup of rooibos infusion (227 mg/200 mL) as basis. The equivalent values in terms of phenolic content and TAC for 1362 mg of hot water soluble solids obtained through extraction, but representing the average amount of soluble solids ingested when drinking six cups of rooibos infusion, were thus calculated. These equivalent values were calculated for the full set of simulated industrial extracts (n = 74), as well as only for the subset of extracts prepared from the same rooibos samples used for the preparation of the infusions (n = 20) (Table 4). Based on this direct comparison 1362 mg extract would deliver the same total polyphenol, aspalathin and nothofagin contents (P  0.05), but a lower flavone content than six cups of rooibos infusion (P < 0.05). Similar chromatographic profiles at 288 nm and 350 nm were obtained for a simulated industrial extract (Fig. 4a and b), prepared

E. Joubert, D. de Beer / Journal of Food Composition and Analysis 27 (2012) 45–51

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Table 3 Soluble solids content, phenolic composition and antioxidant activity values for rooibos infusions (n = 20). Parameter

Value

Soluble solidsa Total polyphenolsb Aspalathinc Nothofaginc Isoorientinc Orientinc TACDPPHd TACORACd

227.0  22.6 (176.2–267.7) 25.78  1.12 (22.98–27.95) 0.525  0.175 (0.264–0.979) 0.063  0.019 (0.033–0.089) 1.199  0.077 (1.110–1.345) 0.936  0.056 (0.856–1.040) 1777  114 (1572–2013) 9038  562 (8186–10175)

a

mg soluble solids per cup of tea (200 mL). g gallic acid equivalents/100 g soluble solids. c g/100 g soluble solids. d total antioxidant activity expressed as mmol Trolox equivalents/g soluble solids. b

Table 4 Phenolic composition and antioxidant activity values for rooibos infusions and industrial extracts per 6 cups (200 mL per cup). Parameter

Infusions (n = 20)

Industrial extracts (n = 20)

Industrial extracts (n = 74)

Total polyphenolsa Aspalathinb Nothofaginb Isoorientinb Orientinb TACDPPHc TACORACc

352.5  36.9 a 7.30  2.90 a 0.86  0.29 a 16.39  1.79 a 12.79  1.31 a 2448  282 b 12317  925 a

368.6  16.5 a 7.30  2.47 a 0.85  0.27 a 11.44  0.94 b 10.08  0.87 b 2792  211 a 11272  772 b

368.8  19.0 7.92  3.59 0.94  0.37 11.35  1.22 10.06  0.96 2968  249 11503  900

Different alphabet letters in a row indicate significant (P < 0.05) differences between means. a mg gallic acid equivalents per 6 cups. b mg per 6 cups. c Total antioxidant capacity expressed as mmol Trolox equivalents per 6 cups.

4. Discussion

Fig. 3. 3D-plots of total polyphenol (TP) content against oxygen radical absorbance capacity (ORAC) against (a) aspalathin, (b) isoorientin, and (c) orientin contents of hot water extract of fermented rooibos. TE, Trolox equivalents; GAE, gallic acid equivalents; black marker, grade B; grey marker, grade C.

from unrefined rooibos, and the infusion of its corresponding refined fraction (Fig. 4c and d). The average TACDPPH of the extract was slightly higher (P < 0.05) than that of the corresponding infusions, while the opposite was observed for TACORAC.

The utilisation of rooibos has moved beyond its traditional use as herbal tea to intermediate value-added products such as extracts for the beverage and food markets (Joubert and De Beer, 2011). Fermented rooibos provides the bulk of the material for production of spray-dried hot water extract, which is used predominantly in beverages and functional foods as an ingredient. The extract powder, mixed with sugar, is also sold as ‘instant’ rooibos, affording the consumer rooibos in a more convenient form. The present study showed that the hot water extract, reconstituted to a ‘cup of tea’ strength, i.e. a one-cup-serving on the basis of soluble solids content, would provide more or less the same total polyphenol and dihydrochalcone contents, but a lower flavone content than a freshly prepared rooibos infusion. The two TAC assays gave opposing trends with the soluble solids of the extract and infusion, respectively, having the higher TACDPPH and TACORAC values. In the case of a food product containing the equivalent of a six-cup-serving of fermented rooibos per portion, its taste and colour may be impaired as at least 1.3 g of hot water fermented rooibos extract would be required. For extract production the fermented plant material is used ‘asis’, i.e. waste material in the form of coarse stems and whole leaves generated during down-stream product refinement for the herbal tea market is normally not removed because the appearance of the plant material is not important. Previously it was demonstrated that the waste material gives a low recovery of soluble solids (8.9% vs. 20.4% for the sieved fraction <10 mesh) (Joubert, 1984). It would explain the correlation, although weak, observed between the percentage of coarse fraction and the soluble solids content of the unrefined fermented rooibos.

E. Joubert, D. de Beer / Journal of Food Composition and Analysis 27 (2012) 45–51

50

450

(a)

1200

Absorbance (mAU)

Absorbance (mAU)

1400

1000 800 600 400 1

200

2

0

(b)

400 350 300

3

250

4

200 150 100 50 0

0

3

6

9

12

15

0

3

Time (min)

(c)

Absorbance (mAU)

Absorbance (mAU)

2500 2000 1500 1000 500

1 2

0 0

3

6

9

6

9

15

12

Time (min)

12

15

1000 900 800 700 600 500 400 300 200 100 0

(d) 3 4

0

3

Time (min)

6

9

12

15

Time (min)

Fig. 4. Example chromatograms of a simulated industrial rooibos extract at 288 nm (a) and 350 nm (b) and a rooibos infusion at 288 nm (c) and 350 nm (d) (1, aspalathin; 2, nothofagin; 3, isoorientin; 4, orientin).

A number of factors may contribute to the quantitative difference in composition between the extract and infusion, specifically with regard to the flavone content. Stem content and parameters affecting mass transfer such as particle size of the raw material and solvent-to-solid ratio during extract and infusion preparation would play a role. Compared with previous data concerning commercial spraydried extracts (Joubert et al., 2009) the simulated industrial extracts contained more aspalathin (0.58% vs. 0.26%) and isoorientin (0.83% vs. 0.64%), but less orientin (0.79% vs. 1.16%). These differences are attributed to natural variation in the phenolic composition of fermented rooibos, and not the process conditions as spray-drying as opposed to freeze-drying did not affect the phenolic composition of extracts (Joubert et al., 2009). The absence or low levels of specifically aspalathin in RTD fermented rooibos beverages on the market (Joubert et al., 2009) is a clear indication that either very poor quality extract or very low quantities were used by manufacturers. The data presented in this paper originated from rooibos classified into two quality grades, B and C. Parameters that are taken into account during grading are the percentage refined rooibos fraction of a production lot, as well as the sensory evaluation of the appearance (colour and brightness) and flavour (aroma, taste and mouth-feel) of an infusion prepared from such a fraction according to a standard protocol. It is thus interesting to note that although none of the extract parameters quantified in the present study is used to determine the grade of rooibos, the combination of total polyphenol content, TACORAC and the content of a single compound, either aspalathin, isoorientin or orientin, could differentiate rooibos production batches according to grade. Grade C rooibos consistently gave extracts with higher TACORAC values. The combination of these parameters could, therefore, be useful in product standardisation or at least in specifying minimum levels. Aspalathin as a marker compound for chemical standardisation has the benefit that it is unique to rooibos and can, in consequence, be used for authentication. On the other hand, isoorientin and orientin are more stable under heat processing (Joubert et al., 2009) and varying pH conditions than aspalathin (De Beer et al., 2011).

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