Sampling procedure and sample preparations

Random samples of brand-specific rooibos and flavoured rooibos herbal tea types and forms (n

= 2 - 3 brands in each case) were sourced from two local retail chain supermarkets (providing for duplicate purchases and samples). A retail chain supermarket located in the central and in the northern regional sectors of the City of Cape Town was used for this purpose as these stores offer a wider product range.

The controls for the different brewing times and the different flavouring additions and flavouring amounts added individually and in combination, were a standard cup of traditional / fermented rooibos herbal tea prepared using tea bags from the different selected brands and prepared according to the specifications of the SA Rooibos Council without any additions. The controls were taken from the same batches of tea prepared for the experimental samples. A batch for each brand of traditional / fermented rooibos from each supermarket was made up. Seven tea bags from each brand were steeped in 1.26 L of boiling water, stirred for 30 seconds and steeped for three minutes. The time for stirring and steeping was checked against a countdown timer. The tea bags were removed with tweezers and caution was taken to remove the least amount of liquid with the tea bags. Samples from these batches were also used for the samples with added household flavourings. For all the samples, except for the analysis of the form and type of rooibos herbal tea, traditional / fermented rooibos herbal tea bags were used as this form is the most widely available in supermarkets, representing the most commonly available rooibos herbal tea form to the typical consumer. The standard procedure was adapted to represent different brewing times as independent variables.

Prepared samples (each sample being 18 mL) were stored in a test tube and marked with a code for identification purposes with a permanent marker. The extracted and diluted samples were stored at 4^oC until analysed.

3.2.2.1 Tea type sample preparations

The different brands of traditional / fermented (n = 5), green / unfermented (n = 1) and organic traditional / fermented (n = 4) rooibos herbal tea purchased from the two retail chain supermarkets (see Addendum B) were prepared using tea bags. The time for stirring and steeping were checked against a countdown timer. The tea bags were removed with tweezers and caution was taken to remove the least amount of liquid with the tea bags.

3.2.2.2 Tea form sample preparations

In addition to the tea bag sample preparations presented above, samples were also prepared from traditional / fermented (n = 1 and n = 1, respectively), green / unfermented (n = 1 and n = 0, respectively) and organic (n = 1 and n = 0, respectively) rooibos herbal tea leaves and powder (see Addendum B). The amount of tea leaves (2.6 g) was weighed on a Sartorius 2006 MP scale and 180 mL boiling water was added, stirred for 30 seconds and steeped for three minutes (checked against the timer). The samples were strained through a sieve to separate the tea leaves from the tea sample.

Only one brand of rooibos herbal tea powder (see Addendum B) could be prepared (done in triplicate) as this form of tea is not yet readily available on supermarket shelves. Green / unfermented rooibos herbal tea powder is not yet available. A small amount of powder (50 mg) was weighed on a Sartorius 2006 MP scale and dissolved in 25 mL boiling water in ratio as per the product preparation instructions. The sample was stirred for 30 seconds and left for three minutes (checked against the timer) before the sampling took place.

RTD, unflavoured iced teas with traditional / fermented rooibos herbal tea (n = 3) (see Addendum B) were sampled directly from the containers. The iced teas in powder form (n = 6) (see Addendum B) were prepared according to the instructions on the sachet or container.

These iced tea powders were properly mixed and dissolved in the sample preparation before the sampling took place.

3.2.2.3 Brewing time sample preparations

Tea samples (n = 24) utilising different brewing times were prepared from traditional / fermented rooibos herbal tea bags. The standard brewing time according to the instructions of the SA Rooibos Council, is three minutes. The three different times that were used to evaluate the different preparation methods were chosen to represent a quick brewing time (one minute), an average (five minutes) and a long (10 minutes) brewing time (Campanella et al., 2003:732).

Samples with a brewing time of 20 and 30 minutes respectively were also prepared to determine the effect of a prolonged brewing time on the total polyphenol content and TAC. Each sample from the different brands were prepared with 180 mL boiling water added to one tea bag, stirred for 30 seconds and then steeped for the various indicated times checked against the timer.

3.2.2.4 Tea addition sample preparations

Various rooibos herbal tea samples with household flavourings / additions and commercially flavoured rooibos herbal tea samples (see Addendum B) were prepared using the batches of rooibos herbal tea as described in 3.2.2, then flavoured and sampled.

3.2.2.4.1 Household flavourings

The different amounts of household flavourings added to each sample had to be predetermined.

The small, medium and large quantities of added milk (10 mL, 20 mL and 30 mL respectively of skim, low fat and whole milk) and added sugar (4 g, 6 g and 10 g respectively of white and brown granulated sugar) to a cup of tea [180 mL as indicated by Langenhoven et al. (1991:148)]

were obtained from the Medical Research Council (MRC) Food Quantities Manual (Langenhoven et al., 1991:9,10,140). The small, medium and large amounts of honey added were determined by the weight of half a teaspoon (5 g), a level teaspoon (9 g) and a heaped teaspoon (15 g) of honey (Langenhoven et al., 1991:139).

Due to logistical reasons, long life milk cartons were purchased. One batch of milk (of skim, low fat and whole milk), sugar (of white and brown granulated sugar) and honey were used for the different samples in order to eliminate any interference through nuisance variables. Each added amount of sugar and honey were weighed on a calibrated Sartorius 2006 MP scale beforehand.

Each added amount of the different types of milk was measured with calibrated Eppendorf pipettes. The tea samples (n = 594) were taken from the prepared tea batches described in

3.2.2.1. All the combinations of added household flavourings undertaken are presented in Addendum B.

Rooibos herbal tea with added sweetener (tablet or powder form) was not sampled due to the wide variety of sweeteners available on the supermarket shelves and the different amounts in which each consumer adds this sweetening agent. There was also no rooibos sample prepared with added lemon juice as using lemon juice as a household flavouring was added to the consumer questionnaire (Phase 2 of the study) only after the preparation and analysis of the samples was completed.

3.2.2.4.2 Commercially flavoured rooibos herbal tea

Camomile, lemon and honey flavoured rooibos herbal tea bag samples (n = 10) were prepared.

Boiling water (180 mL) was added to these commercially flavoured rooibos herbal tea bags and steeped for three minutes (checked against the timer). The tea bags were removed from the infused water with tweezers with caution taken to remove the least amount of liquid. No additional flavourings were added.

Flavoured RTD iced teas were sampled directly from the container. These included iced-teas (n

= 11 samples) flavoured with peach, berry and lemon. The flavoured iced-teas in powder form were prepared according to the instructions on the sachet, whereafter the sample was taken.

Samples (n = 6) prepared from flavoured powdered iced-tea included the peach, apple, lemon and fruit punch flavours.