1. Introduction
Consumers are pushing a phenomenal surge for the potentially probiotic beverage known as Kombucha. The global sales volume reached USD 1779 million in 2020, with a compound annual growth rate (CAGR) projected at an astonishing 28.9% from 2020 to 2026, according to 360ResearchReports [
1]. Kombucha is a sweetened tea that is fermented using a Symbiotic Culture of Bacteria and Yeast (or SCOBY). Examples of SCOBY are a combination of numerous lactic acid bacteria (
Acetobacter, Brettanomyces, and
Gluconacetobacter) and yeast (
Saccharomyces,
Zygosaccharomyces). These microorganisms form a biofilm-like structure at the top of the fermenting vessel. Kombucha is said to be well accepted in a sensory way by providing a unique drink that has an element of sweetness, while giving fruitiness and sour and acidic flavours. Ivanišová et al. [
2] found that their Kombucha had a slightly better sensory preference overall compared to the sweetened black tea control, as well as improvements in taste and flavour intensity. Common flavours that are described by sensory panels seem to include lemon, vinegar, sour, and yeast flavours and aromas [
3,
4]. Mouthfeel is another sensory element to be considered. Gramza-Michałowska and collaborators [
3] made Kombucha with different tea types (e.g., green, black, or yellow) and found that black tea had the poorest sensory acceptability, perhaps due to its lack of clarity and smoothness/mouthfeel in comparison to the other three tea types used, which were white, yellow, and green teas.
Currently, there is a lack of research around the sensory profile of Kombucha using different ingredients, such as the New Zealand native Kawakawa, or even simple Kombucha [
5]. This has driven the need for research into the instrumental quality and flavour profiles of Kombucha made with New Zealand ingredients or other additives.
Black pepper (
Piper nigrum L.) and the New Zealand plant Kawakawa (
Piper excelsum) are both of the Piperaceae or pepper family and, therefore, have a unique spiciness. In New Zealand, Kawakawa has a history of being used as a medicinal plant for a range of small ailments [
6]. In comparison, black pepper is a commonly found seasoning that also has a range of health benefits attributed to it, such as improvement to digestion and the ability to cure colds [
7].
Hops are the flowers of the
Humulus lupulus L. vine that are most commonly known for their role in beer production and flavour. Hops have concentrations of alpha acids that give bitterness to beer when they are boiled and isomerised. Other components of note are essential oils, resins, and phenolics that can be imparted during a process called dry hopping or during boiling [
8]. Beta acids, found in hops, may be an issue for SCOBY growth since they have antibacterial properties. Hops can impart citrus flavours when dry-hopped (added after boiling), which may or may not have similar effects in Kombucha [
9]. The hops variety used for this experiment was Riwaka™, which has a moderately low level of alpha acids and is described as having grapefruit and passionfruit aromas.
Word clouds, also known as tag clouds, are a valid visual tool to highlight the results of consumer studies such as focus groups. Word clouds are applied to qualitative analyses. The principle is to display those words that occur more often in a given description. The occurrence and size of each word reveals the relevance of each specific result, allowing observers to quickly notice differences among samples and the reasons behind them.
The hypothesis of this research is that each ingredient will produce a different response in the panellists, resulting in unique descriptors for each type of Kombucha. These may include acetic tastes, cloudiness or clarity, etc. The different ingredient additions may impact the growth of the microorganisms within, thereby impacting the instrumental quality of the Kombucha.
The aims of this research include testing the sensory characteristics of the Kombucha through the use of a semi-trained focus group consisting of at least eight people. This panel will test four samples of Kombucha, including a control Kombucha. The panellists will look at the various sensory qualities of the Kombucha, including areas such as clarity, bubble and foam formation, and colour. The second aim includes collecting instrumental data, including °Brix, pH, titratable acidity, and alcohol percentage. Instrumental data can be useful to support the discussion of sensory profiles, providing objective information on sensory-affecting parameters such as acidity and sweetness. Word clouds were used to visually highlight the results of the focus group, providing a novel, clear tool to describe food quality.
2. Materials and Methods
2.1. Materials
The Kombucha beverages were prepared using 1 L of boiling water, 70 g of white sugar (Pams, Auckland, New Zealand), 5 g of tea, 75 g of mother liquid, and 30 g of SCOBY (Get Cultured, Tauranga, New Zealand). The composition of this SCOBY can vary based on batches, but it usually comprises the following microorganisms:
Acetobacter aceti,
A. intermedius,
A. nitrogenifigens,
A. pasteurianus,
A. xylinum,
Bacterium gluconicum,
B. xylinum,
Gluconacetobacter kombuchae,
G. oxydans,
Brettanomyces bruxellensis,
B. claussenii,
B. intermedius,
Candida collecolusa,
C. famata,
C. guilliermondii,
C. kefyr,
C. obtusa,
Koleckera apiculata,
Mycoderma sp.,
Mycotorula sp.,
Pichia membranefaciens,
Saccharomyces bisporus,
S. cerevisiae,
S. ludwegii,
Saccharomycodes sp.,
Schizosaccharomyces pombe,
Torulaspora delbrueckii,
Zygosaccharomyces bailii,
Z. kombuchaensis,
Z. lentus, and
Z. rouxii, The adjuncts were ‘Empire’ cracked black pepper and Riwaka hops (
brewshop.co.nz). The Kawakawa leaves used were from PureNature NZ (Auckland, New Zealand) and the black tea was from Bell Tea (Dunedin, New Zealand). The composition of Kawakawa dry leaves was determined in our laboratories, expressed in g/100 g of dry weight, and is as follows: protein, 17.1; minerals, 13.8; lipids, 8.41; soluble carbohydrates, 1.84; and insoluble carbohydrates, 48.2. The mineral profile was the following, expressed in mg/100 g dry weight: potassium, 3433; calcium, 1664; phosphorous, 603; magnesium, 557; sodium, 90.1; and iron, 11.9.
2.2. Kombucha Production
The SCOBY (30 g) and mother liquid (75 g) were from the same batch date, and the mother liquids were mixed, trying to maintain consistency through the four types. The boiling water, tea, and sugar were added to a pot, weighed, and kept boiling on a stove for 15 min, being stirred throughout. After boiling, boiling water was added to make up the initial weight. The tea was then filtered using a cheesecloth in a sieve before being placed in a pre-prepared ice bath to cool to approximately 22 °C. The tea was then transferred to a clean jar that had been sprayed with 70% ethanol, and the mother liquid and SCOBY were then added. The lid of the jar was placed on top but not screwed on in order to let any gas escape. The jars were transferred to a temperature-controlled room at 22 °C for seven days. The newly formed SCOBY as well as the original SCOBY were then removed, and any adjuncts added. The Kombucha was then kept in the fridge (+4 °C) overnight and in the morning was filtered using a sieve and cheesecloth.
2.3. pH Testing
The pH testing was performed using a Metrohm 730 Sample Changer Auto Titrator from MEP instruments, Newmarket (Auckland, New Zealand). The titrant used was 0.1 N NaOH from LabServ (Auckland, New Zealand), and this was administered using a Metrohm 702 SM Tritrino (Auckland, New Zealand). A sample size of 5 mL was used to perform the analysis. A standard pH meter was originally used, but this gave different results to the Auto Titrator; therefore, the Auto Titrator was chosen as the most accurate reading.
2.4. Titratable Acidity
Titratable acidity was performed using the Metrohm 730 Sample Changer Auto Titrator (Auckland, New Zealand). The titrant was 0.1 N NaOH from LabServ, and this was dispensed using a 702 SM Tritrino. A 5-millilitre sample was used. The corresponding software used was Tiamo 1.2.
2.5. Degrees Brix
The °Brix was obtained using a refractometer (Keg King, Springvale, Australia) calibrated with tap water. A drop of room temperature sample was placed on the testing panel, read, and was then cleaned from the panel using soft tissues. During preliminary testing, the °Brix test was performed daily, while in the final brew, °Brix was only obtained from day 0 and day 8, after the adjuncts were added.
2.6. Alcohol Testing
Alcohol testing was performed using a Dujardin-Salleron ebulliometer (Paris, France) that was calibrated using tap water. The boiling point of the tap water set the ebulliometer scale. A measure of the sample was added based on the ebulliometer equipment, and the temperature was read when the sample reached the boiling point. The boiling point was correlated to an equivalent alcohol percentage using the scale. The ebulliometer was cleaned between samples and was rinsed with the new sample.
2.7. Microbial Enumeration
Microbial enumeration was performed to establish the quantity of lactobacilli (probiotic bacteria) present in the beverages. Two serial dilutions were performed to 10
−2. The MRS agar plates (Fort Richard, Auckland, New Zealand) were incubated for 48 h in an anaerobic environment with Thermo Scientific™ Oxoid AnaeroGen 2.5 L sachets (Auckland, New Zealand). Then, colony forming units (log CFU/mL) were counted [
10].
2.8. Sensory Panel
Eight master’s students volunteered to be part of a sensory focus group. They were selected based on the following three criteria: experience in food science and sensory analysis, habit of drinking fermented and non-fermented beverages, and a multicultural background. The students were of different nationalities, including Chinese, Indian, and Vietnamese. They were of young age (twenties), 6 were female and 2 were male. This selection represented the consumer target of Kombucha, being mostly young females according to a recent survey by Mintel in 2019 [
11]. The participants were asked to sign a consent form that included a list of potential allergens that may be in the samples. Sensory descriptors for four different categories, namely appearance, aroma, flavour, and mouthfeel, were collected for three commercial Kombucha products during a 1-h training session. The commercial samples were a control, original Kombucha by Daily Organics (Matakana Village, New Zealand), a spiced Kombucha by Daily Organics called ‘Winter’ that included a chai tea and ginger blend, and a Ginger and Kawakawa Kombucha by Mauriora (Christchurch, New Zealand) that provided a Kawakawa flavour. The most significant descriptors were chosen to use as a reference for the Kombucha samples. A set of sensory reference samples were created to use for training; these included carbonation, bitterness, sourness, sweetness, and colour. The carbonation training was performed using sparkling water, the bitterness using a hop pellet dissolved in roughly 25 mL of water, the sourness using a lemon slice, the sweetness using a 7% sugar solution, and the colour using black tea. With the reference samples as a guide, the semi-trained panel chose and agreed upon different sensory descriptors for each Kombucha sample. One sample of each product was provided in the sessions. In the second session, samples were labelled with 3-random-digit codes. Samples were presented in the following order: control, hops, Kawakawa, black pepper. This session lasted for about one hour. A 10-minute break was allowed between the two sessions (training, samples) to avoid sensory fatigue. Water was provided as a palate cleanser. All data were recorded on a whiteboard to ensure that the panellists could change their answers and could compare to previous samples. The resulting data were tabulated for discussion. The study was approved by the Ethics Committee of Lincoln University (protocol code 2020-60, approved on 11 December 2020).
2.9. Statistical Analyses
Instrumental measurements were taken in duplicate and an average was calculated with Excel 2016. Minitab 18 software was used to calculate statistical significance using a one-way analysis of variance (ANOVA) and a post hoc Tukey test, with an α value of 0.05. An online word cloud generator (
www.wordclouds.com by Zigomatic, Vianen, The Netherlands, accessed on 3 May 2021 was used to summarise the results of the focus group and display the most agreed attributes for each sample.
4. Conclusions
The hypothesis was that each Kombucha would have a unique sensory profile depending on the different ingredients added. This turned out to be true, with distinct differences for the Kawakawa and hops Kombucha beverages, and subtler differences for the black pepper Kombucha. The differences were also shown in the instrumental analyses, with minor differences in the pH and alcohol levels among the fermented products, some of which were picked up in the sensory profiling. For example, the hops Kombucha was described as sour in taste by fewer panellists (3 vs. 5–7 people). This agreed with the significantly higher pH than the other beverages (3.72 vs. 3.49–3.54). In addition, an alcohol flavour was noted only for the Kawakawa Kombucha, which, in fact, was significantly more alcoholic than the other beverages (0.40 vs. 0.15–0.30%).
This experiment provided a range of information and potential explanations as to why the results occurred, though there are many areas that require more research for a surer knowledge. Some of these areas include the discrepancy between the professed antimicrobial, yet prebiotic nature of the Piperaceae family, particularly for Kawakawa, that have not been investigated in depth. The sweetness that black pepper gives could also benefit from a confirmation as to its origin. Finally, the foaminess of the Kawakawa Kombucha could use with further investigation, as there would likely be fairly low protein levels; therefore, a different compound may have a part to play.
What the focus group could not display was the extent of the differences. Word clouds provided this information and put it into context, allowing a visual description of the sensory profiles. Focus groups provide detailed information of scientific significance, while word clouds offer an easily readable overall view. An integration of both methods is a comprehensive approach to descriptive sensory analysis.