CHANGES OF MANGOSTEEN PEEL KOMBUCHA

DURING FERMENTATION

By

Mr. Wei Chieh Liao

ID: 6329601

A special project submitted to the Faculty of Biotechnology, Assumption University

in partial fulfillment of the requirement for the degree of Masters of Science
in Food Biotechnology
2022

I
 1
2 Title: CHANGES OF MANGOSTEEN PEEL KOMBUCHA
3 DURING FERMENTATION
4 By: Wei Chieh Liao
5 Advisor: Dr. Roungdao Klinjapo
6 Level of study: Master of Science
7 Department: Food- Biotechnology
8 Faculty: Theophane Venard School of Biotechnology
9 Academic year: 2022
10
11
12
13
14 …………….……………………………...…………Advisor
15 ( Dr. Roungdao Klinjapo)
16 Chairperson of Food Technology Department,
17 Faculty of Biotechnology, Assumption University
18
19
20 ……….………………………………...…………Co-advisor
21 (Asst. Prof. Dr. Atittaya Tandhanskul)
22 Assistant Dean for Global Academic Development and Industrial Collaboration,
23 Faculty of Biotechnology, Assumption University
24
25
26 ……………………………...………… Internal Committee
27 (Asst. Prof. Dr. Patchanee Yasurin)
28 Dean,
29 Faculty of Biotechnology, Assumption University
30
31
32 ……………………………...………… External Committee
33 (Asst.Prof. Dr.Aussama Soontrunnrudrungsri) ,
34 Product Development Department,
35 Faculty of Agro-Industry, Kasetsart University
36

II
37 CONTENT
38
39 LIST OF TABLES Ⅴ
40 LIST OF FIGURES Ⅵ
41
42 ACKNOWLEDGEMENT Ⅶ
43 ABSTRACT Ⅷ
44 CHAPTER 1: INTRODUCTION 1
45 CHAPTER 2: LITERATURE REVIEW 4
46 2.1) Kombucha 4
47 2.1.1) Microorganisms 4
48 2.1.2) Chemical composition 6
49 2.1.3) Health benefits 7
50 2.2) Mangosteen (Garcinia mangostana L.)
51 2.2.1) Tree and fruit 8
52 2.2.2) Benefits and uses 8
53 2.3) Method for sugar analysis 12
54 2.3.1) Phenol-Sulfuric Acid Method 12
55 2.3.2) Somogyi–Nelson method 13
56 2.3.3) Dinitrosalicylic acid method 13
57 2.3.4) High-performance Liquid Chromatography (HPLC) method 14
58 CHAPTER 3: METHODOLOGY
59 3.1) Materials 15
60 3.2) Preparation of mangosteen peel kombucha 15
61 3.3) Investigation of the chemical changes and sensory quality of
62 Mangosteen peel Kombucha during fermentation 15
63 3.4)Determination of total polyphenolic compound (TPC) by Folin-Ciocalteu
64 Method 16
65 3.5) Microbiological analysis of yeast and acetic acid bacteria 16
66 3.7) Chromatographic condition for sugar determination 17
67 3.8) Statistical analyses 17

III
68 CHAPTER 4: RESULT AND DISCUSSION 18
69 1. Preparation of mangosteen peel kombucha 18
70 2. Investigation of the chemical changes and sensory quality of
71 Mangosteen peel Kombucha during fermentation 19
72 2.1 Color analysis of Mangosteen peel kombucha 19
73 2.2 Physio-chemical change of mangosteen peel kombucha 24
74 2.3 Sensory evaluation 25
75 3. Total polyphenolic compound content in MPK 27
76 4. Microbiological analysis of acetic acid bacteria and yeast 28
77 5. HPLC result of sugar concentration change in MPK during fermentation period 31
78

79
80 CHAPTER 5
81 Conclusion…………………………………………………………31
82 Reference ………………………………………………………… 32
83 Appendix ………………………………………………………… 38
84

IV
 85 LIST OF TABLES
86
87
88 Table 1. Sensory evaluations of mangosteen peel kombucha in 9-point hedonic
89 score…………25
90 Table 2. Characterization of acetic acid bacteria and yeast isolate from MPK……………….27
91 Table 3. Change of sugar content of fructose, glucose and sucrose during 15 days MPK
92 Fermentation…………………………………………………………………………………….28
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115

V
116 LIST OF FIGURES
117
118
119 Figure 1: Main metabolic activity of Kombucha………………………………………………6
120 Figure 2: Cross section of mangosteen fruit showing aril and pericarp………………………..9
121 Figure 3 . Mangosteen peel kombucha and its SCOPY during fermentation………………….18
122 Figure 4: Change of MPK colors as L* , a* , and b* value during fermentation ……………..20
123 Figure 5: Color of mangosteen peel kombucha at various ratios between Thai tea and mangosteen
124 peel after fermentation……………………………………………………………21
125 Figure 6: Change of pH of MPK during fermentation…………………………………………22
126 Figure 7: Acidity changes of MPK during fermentation……………………………………….23
127 Figure 8: Change of total soluble solid of MPK during fermentation………………………….23
128 Figure 9: Change of total phenolic content during MPK fermentation………………………….27
129 Figure 10: Change of sugar content of fructose , glucose and sucrose during 15 days MPK
130 fermentation……………………………………………………………………………………28
131
132
133
134

VI
135 ACKNOWLEDGEMENT
136
137 I would like to express my sincere gratitude and deepest appreciation to my advisor,
138 Dr. Roungdao Klinjapo, co-advisor, Dr. Atittaya Tandhanskul and mentor, for their kindness in
139 suggestion, valuable guidance, enduring patient and their and extensive knowledge in the lab and
140 as well as during the thesis writing has help me enormously, which makes completion of this
141 project possible.
142
143 Beside my advisors, I would also like to thank my committee I do greatly appreciate my
144 committees Asst. Prof. Dr. Patchanee Yasurin and Asst. Prof. Dr. Aussama Soontrunnrudrungsri
145 for their willingness to participate in my research project presentation and for their invaluable
146 comments and insight.
147
148 Sincere thanks are likewise expressed to lab assistants in Faculty of Biotechnology,
149 Khwanrerai Charusith (P’Tuk), for their assistance and help during my lab experiments.
150
151 Last, but never the least, I would like to thank my parents and my family for their solid
152 support and encouragement. The achievement would not have been possible without them.
153
154
155
156
157 Mr. Wei Chich Liao
158 March, 2023
159
160
161
162
163
164
165

VII
166 ABSTRACT
167
168 Every year, a significant amount of mangosteen peel is discarded as waste. Various

169 research reported on its high medical value due to its abundance of bioactive compounds including

170 others utilization in foods and cosmetics. Kombucha is one of the recent trends in probiotics

171 beverage containing high health benefits. Therefore, as the medicinal properties of mangosteen

172 peel and the health benefits of kombucha, the mixture of mangosteen peel powder and Thai tea

173 was used to formulate the mangosteen peel kombucha (MPK) at various ratios (1:2, 1:1, 2:1, and

174 1:0), and evaluated MPK based on color, pH, acidity, total soluble solids (TSS), and sensory

175 attributes. Throughout the fermentation process, mangosteen peel kombucha (MPK) showed an

176 increasing of pH and total phenolic compounds (TPC), while the acidity was decreased. MPK

177 made with a 1:1 ratio exhibited the highest preference score at 7.2. Then, MPK with a 1:1 ratio

178 was studied the chemical and biological changes through fermentation. As the results, MPK had

179 pH 2.85±0.03, an acidity 0.19±0.03 g/L, and TPC content of 72.91 mg GAE/g sample at the end

180 of fermentation. Both yeast and acetobacter were found in the MPK samples. Moreover, MBK

181 was analyzed the change of sugar both sucrose and fructose content using HPLC. During

182 fermentation, sucrose was degraded to fructose and glucose resulting in the increasing of fructose

183 to the highest level on the 9th day. Sucrose was completely degraded within 12 days of

184 fermentation. In the last day of fermentation, MPK has remain fructose 14.12±0.79 g/L and

185 glucose 33.13±1.99 g/L.

186

187 Keywords: Kombucha, mangosteen peel, Thai tea, fermentation, sensory

188
189
190

VIII
191 CHAPTER 1
192 INTRODUCTION
193
194 In the past 15 years, there have increasing population that willing to adopt the trend of

195 eating and drinking healthy in Thailand. Due to the fast lifestyle that modern people have live in

196 people prefer eating healthy food over exercise to combat weight gaining. Recently, various types

197 of tea have become associated with human eating and living habits (Marcia da Silva Pinto, 2012;

198 Martínez Leal et al., 2018), and numerous research papers have been published about how the

199 antioxidants and organic compounds in tea can impact human health. Consumers' demand for

200 "healthy" food and beverages is considered a driving force behind the growth of the functional

201 foods sector (Corbo et al., 2014), and the functional beverage market is one of the fastest-growing

202 segments in the functional food market (Kim and Adhikari, 2020).

203 Kombucha is well-known and popularized in western country such as the U.S.A. and

204 European countries for its health benefits. It is a beverage made by fermenting tea and sugar with

205 a Symbiotic Culture of Bacteria and Yeast (SCOBY) for 7-10 days (Basak, 2013; Villarreal‐Soto

206 et al., 2018; Kapp and Sumner, 2019). Generally, black tea is used, although green and oolong

207 teas may also be used. The Kombucha SCOBY consists of acid-tolerant bacteria (AAB), such as

208 Komagataeibacter, Gluconobacter, and Acetobacter species (Roos and Vuyst, 2018; Villarreal‐

209 Soto et al., 2018), as well as lactic acid bacteria (LAB), including Lactobacillus and

210 Lactococcus(Marsh et al., 2014), and yeast strains such as Saccharomyces cerevisiae (Sreeramulu

211 et al., 2000; Dufresne and Farnworth, 2000). During fermentation, the activity of certain strains

212 of AAB leads to the formation of a floating biofilm on the surface of the growth medium

213 (Villarreal‐Soto et al., 2018). An optimum fermentation time is required to produce drinkable

214 Kombucha (Sreeramulu et al., 2000).

1
215 Kombucha fermentation results in the production of many organic acids, including acetic,

216 gluconic, glucuronic, tartaric, malic, and citric acids, as well as ethanol. These compounds inhibit

217 pathogenic bacteria or mold(Martínez Leal et al., 2018; Kim and Adhikari, 2020). Kombucha also

218 contains 14 amino acids, vitamins, and some hydrolytic enzymess (Villarreal‐Soto et al., 2018),

219 which give it multiple functional properties, such as anti-inflammatory potential and antioxidant

220 activity, the reduction of cholesterol levels and blood pressure, reduction of cancer propagation,

221 improvement of liver, immune system, and gastrointestinal functions (Jayabalan et al., 2011; Kapp

222 and Sumner, 2019).

223 Mangosteen, also known as the "queen of fruit," is a fruit that grows in many Southeast

224 Asian countries, including Thailand, Malaysia, and Myanmar. It is highly valued for its taste,

225 fragrance, nutritional richness, and antioxidant strength. However, the flesh of mangosteen is

226 typically eaten fresh as dessert, resulting in large amounts of discarded peels (Suttirak and

227 Manurakchinakorn, 2012). Interestingly, the peel and seed of the mangosteen have multiple uses

228 in medical, cosmetic, and health-care applications, such as in drugs, soaps, shampoos, and food

229 supplements. This is due to the bioactive compounds found in the mangosteen peel, which are

230 beneficial to the human body (Moopayak, 2020).

231 Carbohydrates have played a vital role in both the human diet and the food industry.

232 Globally, 70% of caloric intake comes from carbohydrates. However, over the past 10-20 years,

233 sugar consumption has increased, which has been linked to the rise of metabolic disorders such as

234 cardiovascular diseases, type 2 diabetes, hypertension, dyslipidemia, gout, and obesity (Şana,

235 2016).

236 Therefore, Mangosteen Peel Kombucha(MPK) was developed to find the best ratio

237 between mangosteen peel powder and Thai tea including the monitoring of physiochemical

2
238 changes during kombucha fermentation process; pH, color, acidity and sugar content (sucrose,

239 fructose, and glucose) using HPLC analysis.

240

241
242

3
243 CHAPTER 2
244 LITERATURE REVIEW
245
246
247 2.1 KOMBUCHA

248 Kombucha tea is a traditional non-alcoholic fermented beverage originating in the East and

249 yet is quite popular today in the West (Teoh et al., 2004; Chakravorty et al., 2016). Kombucha

250 was first introduced into European countries from China by Portuguese and Dutch explorers as a

251 medicinal herb (Hollman et al., 1996). The traditional way of making kombucha consists in the

252 brewing of black tea liquor to which sucrose is added 5 – 8% (Jayabalan et al., 2014; Tran et al.,

253 2020). Being originally homemade, it has become an industrially produced soft drink whose

254 quality standards are poorly defined and whose production process is still not fully controlled.

255 2.1.1 Microorganisms

256 Kombucha is a refreshing health-promoting beverage of sugared tea infusion fermented by

257 a symbiotic consortium of yeast species (Schizosaccharomyces pombe, Saccharomycodes

258 ludwigii, Saccharomyces cerevisiae, etc.), acetic acid bacteria (AAB; Komagataeibacter,

259 Gluconobacter, and Acetobacter xylinum, A. xylinoides, Bacterium gluconicum), and lactic acid

260 bacteria (LAB; Lactobacillus, and Lactococcus) (Dufresne and Farnworth, 2000; Teoh et al., 2004;

261 Sreeramulu et al., 2011, Jayabalan et al., 2014; Villarreal‐Soto et al., 2018). There is no single

262 “culture” or microbial consortium for developing kombucha but instead a multitude of matrix

263 dependent consortia whose origins are unknown. It appears that the only constant element that

264 defines a kombucha culture is the simultaneous presence of yeasts and acetic acid bacteria, lactic

265 acid bacteria not being always present (Tran et al., 2020).

4
266 Bacteria and yeasts present in the medium create a powerful symbiosis capable of

267 inhibiting the growth of contaminating microorganisms. Its fermentation process also leads to the

268 formation of a floating biofilm on the surface of the growth medium due to the activity of certain

269 strains of AAB. Kombucha tea microbial community can be divided into two phases: a floating

270 cellulosic biofilm and the underlying sour liquid phase (Chakravorty et. al., 2016; Villarreal‐Soto

271 et al., 2018). Biofilms are also called tea fungus, but it is not a real mushroom. That name is

272 wrongly given due to the ability of bacteria to synthesize a floating cellulose network which

273 appears like surface mold on the undisturbed, unshaken medium (Jayabalan et. al., 2014).

274 The different yeasts and bacteria species act in parallel producing two different final

275 products: the fermented tea and the biofilm. A. xylinum could synthesize a floating cellulose

276 network which enhances the association formed between bacteria and fungi. The yeast cells

277 convert sucrose into fructose and glucose and produce ethanol. Acetic acid bacteria convert

278 glucose to gluconic acid and fructose into acetic acid. Acetic acid stimulates the yeast to produce

279 ethanol and ethanol in turn can be helpful to acetic acid bacteria to grow and produce acetic acid.

280 Both ethanol and acetic acid have been reported to have antimicrobial activity against pathogenic

281 bacteria thereby providing protection against contamination of biofilm or tea fungus (Dufresne

282 and Farnworth, 2000; Tran et. al., 2020).

283 As shown in Figure 1, at the beginning of the fermentation, yeast hydrolyze sucrose into

284 glucose and fructose, formerly the ethanol is produced and AAB transform ethanol into acetic acid,

285 nonetheless the production of gluconic and glucuronic acids is notable (Villarreal‐Soto et al.,

286 2018).

287

5
288

289 Figure 1: Main metabolic activity of Kombucha (Villarreal‐Soto et al., 2018).

290

291 2.1.2 Chemical composition

292 Chemical analysis of kombucha showed the presence of various compounds including

293 organic acids (mainly acetic, gluconic, glucuronic acid, citric, L-lactic, malic, tartaric, malonic,

294 oxalic, succinic, pyruvic, and usnic); sugars (sucrose, glucose, and fructose), water-soluble

295 vitamins, amino acids, biogenic amines, purines, pigments, lipids, proteins, hydrolytic enzymes,

296 ethanol, carbon dioxide, polyphenols, minerals (manganese, iron, nickel, copper, zinc, plumb,

297 cobalt, chromium, and cadmium), anions (fluoride, chloride, bromide, iodide, nitrate, phosphate,

298 and sulphate), D-saccharic acid-1,4-lactone, and metabolic products of yeasts and bacteria

299 (Jayabalan et al., 2014; Neffe-Skocińska et al., 2017; Tran et al., 2020; Ivanišová et al., 2020).

300 Acetic acid, gluconic acid, and ethanol are the main components in the liquid (Villarreal‐

301 Soto et al., 2018) including tartaric, lactic, glucuronic, citric, malic, folic, malonic, oxalic, succinic,

302 pyruvic, and ascorbic acids (Neffe-Skocińska et al., 2017; Tran et al., 2020). Moreover, the active

303 compounds of kombucha with potential benefits for human health originate from tea polyphenols,

304 in particular epigallocatechin gallate (Tran et al., 2020). Jayabalan et al. (2007) investigated

6
305 epicatechin isomers EGCG ([-]-epigallocatechin-3-gallate), EGC ([-]-epigallocatechin), ECG (-]-

306 epicatechin-3-gallate), and EC ([-]-epicatechin) and demonstrated changeable stability during the

307 fermentation process.

308 The color hue of kombucha is mainly due to the presence of the pigment polyphenols

309 extracted from the tea and the decrease of pH could be the cause of the change of color (Tran et

310 al., 2020). However, color of kombucha tea was lighter in comparison to the color of black tea

311 and this suggested that polyphenols did undergo microbial change in the acidic environment by

312 the enzymes liberated by bacteria and yeast (Jayabalan et al., 2007). Kombucha has slightly sweet

313 and slightly acidic flavor like sparkling apple cider. The taste of the kombucha changes during

314 fermentation from a pleasantly fruity sour-like sparkling flavor after a few days to a mild vinegar-

315 like taste after a long incubation period (Jayabalan et al., 2014; Tran et al., 2020). While the

316 sourness of Kombucha resulted from the organic acid presented during fermentation process, the

317 sweetness of the Kombucha is dependent on the residual of sugars, which is conditioned by the

318 initial amount of sweetener added to the tea and the consumption of this substrate by

319 microorganisms during the elaboration (Dufresne and Farnworth, 2000; Jayabalan et al., 2014;

320 Villarreal-Soto et al., 2018; Tran et al., 2020). Bitterness in kombucha, if not masked by the

321 sweetness, can take origin from tea caffeine and polyphenols (Tran et al., 2020).

322 2.1.3 Health benefits

323 In recent times, Kombucha tea has seen considerable increase in interest worldwide and

324 can easily be said to be an emerging popular beverage. One of the most important reasons behind

325 the rise of the beverage is its claimed health benefits (Jayabalan et al., 2014; Chakravorty et al.,

326 2019). Many benefits for health have been reported based on personal observation and

327 testimonials (Greenwalt et al., 1998). Of the various health benefits of Kombucha tea, its

7
328 antimicrobial, antioxidant, antidiabetic, and anticancer benefits are most attractive and appealing

329 to ever increasing cohort of scientific investigators and entrepreneurs. The last decade saw

330 noteworthy progress toward understanding the beneficial properties of this fermented tea.

331 Scientific reports claim that drinking Kombucha tea can prevent several types of cancer,

332 cardiovascular diseases, invigorate liver functions, and stimulate the immune system (Chakravorty

333 et al., 2019). Allen (1998) reported beneficial effects that were antibiotic properties, regulation of

334 gastric, intestinal and glandular activities, relief of joint rheumatism, gout and haemorrhoids,

335 positive influence on the cholesterol level, arteriosclerosis, toxin excretion and blood cleansing,

336 diabetes, nervousness, and aging problems. Moreover, on the long-term health benefit research.

337

338 2.2 MANGOSTEEN (Garcinia mangostana L.)

339 Mangosteen (Garcinia mangostana L.) is commonly known as the queen of fruit.

340 Mangosteen is an economically important fruit both for domestic consumption and export.

341 Thailand is the world’s largest producer (Te-chato and Lim, 2004). In 2012, 200,000 tons of fruits

342 were harvested, and 150,000 tons were exported (Office of Agricultural Economics, Ministry of

343 Agriculture and Cooperatives, Thailand, 2014).

344 2.2.1 Tree and fruit

345 Mangosteen is an evergreen tropical tree belonging to the family Clusiaceae and the Genus

346 Garcinia that grows in Southeast Asia. (Wanapat et al. 2014; Ansori et al., 2020). Mangosteen

347 has been conventionally propagated by seeds and mangosteen trees begin to flower 7 years after

348 planting (Te-chato and Lim, 2004). It has thick, dark green, glossy leaves, 15–25 cm (6–10 inches)

349 long, borne in opposite pairs along the stem, and large rose-pink flowers. The fruits are the size

350 of a small orange, round or flattened on the ends. Mangosteens have a thick, hard, deep red rind

8
351 surrounding snow-white flesh, which is in segments resembling those of a mandarin orange.

352 Seedlings take 8 to 15 years to bear fruit (Britannica, 2018). The pulp of this fruit is frequently

353 consumed freshly, while the seed and peel are removed and become a waste (Rohman et al., 2019).

354 Fruit weights vary from 75 to 150 g. The edible mangosteen aril is white, soft, and juicy

355 pulp divided in septa with a sweet, slightly acid taste and a pleasant aroma (Pedraza-Chaverri et

356 al., 2008; Jung et al., 2006). Mangosteen fruit is highly valued for its juicy, delicate texture and

357 slightly astringent flavor and is commonly eaten fresh, canned, or dried (Britannica, 2018). The

358 pericarp, rind or skin of mangosteen is dark purple to red purple, smooth, thick, and tough (Yaacob

359 and Tindall, 1995). Sometimes, it is referred to as purple mangosteen because the deep purple

360 color develops when it ripe. Furthermore, mangosteen contains bioactive compounds such as

361 xanthones, terpenes, anthocyanins, tannins, phenols, and some vitamins (Murthy et. al., 2019;

362 Ansori et al., 2020).

Pericarp
(Rind)

Aril (Pulp)

363

364 Figure 2: Cross section of mangosteen fruit showing aril and pericarp (Murthy et. al., 2019).

365

9
366 Mangosteen peel contains certain type of secondary metabolites such as prenylated,

367 oxygenated, xanthones and other bioactive substances. Xanthones are useful in certain application

368 because of its antioxidant, antitumoral, anticancer and antileukemic activity as well as being anti-

369 inflammatory, anti- allergy, antibacterial, antifungal, and antiviral properties. For the mangosteen

370 seed is composed varies amount of bioactive compound such as moreollin, oil, lipid, carbohydrate

371 and gambolic acid. Moreover, there are big amount of fatty acid content in the seed such as

372 unsaturated fatty acid totaling 46.78 to 53.29%.( Pedraza-Chaverri, 2008)

373 Mangosteen peel and seed usually left to waste, but their properties make them an

374 interesting material for pharmaceutical and cosmetic products (Osaka, 2013) and healthcare

375 application such as in drug, facial masks, shampoos, soap and food supplement.

376 2.2.2 Benefits and uses

377 The chemical components contained in mangosteen’s seed and peel, especially xanthones,

378 have been reported as antioxidants. Several traditional medicine products used the extracts of

379 mangosteen as one of its components (Rohman et al., 2019).

380 The mangosteen fruit extract is popularly used as a dietary supplement to improve rumen

381 ecology and rumen productivity (Wanapat et al., 2014), while the fruit peel extract has been used

382 in herbal medicines. It has been used to cure many health problems in different parts of the world

383 (Ansori et al., 2020). The mangosteen peel has been reported to contain a variety of bioactive

384 compounds with potential applications as therapeutic agents or as functional food additives such

385 as phenolic acids (Zadernowski et al. 2009), tannins (Pothitirat et al. 2009), xanthones (Zarena

386 and Udaya Sankar, 2009b), anthocyanins (Palapol et al. 2009) and other bioactive compounds.

387 The anthocyanins are responsible for the purple color of mangosteen peel and the major

388 compound identified was cyanidin 3-sophoroside and cyanidin 3- glucoside as the minor.

10
389 Anthocyanins were found to have therapeutic properties such as anti-inflammatory (Chen et al.,

390 2008), antibacterial (Suksamrarn et al., 2002) and antioxidant actions (Jung et al., 2006);

391 moreover, the consumption of anthocyanins has been related to the decrease in the risk of coronary

392 heart diseases and atherosclerosis. Most of these beneficial functions were correlated to the

393 antioxidant activity of anthocyanins because of their ability of scavenging free radicals and

394 forming chelates with metals (Chisté et al., 2010).

395 Phytochemical screening, based on ethnomedicinal data, is considered as an effective

396 approach for the discovery of new therapeutic agents. The major bioactive secondary metabolites

397 of mangosteen are xanthone derivatives (Ansori et al., 2020). Pinto et. al. (2005) reviewed the

398 pharmacological investigations of xanthones and mentioned that the major constituents from the

399 xanthone fraction in mangosteen were found to be α-mangostin and γ-mangostin. They also

400 reported that xanthones were currently undergoing clinical trials as an

401 antitumor agent including the use as antioxidants with human health promotion properties.

402 Mangosteen rind, which is about two thirds of the whole fruit weight, is usually disposed

403 of as agricultural waste (Zarena et al., 2012). These wastes are rich in cellulose and have been

404 used to produce microfibrillated cellulose or MFC (Winuprasith and Suphantharika, 2013). Early

405 reports of the traditional uses of infusions and decoctions of its peels and seeds to treat

406 gastrointestinal and urinary tract infections, and as anti-scorbutic, laxative and anti-fever agent,

407 date from almost two hundred years ago (Descourtilz et al., 1821; Lilly et al., 1833; Pardo de

408 Tavera, 1901).

409 2.3 METHOD FOR SUGAR ANALYSIS

410 Carbohydrates or sugars are major energy source from food consumption, they can bring

411 crucial textural properties, and as dietary fiber which influences physiological processes. The

11
412 standard method for the determination of carbohydrates in foods is ‘by difference’, that is by

413 deducting the sum of the measured moisture, ash, protein (calculated from the total nitrogen) and

414 fat from the total weight (Southgate, 1969). There are several technics to determine the

415 carbohydrate contain for example phenol-sulfuric acid method, Somogyi–Nelson method,

416 dinitrosalicylic acid method, enzymic methods, and chromatography method

417 2.3.1 Phenol-Sulfuric Acid Method

418 The phenol-sulfuric acid method is a simple, sensitive, accurate, specific, and rapid

419 colorimetric method to determine total carbohydrates in a sample. The method detects virtually

420 all classes of carbohydrates, including mono-, di-, oligo-, and polysaccharides. In this method, the

421 concentrated sulfuric acid breaks down any polysaccharides, oligosaccharides, and disaccharides

422 to monosaccharides. Pentoses (5-carbon compounds) are then dehydrated to furfural, and hexoses

423 (6-carbon compounds) to hydroxymethyl furfural. These compounds then react with phenol to

424 produce a yellow-gold color. For products that are very high in xylose (a pentose), such as wheat

425 bran or corn bran, xylose should be used to construct the standard curve for the assay and measure

426 the absorption at 480 nm. For products that are high in hexose sugars, glucose is commonly used

427 to create the standard curve, and the absorption is measured at 490 nm. The color for this reaction

428 is stable for several hours, and the accuracy of the method is within ±2% under proper conditions

429 (Nielsen, 2010).

430 2.3.2 Somogyi–Nelson method

431 Somogyi–Nelson method is the quantitative determination of reducing sugars. The

432 arsenomolybdate reagent widely used as the chromogenic reagent because it produces more stable

433 and reproducible colors in combination with Somogyi's alkaline copper reagents. The method is

434 based on the reduction of Cu(II) ions to Cu(I) ions by reducing sugars. The Cu(I) ions then reduce

12
435 an arsenomolybdate complex, prepared by reacting ammonium molybdate [(NH4)6Mo7O24] and

436 sodium arsenate (Na2HAsO7) in sulfuric acid. The reaction of arsenomolybdate complex has

437 shown intense, stable blue color then measure in spectrophotometer (Shao and Lin, 2018).

438 2.3.3 Dinitrosalicylic acid method

439 The Dinitrosalicylic acid or DNS method is a colorimetric technique that consists of a

440 redox reaction between the 3,5-dinitrosalicyclic acid and the reducing sugars present in the sample.

441 The reducing power of these sugars comes from their carbonyl group, which can be oxidized to

442 the carboxyl group by mild oxidizing agents, while the DNS (yellow) is reduced to 3-amino-5-

443 nitrosalicylic acid (red brown) which can be quantified by spectrophotometry at 540 nm,

444 wavelength of maximum absorbance. The intensity of the color is proportional to the

445 concentration of sugars. The reaction is carried out in an alkaline medium. The reagent is a

446 solution formed by the following compounds: 3, 5-Dinitrosalicylic acid (2-hydroxy-3,5-

447 dinitrobenzoic acid), which acts as an oxidant, Rochelle salt (Sodium potassium tartrate), which

448 prevents the dissolution of oxygen in the reagent and sodium hydroxide to provide the medium

449 required for the redox reaction to occur (Garriga et. al., 2017).

450 2.3.4 High-performance Liquid Chromatography (HPLC) method

451 Chromatographic methods are the best analytical techniques for the quantification and

452 identification of mono and oligosaccharides in food products with high degree of precision and

453 accuracy. In fact, HPLC is the preferred method for sugar quantification according to the

454 guidelines of the Association of Official Analytical Chemists (AOAC). The amino-bonded phase

455 HPLC with refractive index (RI) detectors has been extensively used for sugar analysis.

456

457

13
458 CHAPTER 3
459 METHODOLOGY
460
461
462 1. Materials

463 Kombucha SCOBY was purchased from the U.S.A. Mangosteen peel powder was

464 purchased from Kom Bang Big Farm Community Enterprise, Chanthaburi, Thailand. Thai tea

465 original was purchased from Makro supermarket Cha Tra Mue brand. Other chemicals used for

466 analysis were available in the laboratory.

467 2. Preparation of Mangosteen peel Kombucha (MPK)

468 Mangosteen peel powder and Thai tea were mixed at 1:2, 1:1, 2:1, and 1:0. The mixture

469 of 6 g was infused in 1L hot water at 85-90℃ for 5 minutes. Sugar seven% (w/v) was dissolved

470 in the mixture and filtered into a sterile jar. When infused tea was cooled down to 25 to 35℃, 80

471 mL of previous fermented kombucha with 30 g-piece SCOPY was added into infused tea. The jar

472 was covered by sterile sheet cloth, and the cultured tea was incubated at room temperature for 15

473 days.

474 3. Investigation of the chemical changes and sensory quality of Mangosteen peel Kombucha

475 during fermentation

476 MPK samples were sampled at 0, 1, 3, 6, 9, 12, and 15th day of fermentation and

477 investigated for pH, and total soluble solid (°Brix). Total acidity was determined by titration with

478 a standard solution of sodium hydroxide and phenolphthalein as an indicator. Color intensity (L*,

479 a*, b*) using colorimeter (MiniscanEZ-4500 spectrophotometer, Hunter Lab, USA), and total

480 phenolic compound using Folin-ciocauteu method were also carried out.

14
481 MPK samples at 0, 1, 3, 6, 9, 12, and 15th day of fermentation were evaluated for overall

482 acceptability, clarity, color, taste, and aroma using 9-point hedonic score by 30 panelists from

483 Assumption University Hua Mak Campus.

484 4. Determination of total polyphenolic compound (TPC) by Folin-Ciocalteu Method

485 MPK with the highest sensory evaluation score and control was prepared. Samples were

486 sampling at 0, 1, 3, 6, 9, 12, and 15th day of fermentation. 10 µl Sample or a standard solution of

487 gallic acid (ranging from 0–500 mg/l) was mixed with 1.58 ml of distilled water and 100 µl of

488 Folin-Ciocalteau reagent. The solution was standed at room temperature for 5 minutes and then

489 followed by adding 300 µl of saturated sodium carbonate solution. The mixture was left at room

490 temperature for 30 minutes before measured at the absorbance of 765 nm (UNICO S1200, NJ,

491 USA).

492 5. Microbiological analysis of yeast and acetic acid bacteria

493 MPK with the highest sensory evaluation score was prepared. The isolation and

494 verification techniques were employed to determine yeast and acetic acid bacteria in fermented

495 MPK using glucose yeast extract agar (GYEA). The inoculated plate was incubated at room

496 temperature for 48 hours. Isolation was carried out on the two different selective media for acetic

497 acid bacteria, Glucose yeast extract calcium carbonate medium (GYC) and Carr medium. To

498 isolate yeast, it was carried out by using three different selective media; sabouraud dextrose agar,

499 Yeast extract-peptone-detroses agar and Hicrome Candida differential agar. All the inoculated

500 plates were incubated at room temperature for 7 days. For yeast isolation, samples were

501 investigated ethanol tolerance test (Guimarães et al., 2006). Acetic acid tolerance test was also

502 performed using acidified media (Kurtzman et al., 2001). The colony on the selective media has

503 been double check by using Gram stain test to check the Gram nature of the bacteria.

15
504 6. Study the changes of sugar profile in MPK during fermentation using HPLC

505 6.1 Sample preparation

506 Mangosteen peel Kombucha samples were sampling at 0, 1, 3, 6, 9, 12, and 15th day of

507 fermentation. All samples were deproteinized with 5%trichloloacetic acid (TCA) and neutralized

508 with 0.1 N NaOH. Then, the supernatant was filtered by 0.45 μm-membrane before subjected into

509 HPLC.

510 6.2 Chromatographic condition for sugar determination

511 The amount of sucrose, glucose, and fructose in the samples were analyzed by HPLC using

512 system employed consisted of a Shimatsu high-performance liquid chromatograph (Shimadzu

513 HPLC HGE system) coupled with a UV-VIS detector, inersil-NH2 column (5 µm, 250 × 4.6 mm)

514 at 40°C. Mobile phase was acetonitrile-water in the ratio of 3:1 at the flow rate of 1 mL/min. The

515 detector used was Refractive Index (RI) detector. Standard sugar solutions were prepared at 2, 4,

516 and 6 g/L.

517 7. Statistical analyses

518 The data was analyzed by using SAS program for Pearson correlation coefficient (P < 0.05)

519 and significant test by using Randomized Completed Block Design with Duncan’s multiple

520 comparison range test.

521

522

16
523 CHAPTER 4
524 RESULTS AND DISCUSSION
525

526 1. Preparation of mangosteen peel kombucha

527 Mangosteen peel powder and Thai tea were mixed at 1:2, 1:1, 2:1, and 1:0. The mixture

528 of 6 g was infused in 1 L hot water at 85-90℃ for 5 minutes. Sugar seven% (w/v) was dissolved

529 in the mixture and filtered into a sterile jar to reduce contamination. When infused tea was cooled

530 down to 25 to 35℃, 80 mL of previous fermented kombucha with 30 g-piece SCOPY was added

531 into infused tea. The jar was covered by sterile sheet cloth to keep insects, especially Drosophila

532 fruit, flying away. The cultured tea was incubated at room temperature (22-26℃) for 15 days

533 (Jayabalan et.al, 2021).

534

535
536 Figure 3 . Mangosteen peel kombucha and its SCOPY during fermentation
537

538 The newly formed daughter culture will start to float and form a clear thin gel-like

539 membrane across the available surface in the next few days. This is the newly formed tea fungus

17
540 available as a new layer above the old tea fungus, which was inoculated to begin fermentation. At

541 this time, the tea will start to smell fermented, and there will be gas bubbles appearing from the

542 carbonic acid produced during the fermentation. The mother culture will remain at its original

543 volume as it sinks to the bottom of the tea broth, where it remains under the newly forming

544 daughter culture (Figure 3).

545

546 2. Investigation of the chemical changes and sensory quality of Mangosteen peel Kombucha

547 during fermentation

548 Mangosteen peel kombucha (MPK) was prepared and the samples were sampled at 0, 1, 3,

549 6, 9, 12, and 15th day of fermentation. The change of pH and total soluble solid (°Brix) was

550 investigated including evaluated for overall acceptability, clarity, color, taste, and aroma using a

551 9-point hedonic score by 30 panelists.

552 2.1 Color analysis of Mangosteen peel kombucha

553 The color of MPK was measured using Hunter Lab colorimeter with L* a* b* value which

554 is based on the Opponent-Color Theory. This theory assumes that the receptors in the human eye

555 perceive color as the following pairs of opposites. L value indicates lightness and darkness where

556 a low number (0-50) indicates dark, and a high number (51-100) indicates light. The a* value

557 showed the scale between red and green color where a positive number indicates red, and a

558 negative number indicates green. For b* scale, it differentiated among yellow and blue color where

559 a positive number indicates yellow, and a negative number indicates blue. All three values are

560 required to completely describe an object‘s color.

18
561 (a)

562 (b)

563 (c)
564 Figure 4: Change of MPK colors as L* value (a), a* value (b), and b* value (c) during fermentation

565 (A: control or Thai tea kombucha; B: 1:2 MPK; C:1:1 MPK; D: 2:1 MPK; and E: 1:0 MPK).

19
566 As the CIELAB color scale, L* is lightness and the scale runs from 0 (bottom black) to 100

567 (white). The a* and b* value have no specific numerical limits. Positive a* is red. Negative a*

568 is green. Positive b* is yellow. Negative b* is blue (Chang et.al., 2012). From figure 3a to c, L*

569 and b* value of all samples gradually increased through fermentation period while a* value

570 decreased. The results implied that the color of kombucha continued to get lighter when the

571 fermentation progressed. Moreover, the increasing of b* value and decreasing of a* value

572 interpreted that when kombucha was fermented, the decreasing of pH affected the color intensity.

573 Chu and Chen (2006) suggested that the color of black tea was mainly from thearubigins. The

574 color became colorless because of the suppression of ionization or destruction of structures. The

575 decrease of pH and the biological activity of the kombucha consortium could be the cause of

576 significant decrease in color intensity (Chakravorty et al., 2016; Ghosh et. al., 2020; Tran et. al.,

577 2020).

578

(a) (b) (c) (d) (e)

579
580 Figure 5: Color of mangosteen peel kombucha at various ratios between Thai tea and
581 mangosteen peel after fermentation (a: control or Thai tea kombucha; b: 1:2 MPK; c:1:1 MPK;
582 d: 2:1 MPK; and e: 1:0 MPK).

20
583 Figure 5 presented the color of MPK at different ratios between Thai tea and mangosteen

584 peel. On the last day of fermentation, sample c exhibited a dark red-brown color, while sample a

585 displayed a light brown color. The remaining samples demonstrated a similar yellowness.

586 Additionally, the turbidity of the samples was influenced by the quantity of mangosteen peel

587 content. However, in Figure 4b and 4c, the MPK with mangosteen peel and Thai tea ratio 1:0

588 showed the lowest a* and b* value (P<0.05). This implied that MPK made from 100% mangosteen

589 peel showed the highest redness and yellow than others. Because mangosteen peel had

590 anthocyanin as predominant pigment and anthocyanin is sensitive to pH changes. Thus, when the

591 fermentation progressed, and pH decreased resulting in the degradation of anthocyanin. Moreover,

592 the storage of MPK at room temperature with fluctuation promoted of anthocyanin degradation

593 (Chisté et. al., 2010).

594

595
596 Figure 6: Change of pH of MPK during fermentation. A: control or Thai tea kombucha, B: 1:2
597 MPK, C:1:1 MPK, D: 2:1 MPK, and E: 1:0 MPK.
598

21
599
600 Figure 7: Acidity changes of MPK during fermentation. A: control or Thai tea kombucha, B: 1:2

601 MPK, C:1:1 MPK, D: 2:1 MPK, and E: 1:0 MPK.

602

603
604 Figure 8: Change of total soluble solid of MPK during fermentation. A: control or Thai tea
605 kombucha, B: 1:2 MPK, C:1:1 MPK, D: 2:1 MPK, and E: 1:0 MPK.
606

22
607 2.2 Physio-chemical change of mangosteen peel kombucha

608 In Figure 4 and 5, the results showed the decreasing of pH value and the increasing of

609 acidity in all systems due to fermentation. The pH decreased rapidly in the first day, then gradually

610 decreased. The pH of MPK at the end of fermentation was approximately 2.85±0.03, while the

611 acidity was 0.19±0.03 g/L. In 2008, Malbaša et. al. studied kombucha fermentation of Indian

612 black tea sweetened with sucrose and molasses. They reported the change of pH and acidity that

613 pH values sharply decrease at the first 3 days of fermentation, then the decrease becomes slower

614 until the end of fermentation, while the acidity showed the opposite with pH value. The higher

615 the acidity, the lower the pH value. The increase in total acidity that occurs during the fermentation

616 process is caused by changes of glucose by bacteria and yeast during the fermentation process to

617 produce organic acids, especially acetic acid. This occurs because the acid in the kombucha will

618 release protons (H+) so that the pH value decreases (Zubaidah et. al., 2019). Ahmed et. al. (2020)

619 described that the decreasing of pH regarding to the presence of sucrose, which metabolized into

620 organic acids by bacteria and yeast, led to increasing the acidity of the beverage. The pH level

621 decreases accordingly to increase of total organic acids content during fermentation.

622 The major organic acids contributing to kombucha’s taste are acetic acid, gluconic acid,

623 and glucuronic acid, whereas the minor ones are lactic acid, malic acid, and succinic acid (Blanc,

624 1996; Chakravorty et al., 2016; De Filippis et al., 2018; Jayabalan et al., 2007; Malbaša et al.,

625 2008; Neffe-Skocińska et al., 2017). Therefore, the ability of acetic acid bacteria to produce

626 organic acid in the kombucha resulting to sourness of kombucha, although the contribution of yeast

627 and lactic acid bacteria should not be neglected. The decreased total soluble solids occurred during

628 the fermentation process (Figure 8) because sugar was used as a carbon source for the growth of

23
629 microorganism cells, in addition to the metabolic processes that produce cellulose and organic

630 acids (Maungma, 2000; Zubaidah et. al., 2019).

631 2.3 Sensory evaluation

632 The attributes of MPK were evaluated at various ratios of Mangosteen peel to Thai tea: 1:2,

633 1:1, 2:1, and 1:0. The attributes examined include color, clarity, flavor, sweetness, sourness, and

634 overall liking. In Table 1, color scores range from 6.6 to 7.7. The highest scores were observed

635 for the 1:1 and 2:1 ratio, indicating a preferred color intensity. The 1:0 ratio had the lowest score,

636 suggesting a less desirable color. The clarity scores range from 7.3 to 7.8. All ratios received

637 relatively high scores, indicating good clarity. However, the 1:0 ratio received a slightly lower

638 score compared to the others. The flavor scores range from 6.6 to 7.1. There is not a significant

639 variation in flavor scores across the different ratios, as all ratios received similar scores. For

640 Sweetness and Sourness have no significant difference in both scores among the ratios, as all ratios

641 received similar scores.

642 Table 1. Sensory evaluations of mangosteen peel kombucha in 9-point hedonic score.
MPK
Attributes at various ratios of Mangosteen peel: Thai tea
1:2 1:1 2:1 1:0
a a a
Color 7.5±0.83 7.7±0.82 7.6±0.91 6.6±0.99b
Clarity 7.8±0.86a 7.7±0.88a 7.6±0.91ab 7.3±1.1b
Flavor 6.6±1.4a 7.1±0.92a 7.1±1.46a 7.1±0.99a
Sweetness 7.0±1.46a 7.2±1.42a 6.8±1.37a 7.0±1a
Sourness 6.7±1.62a 6.8±1.74a 6.7±1.33a 7.1±1.28a
Overall Liking 7.0±1a 7.3±0.88a 6.9±1.49a 7.2±0.94a
643 Different letters in the same column represent significant difference between MPK samples (p<0.05).
644

645 Have multiple panelists reported that the sample MPK 1:1 have similar flavor as apple juice

646 with Thai tea and mangosteen peel powder aroma. Moreover, 1:1 MPK is most balanced on its

647 sweetness and sourness compared to other sample. MPK using mangosteen peel: Thai tea at 1:1

24
648 had the highest score for color, sweetness, and overall linking as 7.7, 7.2, and 7.3, respectively.

649 For clarity (7.7), flavor (7.1), and sourness (6.8), although those attributes were not the highest

650 score but they had no significant difference from other systems. Therefore, MPK using

651 mangosteen peel: Thai tea at 1:1 was selected for further analysis.

652 3. Total polyphenolic compound content in MPK

653 Total polyphenolic compound (TPC) in Thai tea kombucha (control) and MPK with 1:1

654 (ratio of mangosteen peel and Thai tea) showed the different change throughout fermentation. As

655 the results in Figure 7, even though Thai tea kombucha showed the higher TPC than MPK at the

656 beginning of fermentation, TPC of both systems increased with the progress of fermentation. Total

657 polyphenolic content reached to peak value on day 12 with 99.2028±0.3750 GAE/g sample.

658 According to Essawet et al., (2015), the main antioxidants in fermented kombucha tea beverages

659 were polyphones and tea fungus metabolites such as vitamins and organic acids. For this reason,

660 the fermented kombucha tea usually expresses a higher antioxidant potential in comparison with

661 non-fermented tea. The linearly increasing of TPC through fermentation also found in the study

662 of Srihari and Satyanarayana (2012). Moreover, Lee et.al. (2002) reported that black tea contained

663 TPC of 124 mg GAE and Chaovanalikit et.al. (2012) revealed that mangosteen pericarp contained

664 TPC of 40-45 mg GAE. The significant decreased of total polyphenolic content on the last day of

665 fermentation of mangosteen peel kombucha with 72.91±0.2149 GAE/g sample. TPCs of samples

666 decreased during storage due to the acidic nature of the kombucha drink and its enzymes

667 (Jayabalan et al., 2007).

25
668
669 Figure 9. Change of total phenolic content during MPK fermentation
670

671 4. Microbiological analysis of acetic acid bacteria and yeast

672
673 Table 2. Characterization of acetic acid bacteria and yeast isolate from MPK
Characterization Results
Acetic Acid Bacteria
Gram nature Gram negative short rod
Carr medium Isolate acid producing characteristic color
change in media observe (form green to
yellow then green again)
Yeast extract calcium carbonate glucose agar -
Yeast isolate
Gram nature Gram positive oval budding yeast
Sabouraud dextrose agar +
Ethanol tolerance test 5,10, and 15% + (for all concentrations)
Tolerance to acetic acid test +
Growth on hicrome candida differential agar Pink, purple color colony
674
675 As shown in Table 2, the results for acetic acid bacteria isolation showed Gram negative

676 short rod for the gram stain and with negative result for Erwinia spp. On Yeast extract calcium

677 carbonate glucose agar. For the testing with Carr medium, color of agar changed from green to

678 yellow, and then turned to green color again. This implied that the bacteria had produced acid

26
679 during the incubation at room temperature for 48 hours. Yeast isolation confirmed the straining

680 Gram positive oval budding yeast and the positive result for Sabouraud dextrose agar, ethanol

681 tolerance tests 5, 10, and 15% including the tolerance to acetic acid test. Moreover, there were

682 pink, purple colonies growing on Hicrome candida differential agar. From the color of colony can

683 know the candida species are C.krusei ,which is type of candida species that have been isolated

684 from kombucha in multiple research(Jayabalan et al., 2021)

685 5. HPLC result of sugar concentration change in MPK during fermentation period
686

687
688 Figure 10. Change of sugar content of fructose (blue line), glucose (red line) and
689 sucrose (green line) for 15 days MPK fermentation
690
691 Table 3: Change of sugar content of fructose, glucose, and sucrose for 15 days MPK fermentation
Fructose Glucose Sucrose
Day0 13.14±1.77Aa 38.07±6.26Ba 2.57±2.23Ca
Day1 15.34±0.76Ab 38.25±0.81Ba 4.04±0.18Cb
Day3 15.83±3.38Ab 38.76±7.84Ba 3.90±0.14Cb
Day6 14.99±1.89Ab 35.69±4.98Bb 3.72±0.12Cc
Day9 18.33±1.34Ac 33.39±1.74Bc 3.82±0.09Cc
Day12 16.82±2.01Ad 35.60±6.71Bb 0
Day 15 14.12±0.79Aa 33.13±1.99Ba 0
692 Remark: Capital letters in the same column represent significant difference between MPK samples (p<0.05).
693 Lower case letters in column represent significant difference between teas in each day (p>0.05).

27
694 In the figure 8, the amount of fructose, glucose, and sucrose changed during 15-day

695 fermentation of mangosteen peel kombucha. The results have shown glucose was the dominant

696 type of sugar content in the MPK sample since the beginning of fermentation. Glucose was being

697 utilized on day 3, sucrose was being utilized before other types of sugar. Fructose was the second

698 dominant type of sugar content in the sample. It significantly grew after day 6 then reached peak

699 value (18.33±1.34 g/L) on day 9. In the study of Loncar et al. (2000), they concluded that sucrose,

700 glucose, and fructose were not utilized entirely after 21 d of fermentation and confirmed that

701 fructose was metabolized before glucose. The least sugar content in sample was sucrose, the

702 utilization of sucrose started to speed up on day 9 and got fully utilized on day 12. In the study of

703 Yavari et al. (2010), they concluded that sucrose utilization, after the 4th day began to speed up

704 and this trend continued until the 14th day when the lowest sucrose content (2.1 g/L) was

705 determined. However, Muhialdin et al. (2019) reported that kombucha fermented with white

706 refined sugar have fructose of (2.42 g/mL), glucose of (3.88 g/mL), and sucrose of (83 g/mL) at

707 the end of 14-day fermentations. Final sugar concentrations can differ from one fermentation to

708 another, which indicates that the metabolic pathway does not always occur in the same way (Chen

709 and Liu, 2000).

710
711
712
713

28
714 CHAPTER 5
715 CONCLUSION
716

717 The suitable ratio of mangosteen peel and Thai tea to make Mangosteen peel kombucha

718 was 1:1 with the highest sensory evaluation score (7.2). During kombucha fermentation showed

719 the increasing of pH, decreasing of acidity and increasing of total phenolic compounds (TPC). At

720 the end of fermentation, MPK had pH of 2.85±0.03, acidity of 0.19±0.03 g/L, and TPC of 72.91

721 mg GAE/g sample. As the microbiological analysis, the isolation of yeast and acetic acid bacteria

722 showed positive results. The observed changes in mangosteen peel kombucha the amount of

723 fructose, glucose, and sucrose during a 15-day fermentation of mangosteen peel kombucha by

724 using HPLC. Glucose was the dominant type of sugar content in the sample, followed by fructose

725 and then sucrose. Sucrose was utilized first, followed by fructose and glucose. Fructose peaked

726 on day 9 and sucrose was fully utilized on day 12. In the last day of fermentation has remain

727 fructose 14.12±0.79 g/L and glucose 33.13±1.99 g/L with no sucrose detected in the sample.

728

729
730
731
732
733
734
735
736
737

29
738 REFERENCES
739

740 Anonymous (2009) Code of federal regulations, Title 21, Part 101.9 – Food nutrition labeling of

741 food. US Government Printing Office, Washington, DC

742 Ansori, A.N.M., Fadholly, A., Hayaza, S., Susilo, R.J.K., Inayatillah, B., Winarni, D. and Husen,

743 S.A., 2020. A review on medicinal properties of mangosteen (Garcinia mangostana L.).

744 Research Journal of Pharmacy and Technology, 13(2), pp.974-982.

745 BeMiller, J.N., 2010. Carbohydrate analysis. In Food analysis (pp. 147-177). Springer, Boston,

746 MA

747 BeMiller, J.N., 2007. Carbohydrate chemistry for food scientists, 2nd edn. AACC International.

748 Inc., MN, USA.

749 Britannica, T. Editors of Encyclopaedia (2018, August 1). Mangosteen. Encyclopedia Britannica.

750 https://www.britannica.com/plant/mangosteen

751 Chakravorty, S., Bhattacharya, S., Chatzinotas, A., Chakraborty, W., Bhattacharya, D. and

752 Gachhui, R., 2016. Kombucha tea fermentation: Microbial and biochemical dynamics.

753 International journal of food microbiology, 220, pp.63-72.

754 Chakravorty, S., Bhattacharya, S., Bhattacharya, D., Sarkar, S. and Gachhui, R., 2019. Kombucha:

755 a promising functional beverage prepared from tea. In Non-alcoholic beverages (pp. 285-

756 327). Woodhead Publishing.

757 Chisté, R.C., Lopes, A.S. and De Faria, L.J., 2010. Thermal and light degradation kinetics of

758 anthocyanin extracts from mangosteen peel (Garcinia mangostana L.). International journal

759 of food science & technology, 45(9), pp.1902-1908.

30
760 Chen, C., & Liu, B. Y. (2000). Changes in major components of tea fungus metabolites during

761 prolonged fermentation. Journal of Applied Microbiology, 89, 834–839. https://doi.

762 org/10.1046/j.1365-2672.2000.01188.x

763 Chu, S.C. and Chen, C., 2006. Effects of origins and fermentation time on the antioxidant activities

764 of kombucha. Food Chemistry, 98(3), pp.502-507.

765 BeMiller, J.N., 2009. One hundred years of commercial food carbohydrates in the United States.

766 Journal of Agricultural and Food Chemistry, 57(18), pp.8125-8129.

767 Chaverri, J.P., RODRIGUEZ, N.C., IBARRA, M.O. and ROJAS, J.M.P., 2019. Medicinal

768 properties of mangosteen (Garcinia mangostana).

769 Chong, Y., Chang, S., Sia, W. and Yim, H., 2015. Antioxidant efficacy of mangosteen (Garcinia

770 mangostana Linn.) peel extracts in sunflower oil during accelerated storage. Food

771 Bioscience, 12, pp.18-25.

772 Dufresne, C. and Farnworth, E., 2000. Tea, Kombucha, and health: a review. Food research

773 international, 33(6), pp.409-421.

774 Fu, C., Loo, A.E.K., Chia, F.P.P. and Huang, D., 2007. Oligomeric proanthocyanidins from

775 mangosteen pericarps. J. of Agricultural and Food Chemistry, 55(19), pp.7689-7694.

776 Garriga, M., Almaraz, M. and Marchiaro, A., 2017. Determination of reducing sugars in extracts

777 of Undaria pinnatifida (harvey) algae by UV-visible spectrophotometry (DNS method).

778 Desarro. E INNOVACIÓN EN Ing, p.444.

779 Genovese, S., Fiorito, S., Taddeo, V.A. and Epifano, F., 2016. Recent developments in the

780 pharmacology of prenylated xanthones. Drug discovery today, 21(11), pp.1814-1819.

31
781 Gramza-Michałowska, A., Kulczyński, B., Xindi, Y. and Gumienna, M., 2016. Research on the

782 effect of culture time on the kombucha tea beverage? s antiradical capacity and sensory

783 value. Acta Scientiarum Polonorum Technologia Alimentaria, 15(4).

784 Islam, M., Khan, M.Z.H., Sarkar, M.A.R., Absar, N. and Sarkar, S.K., 2013. Changes in acidity,

785 TSS, and sugar content at different storage periods of the postharvest mango (Mangifera

786 indica L.) influenced by Bavistin DF. International journal of food science, 2013.

787 Ivanišová, E., Meňhartová, K., Terentjeva, M., Harangozo, Ľ., Kántor, A. and Kačániová, M.,

788 2020. The evaluation of chemical, antioxidant, antimicrobial and sensory properties of

789 kombucha tea beverage. Journal of food science and technology, 57(5), pp.1840-1846.

790 Jayabalan, R., Marimuthu, S. and Swaminathan, K., 2007. Changes in content of organic acids and

791 tea polyphenols during kombucha tea fermentation. Food Chemistry, 102(1), pp.392-398.

792 Jayabalan, R., Malini, K., Sathishkumar, M., Swaminathan, K. and Yun, S.E., 2010. Biochemical

793 characteristics of tea fungus produced during kombucha fermentation. Food Science and

794 Biotechnology, 19(3), pp.843-847.

795 Jayabalan, R., Malbaša, R.V., Lončar, E.S., Vitas, J.S. and Sathishkumar, M., 2014. A review on

796 kombucha tea—microbiology, composition, fermentation, beneficial effects, toxicity, and

797 tea fungus. Comprehensive Reviews in Food Science and Food Safety, 13(4), pp.538-550.

798 Jung, H.A., Su, B.N., Keller, W.J., Mehta, R.G. and Kinghorn, A.D., 2006. Antioxidant xanthones

799 from the pericarp of Garcinia mangostana (Mangosteen). Journal of agricultural and food

800 chemistry, 54(6), pp.2077-2082.

801 Martínez Leal, J., Valenzuela Suárez, L., Jayabalan, R., Huerta Oros, J. and Escalante-Aburto, A.,

802 2018. A review on health benefits of kombucha nutritional compounds and metabolites.

803 CyTA-Journal of Food, 16(1), pp.390-399.

32
804 Loncar ES, Petrovi ˇ c SE, Malba ´ ˇsa RV, Verac RM. 2000. Biosynthesis of glucuronic acid by

805 means of tea fungus. Nahrung 44:138–9.

806 Maungma, R., 2000. Study on the Maillard Reaction in Thai Soy Sauce. Mahidol University.

807 Moopayak, W. and Tangboriboon, N., 2020. Mangosteen peel and seed as antimicrobial and drug

808 delivery in rubber products. Journal of Applied Polymer Science, 137(37), p.49119.

809 Miranda, B., Lawton, N.M., Tachibana, S.R., Swartz, N.A. and Hall, W.P., 2016. Titration and

810 HPLC characterization of kombucha fermentation: a laboratory experiment in food

811 analysis. Journal of Chemical Education, 93(10), pp.1770-1775.

812 Muhialdin, B.J., Osman, F.A., Muhamad, R., Che Wan Sapawi, C.W.N.S., Anzian, A., Voon,

813 W.W.Y. and Hussin, A.S., 2019. Effects of sugar sources and fermentation time on the

814 properties of tea fungus (kombucha) beverage. International Food Research Journal, 26(2).

815 Ming-Hui, W.A.N.G., Zhang, K.J., Qin-Lan, G.U., Xiao-Ling, B.I. and Jin-Xin, W.A.N.G., 2017.

816 Pharmacology of mangostins and their derivatives: A comprehensive review. Chinese

817 journal of natural medicines, 15(2), pp.81-93.

818 Moongkarndi, P., Kosem, N., Kaslungka, S., Luanratana, O., Pongpan, N. and Neungton, N., 2004.

819 Antiproliferation, antioxidation and induction of apoptosis by Garcinia mangostana

820 (mangosteen) on SKBR3 human breast cancer cell line. Journal of

821 ethnopharmacology, 90(1), pp.161-166.

822 Murthy, H.N., Dandin, V.S., Dalawai, D., Park, S.Y. and Paek, K.Y., 2019. Bioactive compounds

823 from Garcinia fruits of high economic value for food and health. Bioactive Molecules in

824 Food, Reference Series in Phytochemistry; Merillon, JM, Ramawat, KG, Eds, pp.1-27.

33
825 Neffe-Skocińska, K., Sionek, B., Ścibisz, I. and Kołożyn-Krajewska, D., 2017. Acid contents and

826 the effect of fermentation condition of Kombucha tea beverages on physicochemical,

827 microbiological and sensory properties. CyTA-Journal of Food, 15(4), pp.601-607.

828 Nielsen, S.S., 2010. Phenol-sulfuric acid method for total carbohydrates. In Food analysis

829 laboratory manual (pp. 47-53). Springer, Boston, MA.

830 Osaka, I., & Hefty, P. S. (2013). Simple resazurin-based microplate assay for measuring

831 Chlamydia infections. Antimicrobial agents and chemotherapy, 57(6), 2838-2840.

832 Pedraza-Chaverri, J., Cárdenas-Rodríguez, N., Orozco-Ibarra, M. and Pérez-Rojas, J.M., 2008.

833 Medicinal properties of mangosteen (Garcinia mangostana). Food and chemical

834 toxicology, 46(10), pp.3227-3239.

835 Pedraza-Chaverri, J., Cárdenas-Rodríguez, N., Orozco-Ibarra, M. and Pérez-Rojas, J.M., 2008.

836 Medicinal properties of mangosteen (Garcinia mangostana). Food and chemical

837 toxicology, 46(10), pp.3227-3239.

838 Pinto, M.M.M., Sousa, M.E. and Nascimento, M.S.J., 2005. Xanthone derivatives: new insights in

839 biological activities. Current medicinal chemistry, 12(21), pp.2517-2538.

840

841 Pedraza-Chaverri, J., Cárdenas-Rodríguez, N., Orozco-Ibarra, M., & Pérez-Rojas, J. M. (2008).

842 Medicinal properties of mangosteen (Garcinia mangostana). Food and chemical

843 toxicology, 46(10), 3227-3239.

844 Rodrigues, C.I., Marta, L., Maia, R., Miranda, M., Ribeirinho, M. and Máguas, C., 2007.

845 Application of solid-phase extraction to brewed coffee caffeine and organic acid

846 determination by UV/HPLC. Journal of Food Composition and Analysis, 20(5), pp.440-

847 448.

34
848 Rohman, A., Rafi, M., Alam, G., Muchtaridi, M. and Windarsih, A., 2019. Chemical composition

849 and antioxidant studies of underutilized part of mangosteen (Garcinia mangostana L.) fruit.

850 J Appl Pharm Sci, 9(8), pp.47-52.

851 Scherz, H. and Bonn, G., 1998. Analytical chemistry of carbohydrates. Thieme Medical Pub.

852 Shade, A., Buckley, D.H. and Zinder, S.H., 2011. The kombucha biofilm: a model system for

853 microbial ecology. Final report on research conducted during the Microbial Diversity

854 course. Marine Biological Laboratories, Woods Hole, MA.

855 Shao, Y. and Lin, A.H.M., 2018. Improvement in the quantification of reducing sugars by

856 miniaturizing the Somogyi-Nelson assay using a microtiter plate. Food chemistry, 240,

857 pp.898-903.

858 Southgate, D.A.T., 1969. Determination of carbohydrates in foods. I.—Available carbohydrate.

859 Journal of the Science of Food and Agriculture, 20(6), pp.326-330.

860 Suttirak, W. and Manurakchinakorn, S., 2014. In vitro antioxidant properties of mangosteen peel

861 extract. Journal of food science and technology, 51(12), pp.3546-3558.

862 Suttirak, W. and Manurakchinakorn, S., 2014. In vitro antioxidant properties of mangosteen peel

863 extract. Journal of food science and technology, 51(12), pp.3546-3558.

864 Te-chato, S. and Lim, M., 2004. Early fruit setting from tissue culture-derived mangosteen tree.

865 Songklanakarin J. Sci. Technol, 26(4), pp.447-453.

866 Tran, T., Grandvalet, C., Verdier, F., Martin, A., Alexandre, H. and Tourdot‐Maréchal, R., 2020.

867 Microbiological and technological parameters impacting the chemical composition and

868 sensory quality of kombucha. Comprehensive Reviews in Food Science and Food Safety,

869 19(4), pp.2050-2070.

35
870 Upaganlawar, A.B. and Badole, S.L., 2013. Mangosteen (Garcinia mangostana linn.): Role in

871 prevention of skin disorders. In Bioactive Dietary Factors and Plant Extracts in

872 Dermatology (pp. 451-457). Humana Press, Totowa, NJ.

873 Violeta, N.O.U.R., Trandafir, I. and Ionica, M.E., 2010. HPLC organic acid analysis in different

874 citrus juices under reversed phase conditions. Notulae Botanicae Horti Agrobotanici Cluj-

875 Napoca, 38(1), pp.44-48.

876 Wanapat, M., Chanthakhoun, V., Phesatcha, K. and Kang, S., 2014. Influence of mangosteen peel

877 powder as a source of plant secondary compounds on rumen microorganisms, volatile fatty

878 acids, methane and microbial protein synthesis in swamp buffaloes. Livestock Science, 162,

879 pp.126-133.

880 Zarena, A.S. and Sankar, K.U., 2012. Isolation and identification of pelargonidin 3-glucoside in

881 mangosteen pericarp. Food chemistry, 130(3), pp.665-670.

882 Zubaidah, E., Dewantari, F.J., Novitasari, F.R., Srianta, I. and Blanc, P.J., 2018. Potential of snake

883 fruit (Salacca zalacca (Gaerth.) Voss) for the development of a beverage through

884 fermentation with the Kombucha consortium. Biocatalysis and Agricultural Biotechnology,

885 13, pp.198-203.

886

36
 APPENDIX

Material for making mangosteen peel kombucha

Figure 11 .Thai tea leaf ,

Figure 12. Mangosteen peel

Figure 13. SCOBY (Symbiotic Culture of Bacteria and Yeast),

37
 Figure 14 Kombucha culture

Figure 15: Mangosteen peel kombucha after fermentation

38
 Figure 18: Color of mangosteen peel kombucha before (left) and after (right) fermentation.

Table 4. L* value of mangosteen peel kombucha

L* Day0 Day1 Day 3 Day6 Day 9 Day 12 Day 15
value
0:1 MPK 14.52±0Aa 21.92±0.1058Ab 22.95±0.2784Ad 23.66±0.2743Ae 22.59±0.1058Ac 24.21±0.1050Af 23.12±0.1550Ad
1:2 MPK 12.43±0Ca 18.60±0.5048Cb 19.65±0.4750Cc 20.585±0.2701Cd 19.28±0.9521Cc 21.41±0.4782Cde 20.46±0.3350Cd
1:1 MPK 12.25±0Ba 19.54±0.5997Bb 20.18±0.6819Bc 21.36±0.6834Bd 20.11±0.6673Bc 22.10±0.6734Be 21.29±0.5632Bd
2:1 MPK 12.35±0BCa 19.20±0.0404BCb 20.21±0.06BCc 21.43±0.0954BCe 20.05±0.2574BCc 22.03±0.2606BCf 21.01±0.2458BCd
1:0 MPK 15.58±0Aa 22.07±0.6239Ab 23.08±0.4366Ac 24.13±0.3580Ae 22.63±0.7115Ac 24.55±0.5957Ae 23.56±0.6116Ad
Remark: Capital letters in the same column represent significant difference between MPK samples (p<0.05).
Lower case letters in column represent significant difference between teas in each day (p>0.05).

Table 5. a* value of mangosteen peel kombucha

a* value Day0 Day1 Day 3 Day6 Day 9 Day 12 Day 15
Cf Cd Ce Cd Cc Cb
0:1 14.37±0 13.77±0.1493 14±0.2261 13.74±0.1637 13.42±0.2673 13.06±0.1650 12.66±0.2413Ca
MPK
1:2 17.17±0Ad 16.85±0.21Acd 17±0.3105Acd 16.60±0.2343Abc 16.25±0.6274Ab 15.49±0.3061Aa 15.10±0.3683Aa
MPK
1:1 16.96±0BCd 15.46±0.9676BCc 15.79±0.9271BCc 15.15±0.9115BCb 14.78±0.9115BCb 13.92±0.9753BCa 13.28±0.9107BCa
c
MPK
2:1 17.10±0Be 16.07±0.2754Bd 16.01±0.1801Bd 15.34±0.4407Bc 14.58±0.5001Bb 14.25±0.4750Bab 13.86±0.4858Ba
MPK
1:0 12.50±0Dd 8.22±0.4015Dc 8.11±0.4246Dc 7.72±0.4186Db 7.7±0.4813Db 7.3±0.5988Da 7.24±0.5808Da
MPK

Remark: Capital letters in the same column represent significant difference between MPK samples (p<0.05).
Lower case letters in column represent significant difference between teas in each day (p>0.05).

39
 Table 6. b* value of mangosteen peel kombucha
b* value Day0 Day1 Day 3 Day6 Day 9 Day 12 Day 15
0:1 MPK 4.98±0Aa 36.79±1.1505Ac 38.14±0.2050Ae 37.96±0.4304Ade 37.13±0.4310Acd 36.36±0.2335Ac 35.07±0.6646Ab

1:2 MPK 21.41±0Ba 32.01±0.8685Bb 33.80±0.8136Bc 35.02±0.1562Bd 33.04±1.5908Bbc 33.35±0.9457Bc 33.38±0.3553Bc

1:1 MPK 21.09±0Ba 33.48±0.9091Bbc 34.65±1.1378Bd 34.38±0.5221Bcd 33.48±0.6736Bbc 32.78±0.4041Bb 32.52±0.3270Bb

2:1 MPK 21.26±0Ba 32.96±0.01Bb 34.70±0.0643Be 34.24±0.2485Bd 33.58±0.2117Bc 33.41±0.4994Bc 32.91±0.2914Bb

1:0 MPK 24.97±0Ca 29.43±0.2401Cd 28.08±0.5894Cc 27.12±0.5116Cb 26.81±0.7391Cb 25±0.9436Ca 24.98±1.1820Ca

Remark: Capital letters in the same column represent significant difference between MPK samples (p<0.05).
Lower case letters in column represent significant difference between teas in each day (p>0.05).

Table 7. brix value of mangosteen peel kombucha

brix Day0 Day1 Day 3 Day6 Day 9 Day 12 Day 15
Cc Cc Cb Cb Cb Cb
0:1 MPK 7.6±0 7.6±0 6.9±0.1155 7.0±0 7.0±0 7.0±0 6.7±0.1155Ca

1:2 MPK 7.6±0Bc 7.6±0Bc 7.1±0.1155Bb 7.0±0Ba 7.1±0.1155Bb 7.1±0.1155Bab 7.0±0Ba

1:1 MPK 7.2±0Bb 7.2±0Bb 7.0±0Ba 7.0±0Ba 7.1±0.1155Ba 7.0±0Ba 7.0±0Ba

2:1 MPK 7.8±0Bc 7.7±0.1155Bc 7.1±0.1155Ba 7.3±0.1155Bab 7.3±0.2309Bb 7.2±0.2Bab 7.1±0.1155Ba

1:0 MPK 7.8±0Bd 7.5±0.2309Ac 7.1±0.1155Aa 7.2±0Aab 7.3±0.1155Ab 7.2±0.2Aab 7.3±0.1155Ab

Remark: Capital letters in the same column represent significant difference between MPK samples (p<0.05).
Lower case letters in column represent significant difference between teas in each day (p>0.05).

Table 8. pH value of mangosteen peel kombucha

pH Day0 Day1 Day 3 Day6 Day 9 Day 12 Day 15
0:1 7.52±0ABe 3.18±0.032ABd 3.12±0.05ABcd 3.10±0.06ABc 2.96±0.05ABb 2.95±0.035ABb 2.87±0.040ABa
MPK
1:2 7.08±0Ae 3.14±0.017Ad 3.09±0.040Ac 3.06±0.026Ac 2.95±0Ab 2.97±0.023Ab 2.89±0.025Aa
MPK
1:1 7.16±0ABf 3.25±0.01ABe 3.16±0.026ABd 3.10±0.026ABc 2.95±0.032ABb 2.94±0.023ABb 2.84±0.015ABa
MPK
2:1 7.18±0Bf 3.21±0.012Be 3.15±0.03Bd 3.06±0.029Bc 2.92±0.023Bb 2.90±0.029Bb 2.82±0.025Ba
MPK
1:0 7.22±0ABf 3.26±0.021ABe 3.16±0.023ABd 3.06±0.036ABc 2.91±0.032ABb 2.90±0.025ABb 2.83±0.025ABa
MPK

40
 Titratable acidity analysis of mangosteen kombucha
Calculation formula :
Mw of acetic acid=60.05 g/mol
Total acidity= amount of NaOH used×60.05×normolity of NaOH/10ml
Table 9. Acidity value of mangosteen peel kombucha

Acidity Day0 Day1 Day 3 Day6 Day 9 Day 12 Day 15

0:1 0.0121±0.0036Aa 0.0895±0.0051Ab 0.1056±0.0074Ac 0.1241±0.0105Ad 0.1386±0.0105Ad 0.1757±0.0139Ae 0.2402±0.025Af
MPK
1:2 0.0145±0Da 0.1088±0.158Db 0.1257±0.0096Dc 0.1411±0.0221Dc 0.1185±0.0051Dd 0.1330±0.0073De 0.1644±0.0246Df
MPK
1:1 0.0145±0DCa 0.1072±0.0101DCb 0.1209±0.0089DCc 0.1394±0.0094DCc 0.1306±0.0089DCc 0.1435±0.0070DCc 0.1765±0.0036DCd
MPK
2:1 0.0145±0BCa 0.1096±0.0092BCb 0.1241±0.0147BCc 0.1354±0.0089BCc 0.1411±0.009BCc 0.1467±0.0101BCc 0.1886±0.0109BCd
MPK
1:0 0.0145±0Ba 0.1024±0.0197Bb 0.1032±0.007Bc 0.1362±0.0107Bd 0.1419±0.0064Bc 0.1709±0.0382Bd 0.2031±0.0218Be
MPK

Remark: Capital letters in the same column represent significant difference between MPK samples (p<0.05).
Lower case letters in column represent significant difference between teas in each day (p>0.05).

Total phenolic content analysis of mangosteen kombucha by using Folin-Ciocalteau essay

Procedure: 10 µl Sample or a standard solution of gallic acid (ranging from 0–500 mg/l) was
mixed with 1.58 ml of distilled water and 100 µl of Folin-Ciocalteau reagent. The solution was
standed at room temperature for 5 minutes and then followed by adding 300 µl of saturated sodium
carbonate solution. The mixture was left at room temperature for 30 minutes before measured at
the absorbance of 765 nm (UNICO S1200, NJ, USA).

Table 10. Standard formula of garlic acid for Folin-Ciocalteau essay

Gallic acid concentration (μg/m stock solution (ml Distilled water (ml Total volume (ml
l) ) ) )
1000 2 0 2
500 1 1 2
250 0.5 1.5 2
125 0.25 1.75 2

41
 Figure 20: Standard graph to calculate total phenolic content for mangosteen peel kombucha

Table 11. total phenolic content value of mangosteen peel kombucha

Day0 Day1 Day 3 Day6 Day 9 Day 12 Day 15
control 130.8346±0.031Ac 193.1860±0.066Aa 194.2713±0.151Aa 196.9000±0.077Aa 209.1550±0.086Ab 210.2145±0.071Ab 197.73±0.009Aa
1:1
MPK 61.0116±0.1245Ba 66.2442±0.2168Bb 74.2545±0.2829Bc 79.3450±0.1661Bc 94.7558±0.1557Bd 99.2028±0.3750Bd 72.91±0.2149Bc
Remark: Capital letters in the same column represent significant difference between MPK samples (p<0.05).
Lower case letters in column represent significant difference between teas in each day (p>0.05).

HPLC analysis of mangosteen kombucha

42
Standard graph of fructose, glucose and sucrose for HPLC analysis

43
Microbial analysis of mangosteen kombucha
Glucose yeast extract agar(g/L)
Casein Acid Hydrolysate 1.0
Soluble Starch 1.0
Glucose 2.5
Yeast Extract 5.0
Ferrous Sulphate (FeSO4) 0.1
Manganese Sulphate Hydrated (MnSO4 H2O) 0.0001
Agar 15.0

Carr medium
3% yeast extract
0.0022% bromocresol green
2% agar
2% ethanol 95%

Yeast Extract Calcium Carbonate Glucose Agar (g/L)

Yeast extract 10.000
Dextrose (Glucose) 20.000
Calcium carbonate 20.000
Agar 15.000

Sabouraud Dextrose agar (g/L)

Mycological Peptone 10.00
Dextrose 40.00
Agar 15.00

44
Hicrome candida differential agar (g/L)
Peptone 5.000
Malt extract 3.000
Yeast extract 3.000
Glucose 10.000
Chloramphenicol 0.050
Chromogenic mixture 3.000
Agar 18.000

Sensory analysis

45