Scholarly article on topic 'Production of flavor compounds from olive oil mill waste by Rhizopus oryzae and Candida tropicalis'

Production of flavor compounds from olive oil mill waste by Rhizopus oryzae and Candida tropicalis Academic research paper on "Biological sciences"

Share paper
Academic journal
Brazilian Journal of Microbiology
OECD Field of science

Academic research paper on topic "Production of flavor compounds from olive oil mill waste by Rhizopus oryzae and Candida tropicalis"

BJM 1841-11


Brazilian journal of microbiology xxx (2 016) xxx-xxx



1 Biotechnology and Industrial Microbiology

2 Production of flavor compounds from olive oil mill

3 waste by Rhizopus oryzae and Candida tropicalis

4 qi Onur Gunesera'*} Asli Demirkolb, Yonca Karagul Yuceerb, Sine Ozmen Togayc

5 Muge Isleten Hosoglub, Murat Elibold

6 a Usak University, Engineering Faculty, Department of Food Engineering, Usak, Turkey

7 b Canakkale Onsekiz Mart University, Engineering Faculty, Department of Food Engineering, Canakkale, Turkey

8 c Uludag University, Agriculture Faculty, Department of Food Engineering, Bursa, Turkey

9 d Ege University, Engineering Faculty, Department of Bioengineering, Izmir, Turkey

article info

Article history:

Received 2 February 2016

Accepted 12 August 2016

Available online xxx

Associate Editor: Gisele Monteiro de


is Keywords: 19Q3 Olive oil mill waste Biotechnology Microbial fermentation Bioflavor Q2 Agro-waste

The purpose of this study is to investigate the production of flavor compounds from olive oil mill waste (OMW) by microbial fermentation of Rhizopus oryzae and Candida tropicalis. OMW fermentations were performed in shake and bioreactor cultures. Production of flavor compounds from OMW was followed by Gas Chromatography-Mass spectrometry-Olfactometry and Spectrum Sensory Analysis®. As a result, 1.73-log and 3.23-log cfu/mL increases were observed in the microbial populations of R. oryzae and C. tropicalis during shake cultures, respectively. C. tropicalis can produce a higher concentration of d-limonene from OMW than R. oryzae in shake cultures The concentration of d-limonene was determined as 185.56 and 249.54 |xg/kg in the fermented OMW by R. oryzae and C. tropicalis in shake cultures respectively. In contrast, R. oryzae can produce a higher concentration of d-limonene (87.73 |ig/kg) d-limonene than C. tropicalis (11.95 |ig/kg) in bioreactor cultures. Based on sensory analysis, unripe olive, wet towel, sweet aromatic, fermented aromas were determined at high intensity in OMW fermented with R. oryzae meanwhile OMW fermented with C. tropicalis had only a high intensity of unripe olive and oily aroma.

© 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (



The food and agricultural industries produce annually million tons of waste, resulting from production and consumption of food, peels, pulps, aqueous residues and others, many of which raise serious disposal issues and, consequently,

considerable costs to various industries.1 Therefore, using 28

agro-wastes is the most popular aspect in biotechnological 29

processes for production of high value-added products 30

in terms of the reducing production cost.2,3 Recently, 31

agro-wastes have been focused in biotechnological flavor 32

production by using microbial fermentation or biotransfor- 33 mation owing to their high amount of reusable components

* Corresponding author. E-mail: (O. Guneser).

1517-8382/© 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (

BJM 1841-11


2 Brazilian journal of microbiology xxx (2 016) xxx-xxx

34 for microorganisms.1'4-6 Several studies were conducted on 3000 rpm for 5 min. Both microbial suspensions were counted 89

35 the production of natural flavor compounds from agro-wastes with Thoma counting chamber by using light microscope.7,26 90

36 by microbial fermentation. Cassava bagasse, sugar beet, The suspensions contain 107-8 spores or cells/mL for R. oryzae 91

37 beet molasses, coffee husk, soy bean waste, apple pomace, and C. tropicalis. 92

38 cacao bagasse were extensively used in these studies for

39 the production of natural flavors by yeast or molds.7-11 For Experiments of shake cultures and bioreactor cultures 93

40 instance, de Oliveira et al.12 reported that 2-phenylethanol

41 was produced from cassava wastewater by Saccharomyces Initially, 200 g of OMW were weighed and grinded by using 94

42 cerevsiae, Geotrichum fragrans and Kluveromyces marxianus in knife mill Restch GM 200 (Haan, Germany) for 15 min. Then, 95

43 the following yields order: 0.74 g/L, 0.19 g/L and 0.08 g/L. Zheng 2 L of OMW solution (10%, w/v) was prepared for shake cul- 96

44 et al.13 produced vanillin from waste from waste residue of tures. The OMW solution was homogenized at 24,000 rpm by 97

45 rice bran oil by both strains of Aspergillus niger CGMCC0774 Ultraturax (IKA-WERKE GmbH, Germany). The solution was 98

46 and Pycnoporus cinnabarinus CGMCC1115. In the more recently, divided into six groups (300 mL) and initial pH was adjusted to 99

47 Wilkowska et al.14 showed that the production of esters and 5.0, for R. oryzae and pH 7.0, for C. tropicalis by using 1N HCl and 100

48 alcohols including ethyl acetate, isoamyl acetate isoamyl 1N NaOH. Initial pHs were selected based on previous studies 101

49 alcohol and 2-phenylethanol from apple pomace with choke- and these pHs are optimum for microbial growth.27,28 30 mL of 102

50 berry and cranberrys pomace can be achieved by immobilized OMW solution was poured into 100 mL Erlenmeyer and tops 103

51 K. marxianus LOCK0024 in shake culture. Fadel et al.15 reported were closed with cotton wool and aluminum foil. The OMW 104

52 that a high concentration of 6-pentyl-a-pyrone (3.62 mg/g solutions were sterilized in an autoclave (Hirayama, Saitama, 105

53 DM) associated with coconut aroma can be produced from a Japan) at 121 °C for 15 min and then inoculated with C. tropicalis 106

54 sugarcane bagasse by using T. viride EMCC-107. Olive oil mill and R. oryzae at a level of 107-8 cell or spores/mL OMW suspen- 107

55 waste (OMW) and olive oil mill waste water (OMWW) are the sion. The flasks then were incubated at 120 rpm for 288 h at 108

56 most important wastes for olive oil industry in Mediterranean 30 °C in a rotary incubator (Sartorius-Certomat IS, Goettingen- 109

57 regions. One to 2.5 million tons was produced annually during Germany). The control groups without microorganism were 110

58 olive oil season in Andalusia region (Spain)16 while 200-250 prepared by following the same procedure. Duplicate samples 111

59 thousand tons of OMW are produced in Turkey.17 OMW is a were prepared for each treatment. 112

60 solid phase by-product resulting from extraction of olive oil by Bioreactor cultures were conducted in a 5 L stirred tank 113

61 pressure or centrifugation. OMW approximately has 25-55% bioreactor (STR) (Biostat A-plus®, Sartorius, Melsungen, Ger- 114

62 water; 25-50% of fiber with a great degree of lignifications, many) with 4 L working volume. Fermentation conditions used 115

63 5-8% of residual oil, 2-6% of ash and 6-10% of nitrogen for microbial growth and flavor production were based on the 116

64 associated with the insoluble fiber fraction.18-20 Microbial results obtained in shake cultures. The STR was equipped with 117

65 fermentation has been taken over by many researchers two six-blade impellers, pH probe (Hamilton, Easyferm K8/325) 118

66 due to possibility of valorization of agro-wastes, low-cost and PT 100 temperature sensor. The aeration rate, agitation 119

67 production steps and the possibility of using several types of speed and temperature for both microbial cultures were set 120

68 microorganisms.21,22 From this perspective, utilization of the as 0.325 vvm, 120 rpm and 30 °C respectively. 121

69 filamentous fungi such as Rhizopus species and the thermo

70 and ethanol tolerance of Candida species have been widely Specific growth rate and microbial count 122

71 examined in bioenergy and bioproduct industries.23-25 When

72 we take into the consideration the nutritional content of OMW Spore count of R. oryzae and cell count of C. tropicalis dur- 123

73 for microbial growth, OMW might be used as raw material in ing fermentation were determined by pour plate technique 124

74 the production of flavor compounds by both microorganisms with Potato Dextrose agar (PDA).26 The sample was taken from 125

75 via microbial fermentation. This study therefore focuses shake flask and STR intermittently in aseptic conditions. 126

76 to investigate the production of natural flavor compounds

77 from OMW by microbial fermentation of Rhizopus oryzae and Analysis of aroma compounds 127

78 Candida tropicalis.

Flavor compounds from fermented OMW were deter- 128

mined by gas chromatography-olfactometry (GCO), gas 129

Material and methods chromatography-mass spectrometry (GC-MS) and sensory 130

analysis. 131

79 Microorganisms and inoculum preparation

Extraction of flavor compounds 132

80 Strains of Rhizopus oryzae NRLL 395 and Candida tropicalis ATCC

81 665 were obtained from Department of Bioengineering, Ege Flavor compounds in fermented and unfermented OMW were 133

82 University (Izmir, Turkey). Both microorganisms were grown extracted by solid-phase microextraction (SPME).29 Three 134

83 on slant Potato Dextrose Agar (PDA) in Petri plate at 30 °C grams of OMW were weighed in a 40 mL amber colored screw 135

84 for 7 days. Then, R. oryzae spores and C. tropicalis cells were top vial with hole cap PTFE/silicon septa (Supelco, Bellafonte, 136

85 collected by washing with 0.1% (w/v) Tween 80 from agar sur- USA) to which 1 g of NaCl was added to the vial. The vial was 137

86 face, separately. The spore suspension of R. oryzae was filtered kept at 40 °C in a water bath (GFL, Grossburgwedel, Germany) 138

87 through two layers cheesecloth and centrifuged at 3000 rpm for 20 min to equilibrate the volatiles in headspace. Then, 139

88 for 5 min. The cell suspension of C. tropicalis was centrifuged at a SPME (2 cm to 50/30 ^m DVB/Carboxen/PDMS, Supelco, 140


Brazilian journal of microbiology xxx (2 016) xxx-xxx 3

141 Bellafonte) needle was inserted into the vial. The SPME fiber

142 was exposed at a depth of 2 cm in the headspace of the vial for

143 20min at 40°C in water bath. Then, the sample was injected

144 into GC-MS, immediately.30

145 Gas chromatography olfactometry (GCO) analysis

146 The GCO analysis was performed for 5-days fermented OMW

147 to determine the changes of flavor profile. The GCO was con-

148 ducted by HP 6890 GC (Agilent Technologies, Wilmington, DE,

149 USA) equipped with a flame ionization detector (FID), a sniffing

150 port and splitless injector system. A nonpolar column (HP-

151 5 30 m length, 0.32 mm i.d., 0.25 |m df; J&W Scientific) was

152 used for sniffing. Column effluent was split 1:1 between FID

153 and olfactory port using deactivated fused silica capillaries

154 (90cm length, 0.25mm i.d.). Helium was used as the carrier

155 gas. Inlet pressure was 7.07 psi, and flow 1.2mL/min. The GC

156 oven temperature was programmed from 40 to 230 °C at a rate

157 of 10 °C/min, with initial hold of 5 min and final hold time of

158 20 min. The FID and sniffing port were maintained at the tem-

159 peratures of 250°C and 200°C, respectively. GCO procedure

160 was duplicated.30 Post-peak intensity method was used for

161 the determination of aroma intensity by using 10-point scale

162 anchored to the left with 'not' and to the right with 'very'.31

163 Sniffer had 100 h of experience with GCO technique, scale

164 using and odor description. Aroma-active compounds were

165 identified by comparing retention indices (RI) and odor qual-

166 ity of unknowns with those of references analyzed at the same

167 experimental conditions by sniffer during GCO procedure.

168 Retention indices were calculated using n-alkane series.32

169 Identification and quantification of flavor compounds

170 Flavor compounds were determined by gas chromatography-

171 mass spectrometry (GC-MS). Nonpolar HP5 MS column

172 (30m x 0.25 mm i.d. x 0.25 |m film thickness, J&W Scientific,

173 Folsom, CA) was used for separation of flavor compounds.

174 The GC-MS system consisted of an HP 6890 GC and 7895C

175 mass selective detector (Agilent Technologies, Wilmington,

176 DE, USA). The oven temperature was programmed from 40 °C

177 to 230°C at a rate of 10°C/min with initial and final hold

178 times of 5 and 20 min, respectively. Helium was used as the

179 carrier gas with a constant flow of 1.2 mL/min. The Mass Spec-

180 trometry Detector (MSD) conditions were as follows: capillary

181 direct interface temperature, 280 °C; ionization energy, 70 eV;

182 mass range, 35-350 amu; scan rate, 4.45 scan/s.30 Identifica-

183 tion of the flavor compounds was based on the comparison of

184 the mass spectra of unknown compounds with those in the

185 National Institute of Standards and Technology33 and Wiley

186 Registry of Mass Spectral Data, 7th Edition34 mass spectral

187 databases. Quantification of flavor compounds was expressed

188 as relative abundances of flavor compounds by Eq. (1).35

189 2-Methyl pentanoic acid and 2-methyl-3-heptanone were used

190 as an internal standard (IS) for acidic and neutral-basic com-

191 pounds, respectively.

192 Mean relative abundance (|g/kg)

concentration of IS x peak area of compound

Sensory analysis

A roundtable discussion was conducted to determine descriptive sensory properties and changes in aroma profiles of fermented OMW versus control samples (unfermented OMW) for shake cultures.36 Panelists were staff and graduate students in the Department of Food Engineering at Canakkale Onsekiz Mart University. Four female and three male participated for sensory panel. Their ages ranged from 24 to 45 years. The panel received about 300 h of training during generation and definition of descriptive terms. Panelists quantified the attributes using 15-point product-specific scale anchored to the left with 'not' and on the right with 'very'.36

Statistical analysis

Analysis of variance (ANOVA) was conducted to determine the differences in the amount of flavor compounds during fermentation time in shake cultures and bioreactor cultures. ANOVA model37 is shown in Eq. (2).

Y;,- = ß + a; + e;j

peak area of the IS

where Yij is the jth observation value in the ith fermentation time, ^ is the general population mean, ^ is the effect of the ith fermentation, and eij represents the random error term. Tukey's Honestly significant difference (HSD) test was used for separating means; SPSS for Windows (version 15.0) was used for all statistical analyses.

Results and discussion

Microbial growth ofRhizopus oryzae and Candida tropicalis in OMW

The microbial growth of R. oryzae and C. tropicalis in OMW during shakecultures and bioreactor cultures was shown in Table 1. Maximum increase in microbial population of R. oryzae and C. tropicalis was determined as 1.73 log cfu/mL (1.29 fold) and 3.23 log cfu/mL (1.52 fold), respectively. The growths of R. oryzae and C. tropicalis came into stationary phase around 72 h in shake cultures. In stationary phase, micro-bial populations of R. oryzae and C. tropicalis were around 6.5 log cfu/mL and 9.0logcfu/mL, respectively. In bioreactor cultures, it was observed that both microorganisms were attained the exponential growth phase within 24 h. The micro-bial population of R. oryzae increased around 3logcfu/mL (3 fold) through 288 h of fermentation whereas C. tropicalis populations' increased 1.4logcfu/mL (1.22 fold) at the same fermentation time (Table 1).

In the literature, there are several studies on the growth behavior of R. oryzae and C. tropicalis in solid state and submerged fermentation,38-40 and most of researchers have revealed different results for growth behavior and biomass increase for R. oryzae and C. tropicalis on certain agrowastes. The obtained data about the microbial growth of both R. oryzae and C. tropicalis in OMW showed both microorganisms growths were different for shake cultures and bioreactor cultures unexpectedly. These differences could be attributed to

200 201 202

216 217

220 221 222


JM 1841-11

Brazilian journal of microbiology xxx (2 016) xxx-xxx

Table 1 - Growth of R. oryzae and C. torpicalis in OMW during shake cultures.

Fermentation time (h) Microbial count ±S.E. (log cfu/mL OMW solution)

Shake cultures

R. oryzae C. tropicalis

0 0.92 ±0.08 6.15 ± 0.14

24 6.76 ±0.65 7.97 ±1.13

48 7.65 ±0.01 7.61 ± 0.01

72 6.68 ±0.01 9.21 ± 2.25

120 6.83 ±0.04 9.38 ±0.01

168 6.69 ±0.01 9.05 ± 0.14

288 6.84 ±0.03 9.28 ±0.01

Maximum growth (log cfu/mL) 1.73 3.23

Fermentation time (h) Bioreactor cultures

R. oryzae C. tropicalis

0 3.47 ±0.16 6.35 ± 0.01

24 5.0 ±0.01 7.30 ±0.01

48 4.76 ±0.07 7.72 ±0.11

72 4.57 ±0.07 7.69 ±0.01

96 4.72 ±0.11 7.23 ± 0.01

120 —a 7.15 ± 0.14

180 5.04 ±0.07 -a

288 6.47 ±0.07 7.75 ± 0.14

Maximum growth (log cfu/mL) 3.0 1.4

a Microbial analysis could not conducted for this fermentation time. S.E.: standard error, cfu: colony forming unit.

244 the interaction of microorganism with OMW nutrients in fer-

245 mentation conditions and strain of microorganism.2 In our

246 previous study41 on OMW, we observed the microbial growth of

247 Trichoderma atroviride in shake cultures was higher than biore-

248 actor cultures, whereas the yeast Torulaspora delbrueckii acts

249 adverse growth behavior from T. atroviride at the same condi-

250 tions. Jin et al.42 investigated the growth of different Rhizopus

251 strains in potato, corn, wheat starch and pineapple processing

252 waste water streams for lactic acid production. Similar to our

253 results, an increase in fungal biomass was found to be about

254 2 and 4.5 fold for R. oryzae and R. arrhizus in the fermenta-

255 tion of all studied agrowaste. The researchers also indicated

256 that sharp increase in biomass formation of R. oryzae 2062

257 was higher than R. arrhizus 36017 at 30 °C and 150 rpm in all

258 agrowastes. Saracoglu and Cavusoglu43 found that biomass of

259 C. tropicalis' Kuen 1022 in sunflower hull hydrolysate medium

260 (66g/L) increased in 6-7 fold until 20h fermentation at 30°C;

261 stirring at 140 rpm. Oberoi et al.44 investigated enhanced

262 ethanol production via fermentation of rice straw hydrolysate

263 by C. tropicalis ATCC 13803 which adapted and non-adapted

264 for a rice straw hydrolysate medium. They found that the

265 biomass of adapted and non-adapted C. tropicalis ATCC 13803

266 increased about 3 and 2 folds during 6-18 h fermentation

267 at 35 °C and 120 rpm, respectively and both microorgan-

268 isms biomass remained stationary during 18-24h of the

269 fermentation.

270 Flavor production characteristics by Rhizopus oryzae and

271 Candida tropicalis in OMW

272 Aroma active compounds of 5 days (~135h) fermented and

273 unfermented OMW were shown in Table 2.

In total, 17 and 13 aroma active compounds were identified 274

in fermented OMW by R. oryzae and C. tropicalis respectively. 275

Identified aroma-active compounds included acids, alcohols, 276

aldehydes, esters, ketones, terpene, and 4 unknown com- 277

pounds. Among these aroma-active compounds, unknown 278

1 associated with dirty-acid aroma, isovaleric acid, hexyl 279

acetate methional, (E)-2-nonenal and 2,4-nonadienal were 280

detected at higher intensities in unfermented OMW. More- 281

over, it was determined that there were some differences in 282

aroma profile of unfermented OWM. These differences might 283

be related in pH value of the OMV solution; absorption behav- 284

ior of SPME fiber and sensitivity of perception of panelists 285

during GC-O analysis 286 OMW fermented with R. oryzae had higher intensities of 287

2-pentanone, D-limonene and 2-phenylethanol than unfer- 288

mented OMW whereas D-limonene was only found to be 289

at higher intensity in OMW fermented with C. tropicalis. 290

These GCO results revealed that 2-pentanone, D-limonene and 291

2-phenylethanol can be produced from fermented OMW. 292

Limonene is monoterpen which is one of the main 293

compounds of citrus essential oils and 2-phenylethanol 294

is aromatic alcohol with rose like flavor. D-Limonene and 295

2-phenylethanol can be found in many plant sources. Both 296

flavor compounds are greatly used in food, perfume and 297

cosmetic industry. 2-Phenylethanol and D-limonene are natu- 298

rally produced by distillation of citrus peel and rose petals, 299

respectively. From aspect of yeast and fungal metabolism, 300

there are many biochemical pathways for flavor compounds. 301

Among these pathways, production of alcohols and ester type 302

flavor compounds was achieved by yeast via and Acetyl Coen- 303

zyme A/Alcohol Acetyl Transferase reaction and Ehrlich pathway 304

which covered transamination, decarboxylation, oxidation 305


BJM 1841-11

Brazilian journal of microbiology xxx (2 016) xxx-xxx 5

Table 2 - Aroma-active compounds of unfermented and fermented OMW with R. oryzae and C. tropicalis (n = 2).

RIa Volatile compound Aroma quality Identification Aroma intensityb (mean ±SE)

methods -

Control R. oryzae Control C. tropicalis

598 Diacetyl Butter RI,O 1.25 ±0.25 0.60 ±0.10 4.75 ± 0.75 4.50 ±1.50

734 2-Pentanone Oily RI,MS,O ND 1.50 ±0.50 ND ND

802 Hexanal Green grass RI,MS,O 3.0 ±1.0 ND 2.0 ±0.10 ND

856 Unknown 1 Dirty, acid RI,O 5.50 ±0.50 5.25 ± 1.25 0.75 ± 0.75 ND

862 Isovaleric acid Sour, fruity RI,O 5.50 ±0.50 4.75 ± 0.25 1.75 ± 0.25 1.50 ±1.50

869 Sytren Acid, dirty RI,MS,O ND ND 2.75 ± 0.25 ND

898 Methional Boiled potato RI,O 4.50 ±0.50 ND 4.50 ±1.50 3.75 ±0.25

928 2-Acetyl-1-pyroline Popcorn RI,O ND 0.50 ±0.50 ND ND

975 Unknown 2 Metallic RI,O 5.75 ±0.25 3.0 ±0.10 4.50 ±1.50 3.75 ±0.25

999 Hexyl acetate Cologne RI,O 4.50 ±0.50 0.75 ± 0.25 1.50 ±0.50 0.75 ±0.75

1040 d-Limonene Citrus RI,MS,O 1.75 ±0.25 3.00 ±1.0 1.5 ±0.50. 2.50 ±0.10

1050 Benzeneacetaldehyde Rose, flower RI,MS,O 2.50 ±0.50 2.0 ±2.0 ND ND

1058 Unknown 3 Vegetable oil RI,O 2.0 ±1.0 2.0 ±0.10 ND ND

1077 o-Cresol Wet towel RI,MS,O 1.0 ±1.0 1.50 ±0.50 3.0 ±1.0 2.25 ±0.25

1095 Guaiacol Burn sugar RI,MS,O ND ND 3.50 ±1.50 ND

1120 Unknown 4 Sour RI,O 3.0 ±0.10 0.50 ±0.50 ND ND

1138 (E)-2-Nonenal Hay RI,MS,O 5.0 ±0.10 2.0 ±1.0 2.50 ±0.50 ND

1144 2-Phenylethanol Rose RI,MS,O 2.0 ±0.10 3.0 ±0.10 1.0 ±0.50 1.75 ±0.25

1157 (Z)-2-Nonenal Cucumber RI,MS,O 2.0 ±2.0 1.0 ±0.10 2.50 ±0.50 ND

1158 (E,Z)-2,6-Nonadien-1-ol Hay RI,O 3.50 ±0.50 ND 2.75 ± 0.75 1.0 ±1.0

1186 Naphthalene Dirty RI,MS,O ND ND 0.75 ± 0.75 1.0 ±1.0

1191 Carveol Minty RI,MS,O 0.50 ±0.50 ND ND ND

1216 2,4-Nonadienal Burnt oil RI,MS,O 5.75 ±0.25 1.50 ±0.50 3.25 ± 0.75 2.0 ±2.0

1250 Carvenone Lactone, sweet RI,O ND ND ND ND

1268 Geraniol Sweet, flower RI,O 0.50 ±0.50 1.0 ±1.0 2.0 ±0.10 1.0 ±1.0

1322 (E,E)-2,4-Decadienal Burnt oil RI,MS,O ND ND 4.50 ±0.50 1.0 ±0.50

1372 a-Kubebene Sweet RI.MS.O 3.0 ±1.0 ND ND ND

a RI: Retention indices based on HP-5 column.

b Aroma intensity in 5 days (~135 h) fermented and unfermented OMW. SE, standard error; ND, not detected; MS, mass spectrometry; O, odor.

306 and reduction of branched chain amino acids. Moreover, some

307 methyl ketones and lactones were produced by ^-oxidation

308 of long-chain hydroxy fatty acids (e.g. ricinoleic acid) by

309 yeast and mold. It was also known that several fungi pro-

310 duced most of the terpene by the Mevalonate pathway as

311 found in higher plant and biotransformation reactions.45-49

312 Therefore, it was concluded that 2-pentanone, D-limonene

313 and 2-phenylethanol from OMW were produced by R. oryzae

314 and C. tropicalis through aforementioned reactions. Several

315 researchers have been pointed out similar approaches for the

316 production of flavor compounds from agrowaste.7,11,41,50 In

317 most recently, Mantzouridou and Paraskevopoulou50 demon-

318 strated de novo synthesis of fruity esters as isoamyl acetate,

319 ethyl dodecanoate, decanoate, octanoate and phenyl ethyl

320 acetate from orange peel waste was achieved by Saccharomyces

321 cerevisiae at high level. In our previous study41 on OMW, we

322 observed that the filamentous fungus Trichoderma atroviride

323 produces 1-octen-3-ol and 2-octenol at high level, and 2324 phenylethanol and menthol also can be produced by using

325 Torulaspora delbrueckii.

326 Christen et al.7 indicated that R. oryzae can produce

327 acetaldehyde, ethanol (sweet), 1-propanol, ethyl acetate, ethyl

328 propionate and 3-methyl butanol from Amaranth grain sup-

329 plemented with mineral salt solution. In a study by Chatterjee

330 and Bhattacharyya,51 production of a-terpineol (floral) from

331 a-pinene (herbal) by microbial oxidation of C. tropicalis MTCC

332 230 with a yield of 77% was achieved. We did not observe

333 the production of acetaldehyde, ethanol, 1-propanol, ethyl

acetate, ethyl propionate and 3-methyl butanol from OMW 334

by fermentation of R. oryzae and a-terpineol by C. tropicalis by 335

compared with the findings of previous studies.7,51 This could 336

be related to composition of agrowaste and fermentation 337

conditions including aeration, temperature and fermentation 338

scale.52 339

In fermented OMW, methoxy phenyl oxime and methyl 340

butanoate could not be identified by GCO analysis, but we 341

determined these compounds by GC-MS. These results can 342

be attributed to "odor threshold" and "odor recognition thresh- 343

old". Because, the concentration of volatile compounds in 344

matrix has to be higher than both threshold values in order to 345

identify the volatile compound by GCO technique.53 Methoxy 346

phenyl oxime is N-containing compound with both phenyl 347

and methoxy groups. There is little information on flavor 348

characteristics of methoxy phenyl oxime.54 Some researchers 349

identified it as contaminant come from SPME fiber. They point 350

out that is originated from the glue that is used for SPME 351

fiber.55 However, the compound has been found naturally in 352

some food products,56-58 especially bamboo shoots and sec- 353

ondary metabolites of myxobacteria59 by some researchers. 354

Methyl butanoate which is associated fruity flavor is the ester 355

of butyric acid. Likewise most of ester compounds, it was 356

biosynthesized with Ehrlich pathway and reactions of lipase 357

enzyme by microorganism. 358

Table 3 shows the changes in concentration of flavor com- 359

pounds produced by R. oryzae and C. tropicalis from OMW 360

during shake cultures. 361


BJM 1841-11

Brazilian journal of microbiology xxx (2 016) xxx-xxx

Table 3 - Changes in concentration of flavor compounds in OMW during shake cultures of R. oryzae and C. topicalis.

Volatile compound RIa Aroma quality Mean (^g/kgOMW)b ±SE

Rhizopus oryzae

Controlc 72 h 288 h

2-Pentanone 682 Sweet 12.29 ±8.72 14.16 ± 10.04 24.28 ± 3.30

Methoxy-phenyl-oxime 926 - 61.74 ±29.17 64.95 ± 29.73 92.26 ± 3.32

d-Limonene 1032 Lemon, citrus 40.13 ±1.05B 185.56 ± 17.47A 17.22 ± 1.60B

2-Phenylethanol 1118 Rose 1.35 ±0.96B 13.91 ± 6.57A 11.71 ± 8.30A

Volatile compound RIa Aroma quality Mean (^g/kg OMW)b ± SE

Candida tropicalis

Controlc 72h 288 h

Methyl butanoate 717 Fruity Nd 4.67 ± 0.20A 0.40 ± 0.20B

d-Limonene 1032 Lemon, citrus 119.64 ±77.04™ 249.54 ± 30.82A 8.93 ± 6.26B

A,B Means followed by different superscript letter represent significant differences in the same flavor compound through the fermentation time. a Retention index based on HP 5MS column.

b Mean relative abundance = (concentration of internal standard x peak area of compound)/(peak area of the internal standard). c 72h incubation without R. oryzae or C. tropicalis, Nd: not detected, SE: standard error.

362 Fermentation time had a significant effect on the con-

363 centration of volatile compounds which were produced by

364 both microorganisms (p <0.05). It was determined that con-

365 centrations of D-limonene (citrus) and 2-phenylethanol (rose)

366 significantly increased in OMW during 72 h of fermentation

367 of R. oryzae. The maximum concentrations of D-limonene and

368 2-phenylethanol were determined as 185.56 and 13.91 ^g/kg

369 OMW. The concentration of 2-petanone and methoxy phenyl

370 oxime gradually increased during fermentation of R. oryzae.

371 However, no significant changes were determined in the

372 concentration of both compounds (Table 3). It was thought

373 that this observation is related to a high standard error of

374 results for the compounds. During the fermentation of C. trop-

375 icalis, the concentrations of D-limonene and methyl butanoate

376 (fruity) significantly increased similar to the fermentation of

377 R. oryzae. The maximum concentration of D-limonene and

378 methyl butanoate was 249.54 and 4.67 ^g/kg OMW at 72 h

379 of fermentation. The concentrations of flavor compounds

380 produced by both microorganisms decreased after 72 h fer-

381 mentation.

382 Fig. 1 shows changes in concentration of flavor compounds

383 produced from in OMW by R. oryzae and C. tropicalis in biore-

384 actor cultures.

385 In bioreactor cultures, fermentation time had also signif-

386 icant effect on the amount of flavor compounds in batch

387 fermentation (p <0.05). As seen from Fig. 1A, the concentra-

388 tion of 2-pentanone and methoxy phenyl oxime increased

389 in OMW during 72 h of fermentation by R. oryzae, while the

390 concentration of D-limonene and 2-phenylethanol increased

391 during 180 h of fermentation. After these times, a significant

392 decrease in the concentration of all flavor compounds was

393 observed throughout the fermentation (p<0.05). The maxi-

394 mum concentration of 2-pentanone, methoxy phenyl oxime,

395 D-limonene and 2-phenylethanol were 19.46, 34.44, 87.73 and

396 11.25 ^g/kg OMW, respectively. In the case of fermentation

397 by C. tropicalis (Fig. 1B), concentration of methyl butanoate

398 and D-limonene were reached at maximum level in the

fermentation time of 72 h and 180 h and the maximum con- 399

centration of these compounds were determined as 7.26 400

and 11.95 ^g/kg OMW, respectively. Moreover, a significant 401

decrease was also observed in the amounts of all volatile com- 402

pounds produced by C. tropicalis throughout the fermentation 403

similar to the fermentation of R. oryzae (p < 0.05). 404

Mantzouridou and Paraskevopoulou50 investigated the 405

usage potential of orange pulp for microbial flavor produc- 406

tion by S. cerevisiae in under semi-anaerobic and micro aerobic 407

conditions. The researchers observed that acetate esters as 408

isoamyl acetate and phenyl ethyl acetate concentrations in 409

culture media decreased after 48 h the fermentation time 410

in microaerobic conditions whereas concentrations of ethyl 411

hexanoate, ethyl octanoate and ethyl decanoate decreased 412

after 24h in the same fermentation conditions. Rossi et al.60 413

investigated the producing fruity flavor by Ceratocystis fimbri- 414

ata in solid state fermentation using citric pulp as culture 415

media. They found that isoamyl acetate and ethyl acetate 416

were produced as fruity flavor by selected microorganism, 417

and both flavor concentrations have been increased during 418

48 h fermentation time. In our previous study30 on micro- 419

bial flavor production from tomato pomace by Kluyveromyces 420

marxianus, we determined that the concentrations of isoamyl 421

alcohol, isovaleric acid and phenyl ethyl alcohol have been 422

gradually increased during 24, 48 and 72 h fermentation time, 423

respectively. After these times, significant decreases in the 424

concentrations of all volatiles were observed through 120 h fer- 425

mentation. However, Moradi et al.61 observed that a gradual 426

increase in 7-decalactone concentration in synthetic media 427

during 75 h fermentation time when they fed bioreactor with 428

castor oil at a rate of 5 mL/h and used atmospheric air for aera- 429

tion. When Table 3 and Fig. 1 taken into consideration, general 430

decrease was observed in the concentration of all volatile 431

compounds after a particular time in shake cultures and biore- 432

actor cultures similarly the results of previous studies.41'50'60 433

This observation might be related to stripping effect which is 434

unavoidably discharge of the flavor compounds in the shake 435


Brazilian journal of microbiology xxx (2016)xxx-xxx 7


0 12 24 72 135 180 230 ' 280

Fermentation time (hour)

■ 2-petanone "Mehoxy phenyl oxime i D-limonene "2-phenylethanol

0 12 24 72 135 180 230 280

Fermentation time (hour)

| ■ Methyl butanoate ■ D-limonene |

Fig. 1 - Changes in concentration of flavor compounds in OMW during bioreactor cultures of R. oryzae (A) and C. tropicalis (B). A-EMeans followed by different superscript letter on bar represents significant differences in the same flavor compound through the fermentation time.

436 cultures and bioreactor cultures due to flavor compounds

437 volatilities and their dissolving behavior in culture media dur-

438 ing the fermentation period.62 For instance, this stripping

439 process was modeled and investigated experimentally for the

440 production of ethyl acetate from whey by K. marxianus. It was

441 found that the stripping rate of the flavor compounds nearly

442 independent of the stirring and was proportional to the gas

443 flow. They pointed out the stripping rate was governed by the

444 absorption capacity of the exhaust gas rather than the phase

445 transfer in the bioreactor.

The productivities of flavor compounds produced both 446

strains in bioreactor cultures have varied (Table 4). It was 447

determined that productivity of D-limonene produced by R. 448

oryaze was higher than those of C. tropicalis. So, it can be said 449

the production of D-limonene from OMW by R. oryzae were 450

higher than C. tropicalis. Moreover, 2-phentylethanol has the 451

lowest productivity mean while similar productivity values 452

were observed for D-limonene and methoxy-phenyl-oxime 453

in the case of R. oryzae fermentation. By comparing the pro- 454

ductivity results of this study with previous studies, different 455


BJM 1841-11

Brazilian journal of microbiology xxx (2 016) xxx-xxx

A Unripe olive

^^ Control — R. oryzae

Sweet aromatic

Wet bulger




Fig. 2 - Sensory properties of OMW fermented by R. oryzae (A) and C. tropicalis (B).

productivity values were reported for various flavor compounds.30,63 Mantzourido et al.63 investigated the production of some esters from orange peel waste by solid state fermentation of S. cerevisiae. They found that the productivity values varied between 0.10 and 3.23mg/kgh for some ethyl esters including ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate. In a study by Guneser et al.30 1.41 and 5.27 ^g/kgh of productivity values for isoamyl alcohol produced from tomato pomace by K. marxianus and D. hansenii, respectively. Moreover, Guneser et al.41 reported that 8.81 ^g/kgh of productivity value for 1-octen-3-ol produced from OMW by T. atroviride.

Sensory characteristics of OMW fermented with Rhizopus oryzae and Candida tropicalis

Interpretation of relationship between flavor compounds which are produced by biotechnological process and their sensory properties in fermented culture matrix has not been discussed in detail in the previous studies. However, this interpretation is essential to determine the sensory quality of flavor compounds. Here, we conducted sensory analysis to evaluate the relationship between quantity of produced flavor compound and its sensory impact. Eleven aroma terms were developed by panelists for fermented OMW. Fig. 2


Brazilian journal of microbiology xxx (2 016) xxx-xxx 9

Table 4 - The productivity of volatile compounds produced by R. oryzae and C. tropicalis in bireactor cultures.

Volatile compounds Productivitya Mean (^g/kgh) ±S.E.

R.oryzae C. tropicalis

2-Pentanone Methoxy-phenyl-oxime d-Limonene 2-Phenylethanol Methyl butanoate 0.26 ±0.01 0.45 ± 0.01 0.47 ±0.01 0.06 ±0.01 Nc Nc Nc 0.06 ±0.01 Nc 0.1 ± 0.01

a Productivity was calculated based on the maximum concentration of each volatile compounds through the bioreactor culture. Nc, not calculated.

479 shows sensory characteristics of fermented and unfermented

480 OMW.

481 There were significant differences between fermented

482 and unfermented OMW in terms of oily, unripe olive and

483 woody/herbal, wet towel, sweet aromatic (p <0.05). OMW fer-

484 mented with R. oryzae has a higher intensity in terms of unripe

485 olive, wet towel and sweet aromatic than unfermented OMW.

486 Woody/herbal, rancid and fruity aromas were at high inten-

487 sity in unfermented OMW (Fig. 2A). Significant differences

488 were also observed for unfermented and fermented OMW in

489 the fermentation of C. tropicalis. Unripe olive and oily aromas

490 were found to be at high intensities in fermented OMW and

491 unfermented OMW has only a high intensity of wet bulgur

492 aroma. Other sensory characteristics score were found to be

493 a similar in fermented and unfermented OMW (Fig. 2B). It

494 can be said that sweet aromatic are related to 2-pentonone,

495 2-phentylethanol and D-limonene while methoxy phenyl

496 oxime are associated wet towel flavor. Moreover, unripe olive

497 and oily flavors seem to relate to methyl butanoate and

498 D-limonene.


The results show that OMW can be considered as a raw material for biotechnological production of flavors. The microbial production of 2-petanone, D-limonene and 2-phenylethanol were achieved from OMW by fermentation of R. oryzae, while C. tropicalis produced D-limonene and methyl butanoate. In the study, the stripping effect was observed in bioreactor fermentation. Therefore, modeling of stripping effect is required for produced flavor compounds by taking into account temperature, flow rate, agitation speed and volatility in the next step for improved the productivity. Further studies should be conducted to evaluate the production potential of natural flavors from OMW by using other microorganism with biotechnolo-gical approaches.

Conflict of interest

The authors declare that they have no conflict of interest.


This study was funded by The Scientific and Technologi- 513

cal Council of Turkey (TUBITAK, Ankara Turkey; Project No. 514

1100903 COST). The authors would like to thank Bioflavour 515

COST Action FA0907 for technical supporting of this scientific 516

work. 517


1. LaufenbergG, Kunz B, Nystroem M. Transformation of 520 vegetable waste into value added products: (A) the upgrading 521 concept; (B) practical implementation. Bioresour Technol. 522 2003;87(2):167-198. 523

2. Dastager G. Aroma compounds. In: Singh nee'Nigam P, 524 Pandey A, eds. Biotechnology for Agro-industrial Residues 525 Utilisation. Germany: Springer Science +Business Media B.V.; 526 2009:105-127. 527

3. Gounaris Y. Biotechnology for the production of essential 528 oils, flavours and volatile isolates. A review. Flavour Frag J. 529 2010;25(5):367-386. 530

4. Harlender S. Biotechnology for the production of flavouring 531 materials. In: Reineccius G, ed. Source of Book of Flavors. 532 Maryland, USA: Aspen Publisher; 1994:155-175. 533

5. Schieber A, Stintzing FC, Carle R. By-products of plant food 534 processing as a source of functional compounds - recent 535 developments. Trends Food Sci Technol. 2001;12(11), 401-+. 536

6. Sarma S, Dhillion G, Hegde K, Brar S, Verma M. Utilization of 537 agro-industrial waste for the production of aroma 538 compounds and fragrances. In: Brar S, Dhillon G, Soccol C, 539 eds. Biotransformation of Waste Biomass Into High Value 540 Biochemicals. Springer+Business Media: London; 2013:99-115. 541

7. Christen P, Bramorski A, Revah S, Soccol CR. 542 Characterization of volatile compounds produced by 543 Rhizopus strains grown on agro-industrial solid wastes. 544 Bioresour Technol. 2000;71(3):211-215. 545

8. Haffner T, Tressl R. Biosynthesis of 546 (R)-gamma-decanolactone in the yeast Sporobolomyces odorus. 547 J Agric Food Chem. 1996;44(5):1218-1223. 548

9. Medeiros ABP, Pandey A, Christen P, Fontoura PSG, de Freitas 549 RJS, Soccol CR. Aroma compounds produced by 550 Kluyveromyces marxianus in solid state fermentation on a 551 packed bed column bioreactor. World J Microbiol Biotechnol. 552 2001;17(8):767-771. 553

10. Neto RS, Pastore GM, Macedo GA. Biocatalysis and 554 biotransformation producing gamma-decalactone. J Food Sci. 555 2004;69(9):C677-C680. 556

11. Soares M, Christen P, Pandey A, Soccol CR. Fruity flavour 557 production by Ceratocystis fimbriata grown on coffee husk in 558 solid-state fermentation. Process Biochem. 2000;35(8):857-861. 559

12. de Oliveira SMM, Gomes SD, Sene L, et al. Production of 560 2-phenylethanol by Geotrichum fragrans, Saccharomyces 561 cerevisiae and Kluyveromyces marxianus in cassava 562 wastewater. J Food Agric Environ. 2013;11(2):158-163. 563

13. Zheng LR, Zheng P, Sun ZH, Bai YB, Wang J, Guo XF. 564 Production of vanillin from waste residue of rice bran oil by 565 Aspergillus niger and Pycnoporus cinnabarinus. Bioresour Technol. 566 2007;98(5):1115-1119. 567

14. Wilkowska A, Kregiel D, Guneser O, Yuceer YK. Growth and 568 by-product profiles of Kluyveromyces marxianus cells 569 immobilized in foamed alginate. Yeast. 2015;32(1):217-225. 570

15. Fadel HHM, Mahmoud MG, Asker MMS, Lotfy SN. 571 Characterization and evaluation of coconut aroma produced 572 by Trichoderma viride EMCC-107 in solid state fermentation on 573 sugarcane bagasse. Electron J Biotechnol. 2015;18(1):5-9. 574


JM 1841-11

Brazilian journal of microbiology xxx (2016)xxx-xxx

575 16. Alvarez de la Puente JM, Arana JJ, Garcia-Ruiz R. Composting

576 olive mill pomace: the Andalusian experience. Biocycle.

577 2010;51:31-32.

578 17. Elibol M, Ya§a I, Karacanci S, Ozsoy G. Zeytinyagi isletmelerin

579 kati (pirina) ve sivi (karasu) atiklardan mikrobiyal lipaz uretimi (in

580 Turkish). Ankara-Turkey: Scientific project, TUBITAK; 2008.

581 18. Clemente A, Sanchez-Vioque R, Vioque J, Bautista J, Millan F.

582 Chemical composition of extracted dried olive pomaces

583 containing two and three phases. Food Biotechnol.

584 1997;11(3):273-291.

585 1 9. Gogus F, Maskan M. Air drying characteristics of solid waste

586 (pomace) of olive oil processing. J Food Eng.

587 2006;72(4):378-382.

588 20. Valiente C, Arrigoni E, Corrales JR, Esteban RM, Amado R.

589 Composition of dietary fiber in olive cake - amino-acids

590 associated with insoluble, soluble and total dietary fiber.

591 Grasas Y Aceites. 1995;46(2):98-102.

592 21. Longo MA, Sanroman MA. Production of food aroma

593 compounds: microbial and enzymatic methodologies. Food

594 Technol Biotechnol. 2006;44(3):335-353.

595 22. Vandamme E. Agro-industrial residue utilization for

596 industrial biotechnology products. In: Singh nee'Nigam P, A

597 Pandey A, eds. Biotechnology for Agro-Industrial Residues

598 Utilisation. Germany: Springer Science +Business Media B.V;

599 2009:3-11.

600 23. Rattanachomsri U, Tanapongpipat S, Eurwilaichitr L,

601 Champreda V. Simultaneous non-thermal saccharification of

602 cassava pulp by multi-enzyme activity and ethanol

603 fermentation by Candida tropicalis (vol 66, pg 10, 2004). J Biosci

604 Bioeng. 2009;108(4), 357-357.

605 24. Bhuvaneshwari S, Sivasubramanian V. Comparative studies

606 for chitosan yield and chelating ability of Aspergillus niger

607 and Rhizopus oryzae. Indian J Biotechnol. 2013;12(3):429-431.

608 25. Mateo S, Puentes JG, Moya AJ, Sanchez S. Ethanol and xylitol

609 production by fermentation of acid hydrolysate from olive

610 pruning with Candida tropicalis NBRC 0618. Bioresour Technol.

611 2015;190:1-6.

612 26. Atlas MR. Handbook of Microbiological Media. 3rd ed. Boca

613 Raton, USA: CRC Press; 2004.

614 27. Huang LP, Jin B, Lant P, Zhou JT. Simultaneous

615 saccharification and fermentation of potato starch

616 wastewater to lactic acid by Rhizopus oryzae and Rhizopus

617 arrhizus. Biochem Eng J. 2005;23(3):265-276.

618 28. Liu SC, Li C, Fang XC, Cao ZA. Optimal pH control strategy for

619 high-level production of long-chain

620 alpha-,omega-dicarboxylic acid by Candida tropicalis. Enzyme

621 Microbial Technol. 2004;34(1):73-77.

622 29. Pawliszyn J. Theory of solid phase microextraction. In:

623 Pawliszyn J, ed. Handbook of Solid Phase Microextraction. 1st ed.

624 Waltham, MA, USA: Elsevier Inc.; 2012:13-57.

625 30. Guneser O, Demirkol A, Yuceer YK, Togay SO, Hosoglu MI,

626 Elibol M. Bioflavour production from tomato and pepper

627 pomaces by Kluyveromyces marxianus and Debaryomyces

628 hansenii. Bioproc Biosyst Eng. 2015;38(6):1143-1155.

629 31. van Ruth SM. Methods for gas

630 chromatography-olfactometry: a review. Biomol Eng.

631 2001;17(4-5):121-128.

632 32. Van Den Dool H, Kratz P. A generalization of the retention

633 index system including linear temperature programmed gas

634 liquid partition chromatography. J Chromatogr A.

635 1963;11:463-471.

636 33. Mass spectral library National Institute of Standards and

637 Technology Standard Reference Data Program [computer

638 program]. Gaithersburg, 2008.

639 34. Wiley registry of mass spectral Data [computer program]. 2005.

640 35. Avsar YK, Karagul-Yuceer Y, Drake MA, Singh TK, Yoon Y,

641 Cadwallader KR. Characterization of nutty flavor in Cheddar

642 cheese. J Dairy Sci. 2004;87(7):1999-2010.

36. Meilgaard M, Civille G, Carr B. Descriptive analysis 643 techniques. In: Meilgaard M, Civille G, Carr B, eds. Sensory 644 Evaluation Techniques. Boca Raton, USA: CRC Press; 645 1999:173-186. 646

37. Sheskin D. Handbook of parametric and nonparametric statistical 647 procedures. 3rd ed. Boca Raton, USA: CRC Press; 2004. 648

38. Beolchini F, Del RG, Di Giacomo G, Spera L, Veglio F. Biological 649 treatment of agro-industrial wastewater for the production 650 of glucoamylase and Rhizopus biomass. Separation Sci 651 Technol. 2006;41(3):471-483. 652

39. Carillo ML, Zavala D, Alvarado B. Modeling the effects of 653 temperature, water activity and pH on growth of Rhizopus 654 oryzae. Informacion Technol. 2007;18:54-62. 655

40. Panji T, Farida I, Citroreksoko P. Utilization of coffee 656 processing effluent as growth medium Rhizopus oryzae, 657 producing 7-linoleic acid. Menara Perkebunan. 1998;66:47-54. 658

41. Guneser O, Karagül Yüceer Y, Özmen Togay S, Hosoglu 659 Isleten M, Elibol M. Torulaspora delbrueckii ve Trichoderma 660 atroviride kullanilarak prinadan (zeytin kati atigi) biyoaroma 661 üretimi (in Turkish). Gida. 2014:16-25. 662

42. Jin B, Yin PH, Ma YH, Zhao L. Production of lactic acid and 663 fungal biomass by Rhizopus fungi from food processing 664 waste streams. J Ind Microbiol Biotechnol. 665 2005;32(11-12):678-686. 666

43. Saracoglu NE, Cavusoglu H. Fermentative performance of 667 Candida tropicalis Kuen 1022 yeast for D-xylose and sunflower 668 seed hull hydrolysate in xylitol production. Turkish J Biol. 669 1999;23:433-438. 670

44. Oberoi HS, Vadlani PV, Brijwani K, Bhargav VK, Patil RT. 671 Enhanced ethanol production via fermentation of rice straw 672 with hydrolysate-adapted Candida tropicalis ATCC 13803. 673 Process Biochem. 2010;45(8):1299-1306. 674

45. Romero-Guido C, Belo I, Ta TMN, et al. Biochemistry of 675 lactone formation in yeast and fungi and its utilisation for 676 the production of flavour and fragrance compounds. Appl 677 Microbiol Biotechnol. 2011;89(3):535-547. 678

46. Hazelwood LA, Daran JM, van Maris AJA, Pronk JT, Dickinson 679 JR. The ehrlich pathway for fusel alcohol production: a 680 century of research on Saccharomyces cerevisiae metabolism 681 (vol 74, pg 2259, 2008). Appl Environ Microbiol. 2008;74(12), 682 3920-3920. 683

47. Mason AB, Dufour JP. Alcohol acetyltransferases and the 684 significance of ester synthesis in yeast. Yeast. 685 2000;16(14):1287-1298. 686

48. Wache Y, Aguedo M, Nicaud JM, Belin JM. Catabolism of 687 hydroxyacids and biotechnological production of lactones by 688 Yarrowia lipolytica. Appl Microbiol Biotechnol. 689 2003;61(5-6):393-404. 690

49. Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. 691 Engineering a mevalonate pathway in Escherichia coli for 692 production of terpenoids. Nat Biotechnol. 2003;21(7):796-802. 693

50. Mantzouridou F, Paraskevopoulou A. Volatile bio-ester 694 production from orange pulp-containing medium using 695 Saccharomyces cerevisiae. Food Bioprocess Technol. 696 2013;6(12):3326-3334. 697

51. Chatterjee T, De B, Bhattacharyya DK. Microbial oxidation of 698 alpha-pinene to (+)-alpha-terpineol by Candida tropicalis. 699 Indian J Chem-Section B. 1999;38:515-517. 700

52. Tretzel J, Marx S. Biotechnological processes. In: Ziegler H, 701 ed. Flavourings:Production, Composition, Applications, 702 Regulations. Weinheim, Germany: Wiley-VCH Verlag GmbH & 703 Co.; 2007:120-131. 704

53. Belitz H, Grosch W, Schieberle P. Food Chemistry. 705 Berlin-Heildelberg: Germany: Springer-Verlag; 2009. 706

54. Menotta M, Gioacchini AM, Amicucci A, Buffalini M, Sisti D, 707 Stocchi V. Headspace solid-phase microextraction with gas 708 chromatography and mass spectrometry in the investigation 709 of volatile organic compounds in an ectomycorrhizae 710


BJM 1841-11

Brazilian journal of microbiology xxx (2016)xxx-xxx 11

711 synthesis system. Rapid Commun Mass Spectr.

712 2004;18(2):206-210.

713 55. Grimm C, Champagne E. Analysis of volatile compounds in

714 the headspace of rice using SPME/GC/MS. In: Marsilli R, ed.

715 Flavor, Fragrance, and Odor Analysis. USA: CRC Press;

716 2001:229-249.

717 56. Bryant RJ, McClung AM. Volatile profiles of aromatic and

718 non-aromatic rice cultivars using SPME/GC-MS. Food Chem.

719 2011;124(2):501-513.

720 57. Wang WJ, Zhang LW, Li YH. Production of volatile

721 compounds in reconstituted milk reduced-fat cheese and

722 the physicochemical properties as affected by

723 exopolysaccharide-producing strain. Molecules.

724 2012;17(12):14393-14408.

725 58. Zhen J, Zhan FS, Zhou CH, Kan JQ. Comparison of flavor

726 compounds in fresh and pickled bamboo shoots by GC-MS and GC-Olfactometry. Food Sci Technol Res. 2014;20(1):129-138.

59. Xu F, Tao W, Sun J. Identifation of volatile compounds 727 released by myxobacteria Sorangium cellulosum AHB103-1. Afr 728 J Microbiol Res. 2011;5:353-358. 729

60. Rossi SC, Vandenberghe LPS, Pereira BMP, et al. Improving 730 fruity aroma production by fungi in SSF using citric pulp. 731 Food Res Int. 2009;42(4):484-486. 732

61. Moradia M, Asadollahia M, Nahvib I. Improved 7-decalactone 733 production from castor oil by fed-batch cultivation of 734 Yarrowia lipolytica. Biocatal Agric Biotechnol. 2013;2:64-68. 735

62. Urit T, Loser C, Wunderlich M, Bley T. Formation of ethyl 736 acetate by Kluyveromyces marxianus on whey: studies of the 737 ester stripping. Bioproc Biosyst Eng. 2011;34(5):547-559. 738

63. Mantzouridou FT, Paraskevopoulou A, Lalou S. Yeast flavour 739 production by solid state fermentation of orange peel waste. 740 Biochem Eng J. 2015;101:1-8. 741