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0BJM 1841-11
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BRAZILIAN JOURNAL OF MICROBIOLOGY
SOCIEDADE BRASILEIRA DE MlCROBIOLOGIA
http://www.bjmicrobiol.com.br/
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
abstract
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 (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
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: onur.guneser@usak.edu.tr (O. Guneser). http://dx.doi.org/10.1016/j.bjm.2016.08.003
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 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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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
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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
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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
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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
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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
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DD C_D_E^
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
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Brazilian journal of microbiology xxx (2 016) xxx-xxx
A Unripe olive
^^ Control — R. oryzae
Sweet aromatic
Wet bulger
Woody/herbal
Control
C.tropicalis
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
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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.
Conclusion
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.
Acknowledgments
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
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