Gas chromatography olfactometry expresses the relative intensities of flavor compounds and their odor impacts. Therefore, the findings GC-O should be reviewed with the results GC-MS that indicate the quantities of the flavor compounds in the aroma research [12] [28]. Based on the results of GC–MS analysis, it was observed that six flavor compounds including isoamyl alcohol, isovaleric acid, isoamyl acetate, phenyl ethyl alcohol, phenyl ethyl acetate and guaiacol were produced from rice bran by K. marxianus and D. hansenii at higher concentrations in 72 h shake flask fermentation (Table 2). The concentration of 2,4-nonedienal and 2,5-dimethyl-3-ethylpyrazine determined in the fermented rice bran samples did not increase. These findings could be related to the odor thresholds and concentrations of these compounds [29, 30]. Basically, the odor threshold of aroma compounds was affected by their vapor pressures and molecular interactions of other substances which existed in the medium. The odor threshold value was determined by sniffing flavor compound and defined as the lowest concentration of flavor compound which is required for the recognition of its odor. The intensity of flavor compound was directly related with the odor threshold. Therefore, even if the changes in the concentrations of flavor compounds is low, the intensities of them could be perceived as high [31].
The concentrations of higher alcohols and acetate esters in the fermented rice bran samples significantly increased during the shake flask fermentation (p ≤ 0.05) (Table 2). Rice bran fermented by K. marxianus had 522 µg/kg of isoamyl alcohol and 111 µg/kg of phenyl ethyl alcohol while the concentrations of isoamyl alcohol and phenyl ethyl alcohol were determined as 445 µg/kg, and 108 µg/kg in rice bran fermented by D. hansenii respectively, which were higher than those of unfermented rice bran samples (Table 2) Apart from the primary metabolites (as carbon dioxide, ethanol and glycerol), acids, higher alcohols and esters are produced as the main secondary metabolites in the yeast metabolism. These secondary metabolites, which are produced by different metabolic pathways of yeast, contributed the flavor profile of several fermented foods and beverages such as bread and wine [32, 33]. In yeast metabolism, the synthesis of higher alcohols including isoamyl alcohol and phenyl ethyl alcohol are achieved by Ehrlich pathway, which contains several biochemical reactions of branched chain, aromatic and sulfur-containing amino acids such as transamination, decarboxylation, oxidation, and reduction. In the Ehrlich pathway, L-phenylalanine and L‐leucine are the main substrate for the biosynthesis for isoamyl alcohol and phenyl ethyl alcohol, respectively.
Phenyl ethyl alcohol can also be synthesized from carbohydrate via Shikimate pathway in yeast cells [34–36]. Most knowledge of the Ehrlich and Shikimate pathways was obtained from studies on the fermentation behavior of Saccharomyces cerevisiae, several transaminases, decarboxylase, dehydrogenase enzymes are responsible for these pathways in the metabolism of S. cerevisiae, but the secondary metabolites production pathways of K. marxianus and D. hansenii and their enzymes systems are still being investigated [36, 37].
Table 2
Concentration of flavor compounds in rice bran fermented by K. marxianus and D. hansenii
Flavor compounds
|
Aroma Quality
|
Concentration of flavor compoundsa ± S.E
(µg/kg rice bran solution)
|
Control
|
K. marxianus
|
D. hansenii
|
Isoamyl alcohol
|
Banana
|
48.50±31.46B
|
522±140A
|
445±19.58A
|
Isovaleric acid
|
Sour, fruity
|
65.48±8.20B
|
210.17±34.76A
|
285.60±0.56A
|
Isoamyl aceatate
|
Fruity
|
Nd.
|
153.78±13.09A
|
120.10±2.44A
|
Guaiacol
|
Burnt sugar
|
0.31±0.12B
|
7.77±2.10A
|
7.85±0.37A
|
Phenyl ethyl alcohol
|
Rose
|
3.54±2.50B
|
111±13.88A
|
108.44±13.46A
|
Phenyl ethyl acetate
|
Honey, Floral
|
2.32±1.64B
|
205.93±17.14A
|
35.99±13.72B
|
A–B Means followed by different uppercase letters represent significant differences for the same flavor compound in each yeast fermentation (p ≤ 0.05). aRelative abundance of flavor compound at 72 h fermentation time. SE: standard error. ND: not detected.
|
There are two types of ester can be produced by yeast metabolism. They are acetate esters and medium-chain fatty acid ethyl esters. When the examined our findings, K. marxianus and D. hansenii are seen to produce especially acetate esters including isoamyl acetate and phenyl ethyl acetate from rice bran (Table 2). The concentration of phenyl ethyl acetate (205.93 µg/kg) in rice bran fermented by K. marxianus was found remarkably higher than that of D. hansenii (35.99 µg/kg) (p ≤ 0.05). However, no significant difference was observed between isoamyl acetate production rates of both yeasts (p ≥ 0.05). While acetate esters composed of the acid and alcohol groups, medium-chain fatty acid ethyl esters contain the alcohol and medium-chain fatty acids. Both esters can be produced through lipid and acetyl-CoA metabolism and an acyl transferase or ester synthase enzymes are play important role in the formation of esters in yeast. The synthesis of acetate esters were catalyzed by the alcohol acetyltransferases I and II encoded by the genes ATF1 and ATF2 in yeast [32, 33]. Moreover, the concentration of acetyl-CoA and a fusel alcohol and the activities of enzymes affected the rate of acetate ester formation. It was emphasized that the expression levels of the ATF1 and ATF2 during the fermentation are the most important factor for the concentration levels of the acetate ester as well as the substrate concentration in the medium [32, 38]. Therefore, higher phenyl ethyl acetate concentration in rice bran fermented by K. marxianus could be attributed the higher ATF1 and ATF2 expression compared to D. hansenii.
Concentrations of isovaleric acid and guaiacol increased significantly in the fermented rice bran during shake flask fermentation (p ≥ 0.05). The concentrations of isovaleric acids in the rice bran samples fermented by K. marxianus and D. hansenii were found as 210.17 and 285.60 µg/kg, respectively (Table 2). Determination of isovaleric acid at higher concentrations in fermented rice bran samples could be attributed to the usage of the leucine, isoleucine and valine found in rice bran protein by both yeasts and their aminotransferase enzymes activities. Similarly, the synthesis of higher alcohols, it was emphasized that α-keto acids such as isovaleric acid can be also synthesized in the first step of the Ehrlich pathway. In this step, specifically leucine, isoleucine and valine can be converted to α-keto acids and the aminotransferase enzymes catalyzed this reaction [39].
Guaiacol is defined as phenolic off flavor in fermented beverages such as wine. Phenolic volatiles can be synthesized from the phenolic acids of the hydroxycinnamic series (e.g. 4-hydroxycinnamic, p-coumaric and 4-hydroxy-2-methoxycinnamic or ferulic acid) by decarboxylation reactions in yeast metabolism [40, 41]. In the present study, guaiacol was identified as aroma-active compounds at lower intensity in the rice bran samples fermented by D. hansenii and unfermented rice samples by GC-O analysis (Table 1). However, it was determined by GC-MS in all rice bran samples ranged from 0.31–7.85 µg/kg (Table 2). This difference may be attributed to the fact that molecular interactions of guaiacol with other substances which are found in the medium. Sensitivities of both analysis techniques may also lead to this difference [42].
The findings of the present study are in good agreement with the results of several previous studies on the production of aroma compounds from different agro wastes by yeast metabolism [7, 21, 38, 43–47]. In a recent study by Kılmanoğlu et al. [43], it was reported that higher alcohols and acetate esters including isoamyl alcohol, phenyl ethyl alcohol, phenyl ethyl acetate and ethyl acetate could be produced from tomato pomace hydrolysate by the fermentation with K. marxianus similar to our findings. The researchers also expressed that heat-treated dilute acid pretreatment and cellulolytic enzyme hydrolysis led to increase in the amount of higher alcohols and esters by increasing the fermentable sugars in tomato pomace. Similarly, Martinez et al. [48] showed that higher alcohols and esters can be produced from the mixture of sugarcane bagasse with sugar beet molasses by solid state fermentation of K. marxianus ATCC 10022. According to this, the researchers expressed that the maximum cumulative volatile production was achieved at 40°C, 35% molasses rate and specific air flow rate of 0.14 L/h g and the main volatile components composed of 43% alcohols and 18% ester species including ethanol, isoamyl alcohol, isobutyl alcohol, ethyl acetate, isoamyl acetate and isobutyl acetate. It was also observed that production of esters can be increased up to 35% with the working conditions as 30°C of fermentation temperature, 25% molasses rate, and specific air flow rate of 0.11 L/h g. Vong and Liu [44] investigated the flavor production capabilities of ten yeasts originated from wine and dairy foods in the soybean residues (okara) which is a by-product soy milk. The researchers expressed that soybean residues fermented by dairy yeasts including Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis and D. hansenii have musty and moldy aromas at higher intensities owing to the higher content of aldehydes and methyl ketones which are different from our findings about the fermentation of D. hansenii.
The changes in aroma compounds in the rice bran samples were also determined during batch fermentation in the present study. Table 3 shows the changes in the concentrations of aroma compounds in bioreactor conditions. It was observed that the concentration of the aroma compounds produced from rice bran changed significantly depends on the yeast types. In the K. marxianus fermentation, the synthesis of isoamyl alcohol and phenyl ethyl acetate was the highest at 24 h. Amount of both compounds significantly decreased from 48 h to 72 h of fermentation (p ≤ 0.05). Hence, it was observed that the amount of phenyl ethyl alcohol increased from 5.42 µg/kg to 202.01 µg/kg during 96 h of fermentation. In the same manner, the synthesis of isovaleric acid and guaiacol was found to increase during 96h of fermentation. It was observed that the concentration of isoamyl acetate produced by K. marxianus increased during 48 h, then a sharp decrease in the concentration of this compound determined at the end of fermentation.
In case of fermentation by D. hansenii, isovaleric acid was not determined in the samples in bioreactor conditions. Like the fermentation by K. marxianus, the synthesis of isoamyl alcohol and phenyl ethyl acetate was the highest at 24 h and the concentration of these compounds were determined as 41.564 and 135.77 µg/kg, respectively. The highest phenyl ethyl alcohol concentration was determined at 48h. Moreover, it was observed that D. hansenii showed the similar synthesis behavior with K. marxianus in terms of guaiacol and isoamyl acetate production during bioreactor fermentation. However, D. hansenii produced lower amount of isoamyl alcohol than K. marxianus at the same fermentation time (Table 3). When the productivities of aroma compounds were examined during bioreactor fermentation, the highest productivity was calculated to produce isoamyl alcohol in both yeasts’ fermentation followed by phenyl ethyl acetate and phenyl ethyl alcohol (Data not shown). Productivity of isoamyl alcohol for the fermentations of K. marxianus and D. hansenii were calculated as 34.46 µg/kg h and 17.31 µg/kg h. Hence, it was calculated that K. marxianus had a higher phenyl ethyl acetate productivity (9.00 µg/kg h) than that of D. hansenii (5.65 µg/kg h) while the productivity value of phenyl ethyl alcohol for both yeasts was calculated to be relatively close to each other.
Table 3
Changes in the concentration of flavor compounds produced from rice bran by K. marxianus and D. hansenii during bioreactor fermentation
Flavor compounds
|
Aroma Quality
|
K. marxianus
|
Concentration of Flavor Compoundsa (µg/kg rice bran solution) Mean±S.E
|
Fermentation Time (hour)
|
0
|
24
|
48
|
72
|
96
|
120
|
Isoamyl alcohol
|
Banana
|
54.22±9.0C
|
827.27±77.32A
|
482.82±68.98B
|
68.65±1.87C
|
47.21±3.73C
|
58.17±1.55C
|
Isovaleric acid
|
Sour, fruity
|
200.±70.50D
|
245.30±79.70D
|
502±166CD
|
2813±188AB
|
4013±959A
|
1073±283BC
|
Isoamyl acetate
|
Fruity
|
0.94±0.21C
|
36.27±1.63B
|
59.23±5.36A
|
0.49±0.06C
|
0.47±0.07C
|
Nd.
|
Guaiacol
|
Burnt sugar
|
0.23±0.04C
|
11.66±2.58C
|
36.57±2.52BC
|
98.27±4.15AB
|
134.32±28.03A
|
66.88±5.20BC
|
Phenyl ethyl alcohol
|
Rosy
|
5.42±1.10B
|
169.77±42.22A
|
163.17±11.34A
|
118.73±10.67AB
|
202.01±35.10A
|
155.28±2.97A
|
Phenyl ethyl acetate
|
Honey, Floral
|
2.56±0.48B
|
216.08±31.67A
|
46.09±1.80B
|
2.18±0.10B
|
1.96±0.43B
|
0.4±0.03B
|
Flavor compounds
|
Aroma Quality
|
D. hansenii
|
Concentration of Flavor Compoundsa (µg/kg rice bran solution) Mean±S.E
|
Fermentation Time (hour)
|
0
|
24
|
48
|
72
|
96
|
120
|
Isoamyl alcohol
|
Banana
|
0.61±0.06C
|
415.64±101.06A
|
340.52±16.09AB
|
75.89±4.12C
|
99.77±37.35BC
|
37.28±2.25C
|
Isoamyl acetate
|
Fruity
|
0.16±0.04B
|
46.18±10.53A
|
34.16±1.22A
|
0.79±0.54B
|
0.20±0.08B
|
0.28±0.02B
|
Guaiacol
|
Burnt sugar
|
0.28±0.02C
|
3.72±0.92AB
|
3.18±0.06AB
|
4.04±0.01AB
|
5.98±0.79A
|
2.18±0.08BC
|
Phenyl ethyl alcohol
|
Rosy
|
3.09±0.10C
|
106.52±25.54AB
|
133.95±2.10A
|
92.33±0.03AB
|
127.08±25.79A
|
37.94±3.47CD
|
Phenyl ethyl acetate
|
Honey, Floral
|
0.05±0.04B
|
135.77±36.64A
|
30.26±1.87B
|
1.04±0.01B
|
0.58±0.10B
|
0.26±0.05B
|
A–D Means followed by different uppercase letters represent significant differences for the same flavor compound during fermentation in each yeast fermentation (p≤0.05). aRelative abundance of flavor compound. SE: standard error. ND: not detected.
|
The findings of the present study are consistent with previous studies on the production of flavor compounds by fermentation [11, 12, 49–52]. Mederios et al. [51] reported increasing concentration of ethyl acetate until 22 h of fermentation time, and then ethyl acetate concentration significantly decreased in the solid state fermentation of cassava bagasse by K. marxianus on a packed bed column bioreactor. In a study by Guneser et al. [11], the highest production of isoamyl alcohol and phenyl ethyl alcohol in tomato pomace fermented by K. marxianus and D. hansenii was reported at 24 h fermentation and the concentrations of isoamyl alcohol and phenyl ethyl alcohol was determined in the ranges between 0.23-126.72 µg/kg and 3.30-158.72 µg/kg. Similarly, Yılmaztekin et al. [52] reported that the production of isoamyl alcohol from sugar beet molasses fermented by Williopsis saturnus var. saturnus in 5L bioreactor increased gradually during 288 h, while the production of isoamyl acetate gradually increased until about 140 h fermentation. In our previous study [12], we also observed similar production changes for mushroom-like flavors including 1-octen-3-ol, 1-octen-3-one and octanol which were produced by fungal metabolism of T. atroviride and A. sojae in tomato and pepper pomaces in bioreactor fermentation.
After a certain time of fermentation, loss of aroma compounds produced in bioreactor condition can be related to many factors such as nutrients/precursor concentration, secondary metabolite inhibitions and aeration of bioreactor. Depending on these factors, several systems have been reviewed for recovery of aroma compounds from bioreactor system [1, 53, 54]. For instance, Tay [55] performed in situ product removal techniques for the production of isoamyl acetate from sugar beet molasses in bioreactor with batch and feed batch systems to avoid limiting factors. The researcher found that the maximum production of isoamyl acetate was 308.1 mg/L in the presence of macroporous resin H103 where only 38.4 mg/L of isoamyl acetate was produced without resin in batch fermentation. Unavoidably discharge of the aroma compounds from the bioreactor system with the exhaust gas defined as stripping which is considered the main reason for losses of aroma compounds during fermentation in the aerated bioreactors [49]. Urit el al. [49] modelled the stripping process for the production of ethyl acetate by K. marxianus on whey. The researchers expressed that the stripping of ethyl acetate highly depended on the aeration rate but was independent of the phase-transfer coefficient and the cooling the exhaust gas condenser of bioreactor system did not influence the stripping of the ethyl acetate.
Relationship between aroma profile and sensory characteristics of fermented rice bran
Total 13 descriptive flavor terms were defined in fermented and unfermented rice bran samples by the panelists and significant differences were observed between the rice bran samples in terms of boiled corn, sweet aromatic, fermented cereal, wet bran and rose aromas (Fig. 3). According to this, boiled corn was found to be the highest in the unfermented rice bran samples as control and no significant differences were observed between in rice bran samples fermented K. marxianus and D. hansenii regarding this aroma. In the same way, it was determined that both fermented rice bran samples had a higher fermented cereal flavor than control samples. Rose aroma in rice bran fermented by K. marxianus was determined noticeably higher than others.
For revealing of the relationship between sensory properties and flavor compounds in fermented rice bran solution, Principal Component Analysis (PCA) was performed based on the findings of sensory evaluation and GC-O analysis. It was determined that two principal components expressed by PCA the variations of sensory and aroma profiles of the rice bran samples. While the component PC1 was 59.53%, 40.47% of variations was expressed by component PC2 (Fig. 4). It can be confirmed that the fermented and unfermented rice bran samples have different sensory and aroma profiles. While rice bran samples fermented by K. marxianus can be characterized by rose aroma. Volatile compounds including phenyl ethyl alcohol, phenyl ethyl acetate, isoamyl acetate and guaiacol are well associated with rose aroma in fermented rice bran samples by K. marxianus. On the other hand, fermented cereal, polish and wood aromas well characterized in rice bran samples fermented D. hansenii and these aromas was found to related with 2-heptanal, 2,5,dimethyl-3-ethylpyrazine, methanethiol, acetic acid and 2-heptanone presented in the rice bran samples fermented by D. hansenii (PC2). In case of unfermented rice samples, boiled corn, boiled wheat porridge, wet bran and dough aromas well characterized in unfermented rice bran and they are associated with 1-octen-3-ol, isobutyl acetate, diacetyl, 2-acetyl-1-pyrroline, 2-acetyl-2-thiazoline and (E)-2-nonenal (PC1).
Due to limited number of sensory studies in fermented rice bran for production of flavor compounds, the findings in the present study could not compared with the literature results. However, aroma production behaviors of K. marxianus and D. hansenii on tomato and pepper pomace were evaluated in our previous study [11]. Rose aroma was also determined in pepper pomace fermented by K. marxianus and D. hansenii and a higher wet bulgur aroma observed in tomato pomace fermented by D. hansenii. In a study by Kılmanoğlu et al. [43], rose aroma was also determined at high intensity in tomato pomace hydrolysate fermented by K. marxianus during 8.5 hours. Similar to our findings, İşleten-Hoşoğlu [56] reported that K. marxianus produced higher alcohol and acetate esters including isoamyl alcohol, phenyl ethyl alcohol, isoamyl acetate and phenyl ethyl acetate in synthetic culture media supplemented with different carbon and nitrogen sources. The researcher determined that “sourdough”, “flower” and “sweet aromatic” were characteristic descriptors for fermented culture media. Sweet aromatic and caramel aromas were found to associate with isoamyl acetate and phenylethyl propionate based on multidimensional scale analysis.
D. hansenii commonly found as natural floral yeast for some surface ripened cheese and dry meat products and it is considered as potential flavor producer [57, 58]. Sørensen et al. [58] reported that strain of D. hansenii D18335 primarily produced branched-chain aldehydes and their alcohol derivatives including 2-methylpropanal and 2/3-methylbutanal, 2-methyl-1-propanol and 2/3-methyl-1-butanol in a cheese-surface model. While branched-chain aldehydes produced by D. hansenii are associated with green, malty, chocolate, and almond, alcohols are associated fruity, solvent, alcoholic, fusel and pomace aromas. In another study by Leclercq-Perlat et al. [59], methyl ketones with fruity, floral moldy, cheesy, wine aromas and 2-phenyl ethyl alcohol with rose aroma were determined in deacidified model cheese medium generated by D. hansenii. I the present study, 2-methylpropanal and 2/3-methylbutanal and their alcohols derivatives by the metabolism of D. hansenii in rice bran were not determined. This difference might be attributed to nature of raw materials used in the studies and strain differences. On the other hand, the production of phenyl ethyl alcohol associated with rose aroma in fermented rice bran were determined in the present study which was similar to the findings of Leclercq-Perlat et al.[59].