Bioprocess development for the production of novel oleogels from soybean and microbial oils

Food Research International 126 (2019) 108684

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Bioprocess development for the production of novel oleogels from soybean and microbial oils

T

Aikaterini Papadakia, , Nikolaos Kopsahelisb, Athanasios Mallouchosa, Ioanna Mandalaa, Apostolis A. Koutinasa ⁎

a b

Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece Department of Food Science and Technology, Ionian University, Argostoli 28100, Kefalonia, Greece

ARTICLE INFO

ABSTRACT

Keywords: Circular bioeconomy Sugarcane molasses Soybean cake Soybean fatty acid distillate Microbial oil Carotenoids Oleogelation

This study presents the production of novel oleogels via circular valorisation of food industry side streams. Sugarcane molasses and soybean processing side streams (i.e. soybean cake) were employed as fermentation feedstocks for the production of microbial oil. Fed-batch bioreactor fermentations carried out by the oleaginous yeast Rhodosporidium toruloides led to the production of 36.9 g/L total dry weight with an intracellular oil content of 49.8% (w/w) and 89.4 μg/g carotenoids. The carotenoid-rich microbial oil and soybean oil were evaluated as base oils for the production of wax-based oleogels. The wax esters, used as oleogelators, were produced via enzymatic catalysis, using microbial oil or soybean fatty acid distillate as raw materials. All oleogels presented a gel-like behaviour (G′ > G″). However, the highest G′ was determined for the oleogel produced from soybean oil and microbial oil-wax esters, which indicated a stronger network. Thermal analysis showed that this oleogel had a melting temperature profile up to 35 °C, which is favorable for applications in the confectionery industry. Also, texture analysis demonstrated that soybean oil-microbial oil wax oleogel was stable (1.9–2.2 N) within 30-days storage period. This study showed the potential of novel oleogels production through the development of bioprocesses based on the valorisation of various renewable resources.

1. Introduction The microbial production of lipids and carotenoids using crude renewable resources has gain great attention the last years and could lead to the development of an important bioeconomy sector. The production of carotenoids has been studied in fermentations using mostly the yeasts Rhodotorula sp. (Aksu & Eren, 2007; Petrik, Obruča, Benešová, & Márová, 2014) and Rhodosporidium sp. (Bonturi, Crucello, Viana, & Miranda, 2017; Freitas, Parreira, Roseiro, Reis, & da Silva, 2014; Xu & Liu, 2017). Rhodosporidium yeast strains can produce both carotenoids and microbial lipids, whereas high lipid contents (up to 61.8%, w/w) have been reported when cultivated in various agro-industrial residues and side streams, such as confectionery food industry wastes (Tsakona et al., 2016), crude glycerol (Polburee et al., 2016; Xu, Zhao, Wang, Du, & Liu, 2012), lignocellulosic hydrolysates (Xu & Liu, 2017) and molasses (Vieira et al., 2016; Vieira, Ienczak, Rossell, Pradella, & Franco, 2014). The fermentation media used for microbial oil (MO) production are usually supplemented with high yeast extract concentrations. Alternatively, crude hydrolysates produced from oilseed cakes, the main by-products of industrial oilseed processing, can be used as nutrient ⁎

supplements in various fermentation processes to eliminate yeast extract supplementation (Pateraki et al., 2016; Salakkam et al., 2017; Salakkam & Webb, 2018). In the context of Circular Economy, side-streams deriving from food processing should be used as renewable fermentation feedstocks for the production of intermediate products that will be used in diversified novel applications. Within this frame, microbial oil has been used for the production of oleochemicals via enzymatic conversion of triglycerides into wax esters and polyol esters using lipases in solvent free reaction systems (Papadaki et al., 2017; Papadaki et al., 2018; Papadaki et al., 2019). These microbial oil derived wax esters could substitute conventional wax esters, while polyol esters can be used as biolubricants. Natural wax esters have been already applied in oleogelation in order to structure oils and replace the saturated and trans-fat content in processed foods (Singh, Auzanneau, & Rogers, 2017). Recent developments in oleogels production target the production of hardstock fats alternatives with similar sensory and nutritional profiles (Singh et al., 2017). Oleogels could be applied in the production of various food products, such as margarine and spreadable fat products (Yilmaz &

Corresponding author. E-mail addresses: [email protected] (A. Papadaki), [email protected] (A.A. Koutinas).

https://doi.org/10.1016/j.foodres.2019.108684 Received 27 December 2018; Received in revised form 12 September 2019; Accepted 13 September 2019 Available online 14 September 2019 0963-9969/ © 2019 Elsevier Ltd. All rights reserved.

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2.2. Microorganisms The oleaginous yeast Rhodosporidium toruloides DSM 4444 was obtained from the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures and was utilized in fermentations for microbial oil and carotenoids production. The yeast was maintained at 4 °C on agar slopes containing 10 g/L glucose, 10 g/L yeast extract, 10 g/L peptone and 20 g/L agar (YPDA medium). A YPD pre-culture was prepared at 28 °C for 24 h and it was subsequently used as inoculum in batch and fed-batch fermentations. All media were autoclaved at 121 °C for 20 min. 2.3. Microbial oil and carotenoids production Batch fermentations in shake flasks, using molasses as substrate, were carried out in 250 mL Erlenmeyer flasks containing 50 mL of the following medium (g/L): initial total sugar concentration, 60.0–70.0; yeast extract, 0.5; (NH4)2SO4, 0.5. The phosphate salts (7.0 g/L KH2PO4, 2.5 g/L Na2HPO4) and trace elements (in g/L: MgSO4·7H2O, 1.5; CaCl2·2H2O, 0.15; FeCl3·6H2O, 0.15; MnSO4·H2O, 0.06; ZnSO4·7H2O, 0.02) (Papadaki et al., 2019) used in shake flask cultures varied depending on the experiment. The inoculum (10%, v/v) used was a 24 h pre-culture incubated at 28 οC and 180 rpm in an orbital shaker (ZHWY-211C Series Floor Model Incubator, PR China). The pH value was maintained in the range of 5.5–6.2 by adding aseptically 5 M NaOH or 5 M HCl when needed. The results represent the mean values of duplicates. Fed-batch fermentations were conducted in a 2 L bench top bioreactor (Labfors, Infors4) with 1.0 L working volume. The temperature was controlled at 28 °C and the pH was automatically regulated in the range of 5.8–6.2 using 5 M NaOH and 5 M HCl. An agitation cascade (200–500 rpm) was employed in order to maintain the dissolved oxygen concentration at 20% of saturation and the aeration rate was regulated at 1 vvm. The inoculum (10%, v/v) used was a 24 h YPD pre-culture. The initial fermentation medium was diluted molasses at an initial total sugar concentration of around 70 g/L. Two fed-batch fermentations were carried out to evaluate the effect of different nitrogen sources. In the first fed-batch culture, the fermentation medium contained 0.5 g/L yeast extract, 0.5 g/L (NH4)2SO4, phosphate salts and trace elements at the same concentration as those employed in batch shake flask fermentation. In the second fed-batch fermentation, soybean cake hydrolysate at initial free amino nitrogen (FAN) concentration of 300 mg/L was used as the sole source of nutrients. A concentrated solution of very high polarity cane sugar (500 g/L) was used as feeding solution during fed-batch fermentations when the sugar concentration was reduced to ca. 20 g/L. Carotenoids production was also determined during fedbatch fermentations.

Fig. 1. Valorisation of food industry side streams for the production of oleogels.

Öğütcü, 2015), meat products (Moghtadaei, Soltanizadeh, & Goli, 2018) and cream cheese (Bemer, Limbaugh, Cramer, Harper, & Maleky, 2016). Papadaki et al. (2019) demonstrated that microbial oil–derived cetyl wax esters could be used for the production of olive oil–based oleogel that was rheologically and thermally suitable in spreadable fat product applications. This study focuses on the development of novel oleogels using carotenoids-rich microbial oil and soybean oil. Microbial oil enriched in carotenoids was produced via fermentation using sugarcane molasses and soybean cake from a soybean oil extraction process (Fig. 1). Besides soybean cake, the latter process produces also fatty acid distillates, as a by-product stream, which has been studied as raw material in enzymatic catalysis for biodiesel production (Soares, Pinto, Gonçalves, Mitchell, & Krieger, 2013). In this study, soybean fatty acid distillate (SFAD) and microbial oil were enzymatically converted into wax esters, which were used as oleogelators. Oleogels were produced from either soybean oil or microbial oil and their properties (i.e. crystal morphology, color, texture, thermal and rheological behaviour) were evaluated. This study showed the potential production of novel oleogels, which may find applications in various food formulations, using either carotenoids-rich microbial oil or soybean oil and SFAD derived from soybean oil extraction processes. 2. Materials and methods 2.1. Raw materials

2.4. Oleogels production

Sugarcane molasses was provided by the sugarcane industry Cruz Alta (Guarani, São Paulo, Brazil) and was utilized in microbial oil production via fermentation. Sugarcane molasses contained sucrose, glucose and fructose (around 47%, w/w), protein (3.2%, w/w), various minerals (e.g. Ca, S, Mg, K) and phenolic compounds (Papadaki et al., 2018). Soybean cake was provided by the biodiesel production industry BSBios (Passo Fundo, Rio Grande do Sul, Brazil) and was utilized for the production of a nutrient rich hydrolysate via enzymatic hydrolysis as described by Papadaki et al. (2017). In the case of oleogel production, edible soybean oil was purchased from the local food market (Athens, Greece). SFAD was provided by Miracema-Nuodex (Brazil). The main fatty acids contained in SFAD are linoleic (42.6%), oleic (28.4%) and palmitic (18.7%) acids. Cetyl wax esters derived from microbial oil and soybean fatty acid distillate were utilized as oleogelators. They were produced via enzymatic conversion using lipases as described in previous studies (Papadaki et al., 2019; Papadaki, Mallouchos, et al., 2017).

Soybean oil and microbial oil were used as base oils for the production of oleogels. Different concentrations (7%, 10% and 20%, w/w) of SFAD cetyl wax esters and MO cetyl wax esters were evaluated during oleogelation. The base oils and cetyl wax esters were precisely weighted and the mixture was heated at 90 οC under agitation for 10 min until all the components melted (Wijarnprecha, Aryusuk, Santiwattana, Sonwai, & Rousseau, 2018). Oleogels were transferred into screw capped glass vials and cooled at room temperature for 24 h to allow gel formation. The samples were then stored at 4 οC for 30 days for texture analysis. All results reported for oleogels represent the mean values of triplicates. 2.5. Analytical methods 2.5.1. Determination of total dry weight, sugars and free amino nitrogen Fermentation samples taken at regular intervals were centrifuged 2

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(9000 ×g, 4 °C, 10 min) and the precipitate was washed twice with deionized water. The total dry weight (TDW) was estimated by drying the precipitate at 70 °C until constant weight. Sugars were determined by High Performance Liquid Chromatography (HPLC) (Prominence, Shimadzu, Kyoto, Japan) equipped with a Shimadzu RID-10A detector, a Shimadzu SIL-20AXR autosampler and a Shimadzu CTO-10ASvp column oven. Samples were eluted from a Rezex ROA-organic acid H+ column (300 mm length x 7.8 mm internal diameter, Phenomenex, USA). A solution of 10 mM H2SO4 was used as mobile phase with a flow rate of 0.6 mL/min at 65 °C. The sugar consumption was expressed as g per L of fermentation. FAN concentration was also evaluated according to the Ninhydrin method (Lie, 1973).

using a polarized light microscope (Axiolab, Zeiss) equipped with a digital camera (DSC-575 model, Sony, Japan) at the magnification of ×10. The microstructural analysis was employed using freshly prepared oleogels which were stored at 4 °C. The color of the oleogels was analysed with a colorimeter (ChromaMeter CR-400/410, Konica Minolta, Japan). The calibration of the colorimeter was performed with a white and a black plate. The samples were poured in cylindrical tubes with 2 cm diameter and 1 cm height. The color was recorded using CIE-L*a*b* uniform color space (CIE-Lab), where L* indicates lightness, a* indicates the green (−) to red (+) axis, and b* indicates the blue (−) to yellow (+) axis. Also, hue angle (h*) and Chroma (C*) were evaluated using the equations: b 2 + b 2 , respectively. h = tan 1 and C = Discovery HR 3 Hybrid Rheometer (TA Instruments, New Castle, DE, U.S.A.) equipped with a parallel plate geometry system and a measuring gap of 700 μm was utilized for the rheological properties of the oleogels. Samples were transferred from the storage temperature (4 °C) onto the Peltier plate pre-heated at 20 °C and held for 5 min. The parameters of viscosity, storage modulus (G′), loss modulus (G″) and loss tangent (tan δ = G″/G′) versus temperature were determined during a heating cycle up to 80 °C, ramped at a rate of 2 °C/min and a steady shear rate of 100/s. A texture analyser (Instron 1011, Massachusetts, U.S.A.), equipped with a 50-N load cell, was employed for the determination of texture of the oleogels during a storage period of 1–30 days. Glass tubes with internal diameter of 2 cm were filled with 15 ml of oleogels and stored at 4 °C. The penetration force was measured at 2 cm depth of samples after plunging a cylindrical probe with a penetration speed of 100 mm/ min. The maximum force was reported as a penetration force (N). DSC analysis (Q100 model, TA Instruments, DE, U.S.A.) was employed to determine the thermal properties of oleogels. Around 5–10 mg of samples were precisely weighted and hermetically sealed in an aluminum pan. The samples were heated to 140 °C at a 10 °C/min heating rate, followed by cooling at −20 °C at a 10 °C/min heating rate and then reheated to 140 °C at a rate of 5 °C/min (Yilmaz & Öğütcü, 2015). An empty pan was used as reference. All analyses for the oleogels characterisation were performed in triplicates.

( )

2.5.2. Determination of microbial oil and carotenoids The dried precipitate obtained from fermentation samples was used for the determination of microbial oil. The solid precipitate was solvent extracted as previously described (Papadaki, Mallouchos, et al., 2017). The microbial oil concentration was expressed as g of oil per L of fermentation broth and the intracellular oil content was expressed as g of oil contained in 100 g of TDW. Total carotenoid concentration in 5 mL fermentation samples was determined by separating the solid precipitate by centrifugation (9000 ×g, 4 °C, 10 min) followed by washing with deionized water and lyophilization. All samples treatments were carried out at low light intensity conditions. Total carotenoids were extracted from the lyophilized solids by adding 20 mL chloroform:methanol solution (2:1, v/ v) containing 20 mg/L butylated hydroxytoluene (BHT, Sigma-Aldrich) as antioxidant agent. The samples were left in dark and dry conditions for exactly 3 days and then the solvent was removed by vacuum evaporation at temperatures lower than 40 °C. The total carotenoids were finally collected by adding specific quantity of light petroleum ether (boiling point 40–60 °C). Carotenoids extract was passed through a 0.22 μm syringe filter and the absorbance was then recorded at 450 nm (U-2000, Spectrophotometer, Hitachi) against a blank sample. The total carotenoids concentration was expressed as μg of β-carotene equivalents per g of TDW using the equation reported by Lopes, Remedi, dos Santos Sá, Burkert, and de Medeiros Burkert (2017):

Total carotenoids (µg/g) =

A × V × 10000 E×w

2.6. Statistical analysis

where, A is the absorbance at 450 nm, V is the volume of petroleum ether in mL, E is the 1% extinction coefficient (2592 for petroleum ether) and w is the TDW of the sample used.

The statistical differences among treatments were estimated by analysis of variance (ANOVA). Whenever ANOVA indicated a significant difference between variables at a significance level of 5% (P < 0.05), the Tukey's HSD (honest significant difference) test was carried out using the Excel software.

2.5.3. Fatty acid composition The fatty acid profile of microbial oil and soybean oil was determined in a Fisons GC-8060 gas chromatography (GC) equipped with a CPWAX 52CB column (30 m × 0.32 mm i.d., 0.25 μm film thickness, Chrompack), a split/splitless injector and a flame ionisation detector as described by Papadaki, Mallouchos, et al. (2017). Fatty acid methyl esters (FAMEs) were produced by a two-step derivatization reaction with methanol using sodium methoxide (MeONa) and HCl as catalysts. Samples of 1 μl were inserted in the injector, which was controlled at 250 °C, using a split ratio of 1:50. The oven temperature was programmed initially at 100 °C for 1 min, then increased to 200 °C (at a ratio of 25 °C/min) where it was maintained for 1 min, ramped to 230 °C (3 °C/min) for 6 min, subsequently increased to 250 °C (30 °C/ min) where it was kept constant for 2.5 min. Helium was used as carrier gas at flow rate of 2 mL/min and the temperature of the detector was set at 270 °C. Peak identification was carried out using a certified reference FAME mixture (Supelco 37 Component FAME mix, Sigma-Aldrich). Fatty acid data were expressed as area percentage of FAMEs.

3. Results and discussion 3.1. Shake flasks fermentations Microbial oil production by the oleaginous yeast R. toruloides was initially evaluated in shake flask fermentations using molasses as the sole carbon source at an initial total sugar concentration of 60–70 g/L. The sugars contained in molasses were predominantly sucrose with lower amounts of glucose and fructose. Microbial oil production and fatty acid composition were evaluated when shake flask fermentations were supplemented with different nutrients (e.g. nitrogen sources, phosphate salts, trace elements). The initial carbon to FAN ratio (C/ FAN) was around 230 g/g in all cases, except for the fermentation conducted without any additional nitrogen source, neither yeast extract nor (NH4)2SO4, in which the C/FAN ratio was around 400 g/g. Table 1 shows that the addition of trace elements or phosphate salts affect lipid and microbial mass production. The fermentation carried out without any addition of trace elements and phosphate salts led to a

2.5.4. Characterisation of oleogels The crystal morphology of wax esters and oleogels was studied 3

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Table 1 Effect of trace elements (TE) and phosphate salts (P) supplementation on lipid production by R. toruloides in shake flask fermentations using sugarcane molasses. Nutrient supplementation a

Medium free of TE and P Mediuma with TE Medium a with P Medium a with TE and P Medium without yeast extract and (NH4)2SO4, but with TE and P

Fermentation duration (h)

b

So

90 73 97 100 90

c

60.0 69.4 64.0 69.0 69.4

(g/L) ± ± ± ± ±

Sr

0.0 0.2 0.3 1.8 3.0

c

(g/L)

14.0 ± 1.1 22.1 ± 4.7 12.2 ± 1.4 6.0 ± 0.2 13.7 ± 0.3

TDW 18.3 14.0 19.9 25.3 21.5

c

(g/L)

Lipids (g/L)

a

a

± ± ± ± ±

0.3 1.1a 0.1a 0.3d 1.0e

YL/TDW

4.0 ± 0.5 3.7 ± 0.4a 4.7 ± 0.3a 9.8 ± 0.1a 12.7 ± 1.8d

c

26.4 23.4 38.9 59.0

(%, w/w) ± ± ± ±

0.5 1.6 1.0 5.7

Different letters indicate significant differences between the different supplement strategies (P < 0.05). a Fermentation medium contained 0.5 g/L yeast extract and 0.5 g/L (NH4)2SO4. b Fermentation duration: It indicates the time where sugar consumption stopped. c So: initial total sugar concentration; Sr: residual total sugar concentration; TDW: total dry weight; YL/TDW: intracellular lipid content.

final lipid production and yeast cell concentration of 4 g/L and 14.3 g/ L, respectively, with an intracellular lipid content of 22.1%. Similar lipid concentration (3.7 g/L) and intracellular lipid content (26.4%) were obtained in the fermentation carried out with supplementation of trace elements, while the yeast cell concentration was lower (10.3 g/L). In the fermentation carried out with supplementation of phosphate salts, higher yeast cell concentration (15.2 g/L) with similar lipid production (4.7 g/L) and intracellular lipid content (23.4%) were observed. The fermentation carried out with simultaneous addition of nitrogen sources, trace elements and phosphate salts showed an increased (P < 0.05) lipid concentration (9.8 g/L) and lipid content (38.9%). Yeast cell concentration (15.5 g/L) was similar to the fermentation where nitrogen sources and phosphate salts were used. Phosphorus is an essential nutrient for R. toruloides growth (Gientka, Kieliszek, Jermacz, & Błażejak, 2017; Wu, Hu, Jin, Zhao, & Zhao, 2010). However, Gientka et al. (2017) reported that phosphorus did not affect lipid production, but only the fatty acid profile of the lipids. The trace element mixture contained MgSO4, which may induce key enzymes for lipid synthesis, since ATP-citrate lyase is strongly dependent on the presence of cations, such as Mg2+ (Gientka et al., 2017). The final shake flask fermentation presented in Table 1 has been carried out without nitrogen source supplementation, because molasses contains around 0.5% (w/w) total Kjeldahl nitrogen (Papadaki et al., 2018). In this case, the highest lipid concentration of 12.7 g/L (P < 0.05) and lipid accumulation of 59% (w/w) was observed with a lower yeast cell concentration (8.8 g/L). Fermentations conducted without supplementation of commercial nitrogen sources showed that molasses may partially provide a nitrogen source for yeast growth. The intracellular lipid content was the highest observed in this set of fermentations due to the higher carbon to FAN ratio used in this case, as compared to the other batch fermentations where commercial nitrogen sources were added. The results presented in Table 1 indicate that phosphate salts and trace element addition was necessary in order to enhance yeast growth, while the nitrogen content of molasses could lead to reduced yeast extract supplementation in bioreactor cultures. The main fatty acids in the microbial oils, produced in shake flask fermentations, were oleic acid, palmitic acid, stearic acid and linoleic acid (Table 2). Oleic acid content ranged from 50.6 to 56.6%, with the

highest content observed in the fermentation media that contained trace elements and phosphate salts supplements. The highest linoleic acid content (6.7%) was observed under nitrogen starvation conditions as has been also reported by Sitepu et al. (2013). The results presented in Table 2 indicate that nutrient composition affect the individual fatty acid content. Variation in sulfur supplementation caused substantial changes in fatty acid composition of the microbial oil produced by R. toruloides (Wu, Zhao, Shen, Wang, & Zhao, 2011). Fermentations of Trichosporon capitatum showed that optimization of medium composition, concerning Mn2+, Mg2+ or Cu2+, resulted to the highest lipid production (6.6 g/L) and lipid content (43.1%, w/w) (Wu, Li, Chen, & Zong, 2011). Thus, optimization of nutrient addition during fermentation could lead to the production of microbial lipids with tailor-made fatty acid composition suitable for case-specific applications. In this study, the evaluation of fatty acid composition was considered essential, as fatty acids influence the crystal formation of oleogels (O'Brien, 2008). 3.2. Bioreactor fermentations A fed-batch fermentation was initially carried out with R. toruloides cultivated on sugarcane molasses that was supplemented with trace elements, phosphate salts and commercial nitrogen sources. The initial C/FAN ratio was 259 g/g. The FAN concentration was sharply decreased until 20 h, but lipid production was mainly observed after 50 h due to the presence of inorganic nitrogen. The initial total sugar concentration was reduced to 27 g/L at 47 h, when continuous feeding was initiated using a concentrated cane sugar solution (500 g/L). The highest microbial lipid concentration (13.5 g/L) was reached at 121 h with an intracellular content of 54.6% (w/w). Sucrose was hydrolysed into glucose and fructose during fermentation with the consumption rate of glucose being higher than fructose. Thus, fructose was the main remaining sugar towards the end of fermentation (data not shown). The sugar to lipid conversion yield based on the consumed sugars was 0.13 g/g. Fed-batch fermentation of R. toruloides CCT 0783 carried out on molasses with different C/N ratios at the growth phase (11.6) and the lipid accumulation phase (25) resulted in the production of 16 g/L microbial oil and a lipid content of 44% (w/w) (Vieira et al., 2014).

Table 2 Effect of trace elements (TE) and phosphate salts (P) supplementation on fatty acid composition of R. toruloides microbial oil produced in shake flask fermentations using sugarcane molasses. Nutrient supplementation

Fermentation duration (h)

b

Fatty acids (%) C 16:0

Mediuma free of TE and P Mediuma with TE Medium a with P Medium a with TE and P Medium without yeast extract and (NH4)2SO4, but with TE and P a b

90 73 97 100 90

32.2 35.0 29.2 28.2 29.8

Fermentation medium contained 0.5 g/L yeast extract and 0.5 g/L (NH4)2SO4. Fermentation duration: It indicates the time where sugar consumption stopped. 4

± ± ± ± ±

2.6 1.8 0.6 0.8 0.6

C 18:0 11.5 ± 0.7 7.2 ± 0.8 13.2 ± 0.8 9.8 ± 0.2 6.6 ± 0.5

C 18:1 51.2 52.6 50.6 56.6 54.8

± ± ± ± ±

2.1 1.8 0.9 2.6 1.3

C 18:2 2.8 2.3 4.5 3.8 6.7

± ± ± ± ±

0.2 0.9 0.4 0.3 0.3

Others 2.4 2.9 2.2 1.5 2.1

± ± ± ± ±

0.2 0.3 0.2 0.1 0.2

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than palmitic acid content, reaching 51.4% and 54.8% at the end of fermentations carried out with commercial nitrogen sources and soybean cake hydrolysate, respectively. Total carotenoids production was also determined during fed-batch fermentations (Fig. 2). The highest total carotenoids concentration was 83.1 μg/g and 89.4 μg/g in the fermentations carried out with commercial nitrogen sources and soybean cake hydrolysate, respectively. Buzzini et al. (2007) reported that the species R. diobovatum and R. toruloides produce carotenoids up to 69 μg/g and 122.6 μg/g when glucose is used as carbon source. Carotenoids production by R. toruloides of 91.2 μg/g has been reported when 100 g/L initial sugar concentration of carob pulp syrup was used (Freitas et al., 2014). Moreover, Bonturi et al. (2017) reported the production of 1.2 mg/L carotenoids by R. toruloides cultivated on sugarcane bagasse hemicellulose hydrolysate. It is evident that the highest total carotenoids concentration occurred when the highest lipid concentration was achieved (Fig. 2). This allows the recovery of microbial lipids enriched in natural carotenoids providing a useful raw material for food applications. Lipids and carotenoids are secondary metabolites which means that their production is triggered by nitrogen limitation (Braunwald et al., 2013). Some carotenoids, such as β-carotene which is produced by R. toruloides, are highly lipophilic non-polar compounds due to their conjugated hydrocarbon structure lacking polar functional groups (Sani & Keum, 2018). Previous studies have demonstrated the concomitant production of lipids and carotenoids by Rhodosporidium yeast strains. (Dias, Sousa, Caldeira, Reis, & da Silva, 2015; Singh et al., 2016). Moreover, Braunwald et al. (2013) showed that lipid and carotenoids production followed the same pattern under different carbon to nitrogen ratios. 3.3. Production and characterisation of oleogels Among the different concentrations of wax esters (7%, 10% and 20%, w/w) used as oleogelators, 20% was sufficient for oleogelation. The wax esters concentrations of 7% and 10% were insufficient to form an oleogel, thus the concentration of 20% was used for oleogels production and further evaluation of their properties. This is a high wax concentration for oleogel formation that may result to a waxy mouthfeel with negative effects on sensorial properties (Doan, Tavernier, Danthine, Rimaux, & Dewettinck, 2018). Wijarnprecha et al. (2018) have also reported oleogel formation with high rice bran wax concentrations (up to 25%, w/w) stressing that this would be useful for the future incorporation of small proportions of oleogels (i.e. < 5%) in processed foods. A blend of waxes and hardstocks could overcome the waxy mouthfeel effect and lead to products with desired properties and sensory appeal (Doan et al., 2018).

Fig. 2. Time course of total sugars (●) and free amino nitrogen (FAN) (■) consumption as well as production of total dry weight (TDW) (□), lipids (○) and total carotenoids (▲) during fed-batch bioreactor fermentations of R. toruloides cultivated on molasses-based medium supplemented with (a) commercial nitrogen sources and (b) soybean cake hydrolysate.

The fed-batch fermentation presented in Fig. 2b was carried out with soybean cake hydrolysate as the sole nutrient-rich supplement. The purpose of this fermentation was to demonstrate the feasibility of microbial oil production and R. toruloides growth using the crude hydrolysate. An initial FAN concentration of 300 mg/L was used as previous fermentations of R. toruloides on flour-rich hydrolysates showed optimal lipid accumulation at a similar initial FAN concentration and an initial C/FAN ratio of 80.2 g/g (Tsakona et al., 2016). In this study, the initial C/FAN ratio was 96 g/g. The initial FAN concentration was almost entirely consumed at 20 h. Continuous feeding was initiated at 39.5 h when the total sugar concentration was reduced to 17 g/L. The highest microbial lipid concentration (18.4 g/L) was observed at 91.5 h, which corresponds to an intracellular oil accumulation of 49.8% (w/w). The sugar to lipid conversion yield based on the consumed sugars was 0.15 g/g. Comparing the two fermentations presented in Fig. 2, it could be concluded that the soybean cake hydrolysate can provide all essential nutrients for yeast growth and also support efficient microbial oil production. Besides being a rich nitrogen source, soybean cake hydrolysate also contains phosphorus and various minerals, such as Mg, Ca, Fe and Zn (Papadaki, Androutsopoulos, et al., 2017). The fatty acid profiles of microbial lipids (Table 3), which were produced in the fed-batch bioreactor fermentations, were similar in both cultures. The main fatty acids were palmitic acid and oleic acid. Palmitic acid content was 35.2% and 32.5% at the end of fermentations carried out with commercial nitrogen sources and soybean cake hydrolysate, respectively. The oleic acid content was significantly higher

3.3.1. Crystal morphology Analysis of microstructure demonstrated that the morphology and the size of crystals varied depending on the oleogel used (Fig. 3a–c). The use of SFAD wax produced larger crystals with length higher than 100 μm and flaked-like shape (Fig. 3a). The microbial oil-based oleogel exhibited spherulite-shaped crystals with a tendency to form clusters, with the majority of crystals being up to 100 μm (Fig. 3c). The SFAD and MO wax esters presented different crystal morphology in polarized light microscopy analysis (data not shown). Therefore, the differences in the microstructure of the wax-based oleogels could be attributed to the use of wax esters derived from different oil sources (SFAD or MO). Wax esters are the building blocks of the oleogel crystal network and their composition, such as alkyl ester chain length and degree of saturation, influences the molecular organization of wax oleogels (ToroVazquez, Charó-Alonso, Pérez-Martínez, & Morales-Rueda, 2011). For instance, SFAD and microbial oil have different saturation degrees, as SFAD contains mainly 55.8% C18:2 and 23.1% C18:1, whereas the major fatty acids of microbial oil are 54.8% C18:1 and 32.5% C16:0. Furthermore, the use of different oils affect the crystal morphology of 5

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Table 3 Fatty acid composition of microbial oils produced by R. toruloides in bioreactor fed-batch fermentations using sugarcane molasses and different nitrogen sources. Nitrogen source

Fermentation duration (h)

Fatty acids (%) C 16:0

a

Commercial

Soybean cake hydrolysate

a b

b

86 121 133 20 61 91.5

28.6 28.2 35.2 30.6 33.9 32.5

± ± ± ± ± ±

C 18:0

0.2 0.3 0.1 1.1 0.7 1.4

8.0 5.6 5.1 4.9 4.6 4.3

± ± ± ± ± ±

C 18:1

0.4 0.3 0.3 0.2 0.3 0.4

54.6 58.7 51.4 56.0 55.8 54.8

± ± ± ± ± ±

1.8 0.8 2.8 0.9 0.8 1.3

C 18:2 6.2 5.4 5.2 5.8 5.3 5.6

± ± ± ± ± ±

Others

0.2 0.2 0.4 0.2 0.1 0.2

2.5 2.1 3.1 2.6 1.4 2.8

± ± ± ± ± ±

1.5 0.9 1.6 0.1 0.1 0.3

0.5 g/L yeast extract and 0.5 g/L NH4)2SO4. Soybean cake hydrolysate was used at initial FAN concentration of 300 mg/L.

the oleogel. Ferro, Okuro, Badan, and Cunha (2019) reported that coconut oil and sunflower oil oleogels presented different crystal formation, mainly due to saturation degree, concluding that oils with high saturation degree provide high level of organization.

Table 4 Evaluation of color, melting temperatures and texture of the oleogels produced from soybean oil and microbial oil using soybean fatty acid distillate (SFADwax) and microbial oil (MO-wax) wax esters as oleogelators. Parameters

3.3.2. Color analysis Color analysis of the produced oleogels is presented in Table 4. The L* value, which express the luminosity of the oleogels, was lower (P < 0.05) in oleogels prepared with MO-wax than those prepared with SFAD-wax. Moreover, a* measurements were higher (P < 0.05) in oleogels prepared with MO wax. The reddish color of carotenoids, contained in the microbial oil and the derived wax esters, contributed to lower L* and higher α* as compared to SFAD-wax based oleogel. The b* values varied significantly (P < 0.05) with soybean oil derived oleogels exhibiting higher values than microbial oil derived oleogels due to the higher intensity of yellow color. The high C* values of soybean oil-MO wax (24.0) and microbial oil-MO wax (20.6) oleogels indicated the more saturated color as compared to the soybean oil-SFAD wax (5.5). The h* value refers to the hue's location in the CIE-L*C*h color range, where red is 0°, yellow is 60°, green is 120°, and blue is −120°. As it was expected, the reddish color of microbial oil-MO wax resulted to low h* value (9.2°). The other oleogels had higher h* values with the highest one belonging to soybean oil-SFAD wax (89.7°) due to its yellowish color.

Color analysis

a

Thermal analysis (°C) b Texture analysis (N) c

Oleogels

L* a* b* Ton Tp Tcom 1d 10 d 20 d 30 d

Soybean oil SFAD-wax

Soybean oil MO-wax

Microbial oil MO-wax

27.1 ± 0.1a 0.1 ± 0.0 a 15.5 ± 0.0 a 24.8 ± 1.6 a 30.1 ± 2.1 a 32.8 ± 1.9 a 2.9 ± 0.0 a 2.8 ± 0.2 a 0.5 ± 0.0 b 0.5 ± 0.0 b

11.7 ± 0.9 b 20.1 ± 1.2 b 12.0 ± 1.4 b 24.3 ± 1.3 a 32.3 ± 2.5 a 35.3 ± 2.6 a 1.9 ± 0.2 b 2.2 ± 0.1 a 2.2 ± 0.1 a 2.1 ± 0.2 a

11.5 ± 0.1 b 20.3 ± 0.1 b 3.3 ± 0.2 c 23.9 ± 0.9 a 28.5 ± 1.8 a 33.5 ± 1.4 a 14.5 ± 0.1 a 15.6 ± 0.4 b 16.2 ± 0.2 b 15.4 ± 0.6 b

a

Melting temperatures corresponds to Ton: onset melting temperature; Tp: maximum peak temperature; Tcom: completion of melting. b Melting temperatures corresponds to Ton: onset melting temperature; Tp: maximum peak temperature; Tcom: completion of melting. Different letters indicate significant differences between the different oleogels for each melting temperature (P < 0.05). c Texture analysis was evaluated over a storage period of 1–30 days. Different letters indicate significant differences between the different days for each oleogel (P < 0.05).

3.3.3. Thermal analysis DSC analysis showed that melting temperatures were similar for the oleogels produced in this study (Table 4). The melting point temperatures (23.9–35.3 °C) were influenced by the melting points of the oleogelators used (Öğütcü & Yılmaz, 2014), which were 43.8 °C for SFAD-wax and 46.3 °C for MO-wax. DSC analysis showed that the produced oleogels can be applied in the production of food products, since the melting temperatures were lower than the temperature of the human body (Hartel, von Elbe, & Hofberger, 2018). It has been suggested that the final melting temperature of fats is an important parameter in the confectionery industry, mainly due to human mouth temperature. A high melting temperature (> 35 °C) will not melt in the

mouth leading to a waxy mouthfeel (Hartel et al., 2018). 3.3.4. Rheological analysis Fig. 4 shows that the viscosity was decreased with increasing temperature for all oleogels. A shift from the solid-state to a viscous-state was observed by a change of the slope in a temperature range of 33–35 °C. The slope indicates the change of the crystal network. This shift is related to the melting profile of DSC analysis (Papadaki et al., 2019; Wijarnpiecha et al., 2018) and specifically with Tcom values (32.8–35.3 °C). The curve slope was different among oleogels with the

Fig. 3. Polarized light microscopy micrographs of crystal formation of oleogels produced from (a) soybean oil with soybean fatty acid distillate wax, (b) soybean oil with microbial oil wax and (c) microbial oil with microbial oil wax. 6

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Fig. 4. Profile change of viscosity versus temperature of oleogels produced from soybean oil with soybean fatty acid distillate wax (□), soybean oil with microbial oil wax (○) and microbial oil with microbial oil wax (●).

highest slope observed in the case of soybean oil-MO wax oleogel and the lowest slope observed in the case of microbial oil-MO wax oleogel. The non-linear inverse relationship between viscosity and temperature

has been evaluated by the Arrhenius equation ( = e( RT ) ), which describes the decrease of viscosity with increasing temperature (Lim, Hwang, & Lee, 2016). In particular, the activation energy (Eα) evaluates the thermal dependence of the oleogels and depicts the sensitivity to temperature changes. The soybean oil-MO wax oleogel had the highest Eα value, which is also indicated by the curve slope. This means that its viscosity was highly dependent on the temperature change, as compared to the microbial oil-MO wax oleogel, which had lower Eα. It has been suggested that Eα is affected by the amount and the type of wax esters. However, in this case, the different chemical composition of oils seemed to be a critical parameter for the flow behavior. The highest initial values of G′ and G″ were obtained in the case of soybean oil-MO wax and the lowest values in the case of microbial oilMO wax (Fig. 5). High G′ values indicate high gel strength, thus a more stable and compact crystalline network, which is strongly dependent on the chemical composition. The comparison of soybean oil-based oleogels shows that soybean oil-MO wax oleogel presented higher values of G′ and G″, because the presence of both C16 and C18 provides better structuring properties than C18 alone (da Silva, Arellano, & Martini, 2019). This suggests that the type of the oleogelator affects the rheological behavior of the oleogel. The rheological patterns of soybean oilMO wax and microbial oil-MO wax confirms that the type of oil is also an important factor in oleogelation (Wijarnprecha et al., 2018) which is strongly related to their chemical composition (da Silva et al., 2019). Specifically, da Silva et al. (2019) proved that the presence of both C16 and C18 in hardfats were responsible for the good interaction with the oleogelator (candelilla wax). Moreover, the use of fats rich in C16 triacylglyceroles provided stronger crystalline networks, whereas shorter carbon chains (C12, C14) resulted in poor interactions with candelilla wax (da Silva et al., 2019). Furthermore, Ferro et al. (2019) reported that the degree of unsaturation is critical for the rheological behavior of the oleogel, as sunflower oil formed stronger oleogels than coconut oil, which could be related to the greater degree of conformational freedom of the oils, facilitating thus the formation of gelator network. G′ was higher than G″ (G′ > G″) at a specific temperature range (up to 33 °C for soybean oil oleogels and up to 25 °C for microbial oil oleogel) indicating that oleogels presented more solid-like properties at these temperatures. At higher temperatures a cross over point (G′ = G″) was observed indicating the transformation of the gel into a sol (Luo et al., 2019). The highest cross over point at 35 °C was determined for soybean oil-MO wax oleogel, which indicated the greatest thermal stability of the gel network, as compared to the other types of oleogels a

Fig. 5. Storage modulus (G′) (●) and loss modulus (G″) (○) versus temperature of the oleogels: (a) soybean oil with soybean fatty acid distillate wax, (b) soybean oil with microbial oil wax and (c) microbial oil with microbial oil wax.

produced in this study. Tan δ values ranged from 0.38 to 0.66 at 20 °C suggesting that oleogels behaved more like a solid at this temperature. The lowest tan δ value was determined in soybean oil-MO wax oleogel, which demonstrated a more stable three-dimensional network structure compared to the other oleogels (Si, Cheong, Huang, Wang, & Zhang, 2016). However, none of the oleogels were characterized as a true gel (tan δ ≤ 0.1). The different rheological behavior of the produced oleogels may be attributed to the different crystal interactions caused by the chemical compatibility between waxes and oils. Also, the different chemical compounds present in waxes can influence the crystalline network and the rheological parameters (Doan et al., 2017). 3.3.5. Texture analysis Results concerning the firmness of the oleogels are presented in Table 4, which were measured after 24 h of storage at 4 °C. In the first day, the highest firmness was determined for microbial oil-MO wax 7

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(14.5 N) and the lowest firmness for soybean oil-MO wax (1.9 N). After 30 days, only the soybean oil-MO wax oleogel was stable (P > 0.05). In the case of soybean oil-SFAD wax, the firmness was decreased by 82% until the 20th day (P < 0.05) and then it was kept constant until the 30th day (P > 0.05). The decreased firmness could be affected by the large crystal formation (O'Brien, 2008) as determined by polarized light microscopy for the SFAD-wax derived oleogel after 30 days storage period (data not shown). This study showed that different firmness levels could be obtained by using different oils and oleogelators, which may be applied in a wide range of food products. It should be stressed that the successful application of oleogels in food products depends on the oleogel properties, which could be regulated by the addition of other food ingredients. Specifically, the firmness of soybean oil-MO wax oleogel could be more stable over a long storage period by the addition of emulsifiers (Cramer, 2016). Furthermore, Cerqueira et al. (2017) reported that firmness level can be improved by combining different oleogelators and oils.

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