Physicochemical and Morphological Study of the Saccharomyces cerevisiae Cell-Based Microcapsules with Novel Cold-Pressed Oil Blends

Abstract

Vegetable oils rich in polyunsaturated fatty acids are a valuable component of the human diet. Properly composed oil blends are characterized by a 5:1 ratio of ω6/ω3 fatty acids, which is favorable from a nutritional point of view. Unfortunately, their composition makes them difficult to use in food production, as they are susceptible to oxidation and are often characterized by a strong smell. Encapsulation in yeast cells is a possible solution to these problems. This paper is a report on the use of native and autolyzed yeast in the encapsulation of oils. The fatty acid profile, encapsulation efficiency, morphology of the capsules obtained, and thermal behavior were assessed. Fourier transform infrared analysis and low-field nuclear magnetic resonance relaxation time measurements were also performed. The process of yeast autolysis changed the structure of the yeast cell membranes and improved the loading capacity. Lower encapsulation yield was recorded for capsules made from native yeast; the autolysis process significantly increased the value of this parameter. It was observed that NY-based YBMCs are characterized by a high degree of aggregation, which may adversely affect their stability. The average size of the AY capsules for each of the three oil blends was two times smaller than the NY-based capsules. The encapsulation of oils in yeast cells, especially those subjected to the autolysis process, ensured better oxidative stability, as determined by DSC, compared to fresh blends of vegetable oils. From LF NMR analysis of the relaxation times, it was shown that the encapsulation process affects both spin-lattice T₁ and spin-spin T₂* relaxation times. The T₁ time values of the YBMCs decreased relative to the yeast empty cells, and the T₂* time was significantly extended. On the basis of the obtained results, it has been proven that highly unsaturated oils can be used as an ingredient in the preparation of functional food via protection through yeast cell encapsulation.

1. Introduction

Fats and fatty acids are important nutrients that have a significant impact on growth and development at a young age but also have an impact on chronic nutrition-related diseases later in life. Some of the fatty acids cannot be synthesized by humans and must therefore be obtained from the diet. These include polyunsaturated fatty acids (PUFA), ω6 linoleic acid (LA, 18:2 ω6) from vegetable oils, and ω3 α-linolenic acid (ALA, 18:3 ω3) from plant sources. PUFA ω3, eicosapentaenoic acid (EPA, 20:5 ω3), and docosahexaenoic acid (DHA, 22:6 ω3) can be found in fish and crustaceans. In the case of plant-based diets, the supply of these fatty acids is therefore severely limited. However, it is possible to synthesize DHA and EPA from ALA, as long as its supply is at a sufficient level [1].

Vegetable oils are obtained from the seeds of plants containing a minimum of 15% fat. Depending on the type of seed and their nutritional value and pressing ability, various technologies for obtaining oils are used, among which cold pressing is becoming more and more popular [2,3]. The use of low temperature during the entire process (40–50 °C) makes such oils a rich source of many nutrients, such as vitamins or biologically active compounds [4] but most of all, they provide monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) [5]. Cold-pressed oils are characterized by a specific, distinct taste and smell, and a greater possibility of auto-oxidation, i.e., spontaneous attachment of the O₂ molecule to unsaturated fatty acids, which causes them to become rancid more quickly [6]. Access to sunlight and oxygen are usually the main factors causing the oxidation of vegetable oils; this is due to the increased reactivity of methylene groups and unsaturated bonds [7,8]. Vegetable oils are also a frequent ingredient in recipes and are subjected to thermal treatment. Susceptibility to oxidation increases with the number of double bonds in fatty acid molecules. The more PUFAs in the oil, the greater the susceptibility to unfavorable changes or losses of valuable bioactive ingredients (tocopherols, polyphenols, sterols, phytosterols, squalene, carotenoids, and chlorophylls) when using high temperatures [9].

Oils available on the market are characterized by a different composition of polyunsaturated fatty acids. Very often, these products are characterized by a high ω6/ω3 acid ratio. Depending on the diet, this may have an adverse effect on human health. From a health point of view, the recommended optimal ratio of ω6/ω3 fatty acids ranges from 5:1 to 10:1 [10]. One of the ways to obtain the optimal ω6/ω3 ratio may be to mix oils and obtain blends with specific health properties [11]. The high content of unsaturated fatty acids is also one of the problems with the use of high nutritional oils in the production of foods subjected to cooking or foods with extended shelf life. As a result of food storage, fatty acids can be oxidized, and the formation of volatile compounds can significantly affect the deterioration of the sensory properties of food. During heating, oils are susceptible to oxygen in the air, water from the products (e.g., during frying), and the high temperature of the overall process. Consequently, they undergo changes in the processes of oxidation, hydrolysis, and polymerization [12]; thus, a number of compounds with different molecular weights are formed, which affect the health safety of food and its sensory acceptability [13,14].

Among the many methods of protecting fats against negative changes and degradation is the use of antioxidants that protect fatty acids against oxidation, e.g., synthetic (butylated hydroxytoluene-BHT, butylated hydroxyanisole-BHA, tert-butylhydroquinone-TBHQ, gallates, natural tocopherols, and phenolic compounds obtained as an extract from different plants) [15], and also encapsulation, which allows for the protection of compounds against a potentially aggressive environment yet this also often releases them in an active form in certain places of the gastrointestinal tract. In addition to the commonly used encapsulation method in saccharide matrices (e.g., using maltodextrin) [16,17], yeast cells are also known for their ability to absorb large amounts of both hydrophobic and hydrophilic substances. Yeast-based microcapsules (YBMCs) significantly increase the oxidative stability of the active ingredient compounds encapsulated in them compared to the methods of traditional encapsulation in polysaccharide shells [18]. Both native yeast and yeast subjected to technological processes, improving their ability to bind active substances, can be used for the encapsulation process. The main techniques used to empty yeast cells are plasmolysis, autolysis, and enzymatic hydrolysis [19].

Cold-pressed oils contain significant amounts of MUFA, PUFA, but also other biologically active compounds, which makes them difficult to store and use in food production, especially during thermal processing. Bearing the aforementioned in mind, the aim of this study was to evaluate the possibility of using S. cerevisiae yeast cells as an encapsulating carrier for cold-pressed oil blends with a 5:1 ratio of ω6/ω3 fatty acids to protect against adverse oxidative changes and in order to maintain high biological activity. To date, this method has not been used to protect oil blends with such a ratio of nutritionally important fatty acids. The obtained microcapsules were characterized in terms of their morphology and particle size distribution, but also their properties were examined using DSC, FT-IR, and low-field NMR techniques.

2. Materials and Methods

2.1. Oil Blends

Cold-pressed rapeseed, black cumin, wheat germ, evening primrose, and camelina oils, as well as refined rice bran oil, were used to obtain blends of oils which were further used for encapsulation. The oils were ordered directly from the oil producer (Semco Sp.z o.o. Sp.k., Smiłowo, Poland). Recipes of 16 blends were developed, the ω6/ω3 fatty acid composition of which was characterized by a ratio of 5:1. The composition of the oil blends was developed at the earlier stages of the research project and was submitted for patent protection [20,21]. Cold-pressed oils were used due to the higher content of antioxidants, including phenolic compounds. In this study, three oil blends were used (

Table 1. Composition of oil blends.

Code	Type of Oil			Share (%)
RBWg	Rapeseed oil	Black cumin oil	Wheat germ oil	50	30	20
REp	Rapeseed oil	Evening primrose oil		65	35	-
CRb	Camelina oil	Rice bran oil		12	88	-

), which in previous studies were characterized by high biological activity [11], in order to protect them during storage and use in food production.

2.2. Fatty Acid Composition

The fatty acid composition was determined according to the AOCS Official Method Ce 1h-05 [22] using an Agilent 7820A GC equipped with a flame ionization detector (FID) (Agilent Technologies, Santa Clara, CA, USA). The oil samples were first dissolved in n-hexane and transesterified with sodium methylate. After transesterification, the fatty acid methyl esters (FAME) were separated using the SLB-IL111 capillary columns (Supelco, Bellefonte, PA, USA) (100 m, 0.25 mm, 0.20 mm).

2.3. Baker’s Yeast Cell Pretreatment

In order to remove residue of the culture medium and cell fragments, purchased in a store, commercially available Saccharomyces cerevisiae baker’s yeast (Lesaffre Polska S.A., Wolczyn, Poland) was rinsed three times with 0.01 M phosphate buffer [23]; the buffer to yeast ratio was 5:1. Each time the obtained yeast suspension was centrifuged (20 min, 3500× g) in order to separate the biomass, and then the buffer was decanted. After the final rinsing and centrifugation, the yeast was frozen (−22 °C), freeze-dried, and ground using a laboratory grinder. The obtained native yeast (NY) from this process was further used for the encapsulation process.

In order to maximize the capacity of yeast shells, the washed NY wet biomass was subjected to autolysis. First, the pH of the yeast suspension was adjusted to 5.5 with 0.5 N HCl. It was then incubated for 48 h at 55 °C in a water bath with stirring set at 150 rpm [24]. After this time, the yeast was rinsed five times with 0.01 M phosphate buffer, centrifuged (20 min, 3500× g), frozen, and freeze-dried. Autolyzed yeast, denoted as AY, was used then for the encapsulation process.

2.4. Microencapsulation

The microencapsulation of the oil blends (according to Table 1) in yeast biomass was carried out based using the methodology used by Czerniak et al. [24], with a few modifications as described in the Polish patent application [25]. The microencapsulation was carried out in triplicate for two parallel tests (n = 6). The binding of oil with biomass (both NY and AY) was carried out in glass bottles with a capacity of 250 mL, with an amount of 40 g of deionized water containing 3% wt. addition of the emulsifier Tween 80 (polyoxyethylene sorbitan monooleate). Then, 10 g of the blend of oils was added to the solution and homogenized (CAT X120 homogenizer with TN10 tip, Ingenieurbüro CAT, M. Zipperer GmbH, Ballrechten-Dottingen, Germany) for 5 min at 16,000 min⁻¹ to prepare a pre-emulsion. The final emulsion was prepared with an ultrasonic homogenizer (Sonoplus HD 3200 with probe VS 70/T, BANDELIN electronic GmbH & Co. KG, Berlin, Germany), using the following process parameters: homogenization time 5 min, pulse length 20 s, pause 10 s, ultrasound frequency 10 kHz, and power 200 W with an amplitude of 150 μm. During the homogenization, the vessel was cooled in an ice bath to limit the temperature increase, which during the entire emulsion formation process did not exceed 35 °C. The mixture was incubated in a shaker (New Brunswick™ Excella E24, Eppendorf SE, Hamburg, Germany) with 180 min⁻¹ agitation for 8 h at 45 °C. After incubation, the contents of the vessels were poured into falcon tubes and centrifuged at 3500× g (Rotina 380R, Andreas Hettich gmbH & Co. Kg, Tuttlingen, Germany) for 10 min to separate the biomass. The separated mass of microcapsules was then washed three times with deionized water to remove the remnants of the mixture. The wet biomass was transferred to lyophilization vessels and frozen at −20 °C for 12 h, and then freeze-dried at a pressure of 0.125 mbar for min. 48 h. The microcapsules prepared in this way were stored at −20 °C until analysis.

2.5. Determination of Oil Content

An amount of 15 g of microcapsules was weighed into a filter paper thimble, which was then sealed with cotton wool and placed in a Soxhlet extractor. Extraction was performed with 200 mL of petroleum ether at the reflux temperature of the solvent for 2 h. After extraction, the oil-containing extract was transferred to a pre-weighed dry round bottom flask, and the solvent was evaporated using a vacuum rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) at 40 °C and 200 mbar, and then dried in a laboratory oven for 30 min at 95 °C. Total oil content was calculated from the weight difference between the flask containing the oil and the dry flask.

2.6. Encapsulation Yield (EY)

The EY was calculated according to formula:

$$EY [\%]=\frac{M_{OB}}{M_{MC}}\times 100$$

where: M_OB—the mass of the total oil encapsulated in yeast shells, and M_MC is the mass of the capsules.

2.7. Microcapsule Morphology and Size

The morphology of the microcapsules was evaluated using a scanning electronic microscope (SEM) (Zeiss EVO 40, Carl Zeiss AG, Oberkochen, Germany). The size distribution of the microcapsules was measured using a Mastersizer 2000 (Malvern Panalytical GmbH, Herrenberg, Germany) using isopropanol as the dispersion medium.

2.8. Fourier Transform Infrared (FT-IR) Spectroscopy

FT-IR spectra were obtained via a spectrophotometer of the Perkin Elmer company (Waltham, MA, USA), equipped with an ATR device with diamond as the internal reflection element [26]. Data were collected over a spectral range of 4000–400 cm⁻¹.

2.9. Melting Characteristic

DSC measurements of the melting characteristics of the oils were carried out with a TA DSC Q200 (TA Instruments Corporate, New Castle, DE, USA). Calibration was done with indium standards. Fat samples of 3–4 mg were placed into aluminum pans with a lid and were hermetically sealed. An empty sealed aluminum pan was used as a reference and the experiments were performed under a nitrogen atmosphere at normal pressure. Melted samples were heated to 80 °C and held for 10 min in order to melt all the crystals and to erase the thermal memory. The samples were then cooled to −80 °C at 10 °C/min and maintained at −80 °C for 30 min. Then the melting profiles were obtained by heating the samples to 80 °C at a heating rate of 15 °C/min [27].

2.10. Differential Scanning Calorimetry (DSC) Studies

The lyophilized capsules were studied by DSC (TA DSC Q 200, TA Instruments Corporate, New Castle, DE, USA) in a normal pressure cell. The cell was purged with dry nitrogen at 50 mL/min and calibrated for the baseline of an empty oven temperature using standard pure indium. Specific heat capacity was calibrated using a sapphire. The powders were cooled with a mechanical refrigeration cooling system (intracooler). An empty hermetically sealed aluminum pan was used as a reference in every test. The powders (10–15 mg) were hermetically sealed in aluminum pans (volume 30 μL). The samples were heated from −80 °C up to 200 °C with a heating rate of 5 °C/min. The DSC technique was used to obtain curves of heat flow (W/g) versus temperature curves [28].

2.11. Water Activity

Measurements of water activity were performed using an analyzer of water diffusion and activity ADA-7 (COBRABID, Poznań, Poland), with a system of automatic time recordings of the water evacuation runs from individual samples. The process was carried out for six repetitions (n = 6). Detailed characteristics of the experimental method were performed as described by Le Thanh-Blicharz et al. [29]. Before the measurement, the chamber was dried to a water activity of 0.05. The temperature was stabilized at 20.0 ± 0.1 °C using Peltier modules. The duration of one measurement was set at 1000 s.

2.12. Low-Field Nuclear Magnetic Resonance (LF NMR) Measurements

Measurements of relaxation times were performed using a pulse NMR spectrometer PS15T, operating at 15 MHz under a system-controlled temperature (Ellab, Poznań, Poland). The pulse sequence (180-t-90) [30] was applied for measurements of the spin-lattice relaxation times. Distances between RF pulses (t) were changed within the range from 0.1 to 120 ms, and the repetition time was approximately 13 s. Each time, 32 FID signals and 100 points from each FID signal, were collected.

Five accumulation signals were employed. All samples were measured at 20.0 ± 0.5 °C. All presented results are mean values and standard deviations, recorded from five repetitions of each sample.

Calculations of the spin-lattice relaxation time values were performed with the assistance of the CracSpin program [31], designed for calculating relaxation parameters from experimental data using the “spin grouping” approach. The Marquardt method of minimization was applied for fitting multiexponential decays. Standard deviation was used to determine the accuracy of the relaxation parameter measurement. Time changes of the current value from the FID signal amplitude used in the employed frequency of impulses are described by the following formula:

$$M_z=M_0\left(1-2e^{\frac{-t}{T_1}}\right)$$

where: M₀ and M_z were equilibrium and accuracy magnetization amplitude, respectively.

2.13. Statistical Analyses

All measurements were repeated in triplicate, unless otherwise stated. Statistica 13 software (Dell Software Inc., Round Rock, TX, USA) was used to perform a one-way analysis of variance (ANOVA). A post-hoc Tukey HSD multiple comparison test was used to identify statistically homogeneous subsets at α = 0.05.

3. Results and Discussion

3.1. Oil Blends Characteristics

The main determinant of the quality, nutritional value, and oxidative stability of edible oils is their fatty acid profile.

Table 2. Fatty acid composition (%) and ω6/ω3 fatty acid ratios of oil blends.

Fatty Acid	RBWg	REp	CRb
C16:0	9.36 ± 0.01 a	5.01 ± 0.23 b	15.55 ± 0.25 c
C16:1	0.19 ± 0.03 a	0.11 ± 0.01 a	0.13 ± 0.01 a
C18:0	1.89 ± 0.02 a	2.66 ± 0.06 b	1.65 ± 0.07 a
C18:1	42.79 ± 1.38 a	42.76 ± 1.15 a	37.14 ± 0.68 b
C18:2	37.19 ± 1.11 a	38.16 ± 0.22 a	36.71 ± 0.95 a
C18:3	7.37 ± 0.17 a	7.49 ± 0.68 a	7.18 ± 0.14 a
C20:0	0.11 ± 0.01 b	0.05 ± 0.01 a	0.69 ± 0.02 c
C20:1	1.02 ± 0.05 b	3.75 ± 0.08 c	0.57 ± 0.03 a
C22:0	0.02 ± 0.01 a	0.02 ± 0.01 a	nd
C22:1	0.06 ± 0.01 a	nd	0.39 ± 0.03 b
SFA	11.38 ± 0.01 b	7.73 ± 0.16 a	17.89 ± 0.34 c
MUFA	44.06 ± 1.30 b	46.61 ± 1.06 b	38.23 ± 0.75 a
PUFA	44.56 ± 1.28 a	45.66 ± 0.90 a	43.89 ± 1.09 a
ω6/ω3	5.05 ± 0.03 a	5.11 ± 0.44 a	5.11 ± 0.03 a

Composition of blends as shown in Table 1. Values are means of three determinations ± SD. Means in the same row followed by different letters indicate significant differences (p < 0.05) between samples of each type of oil blend. nd—not detected. SFA—saturated fatty acid, MUFA—monounsaturated fatty acid, PUFA—polyunsaturated fatty acid.

shows the percentage of fatty acid composition of the tested oil blends, as well as photographs of their color. It can be seen that both RBWg and REp were yellow, with the RBWg’s color appearing much more intense (darker). CRb was characterized by a straw color. Chromatographic analysis of the fatty acid profile showed that the ω6/ω3 fatty acid ratios were 5.05, 5.11, and 5.11 for RBWg, CRb, and REp, respectively. The fatty acids from ω3 and ω6 groups are extremely important for the proper development of the body, but from a nutritional point of view, the most important thing is their relationship to each other because both these groups of essential fatty acids compete for the same enzymes during metabolic processes in the human body [32]. Consuming excessive amounts of ω6 fatty acids promotes inflammation, which in turn can lead to a wide variety of health conditions, including heart disease [33]. The WHO/FAO has suggested an optimal ω6/ω3 ratio range from 5:1–10:1 [10].

All analyzed blends were characterized by a favorable nutritional profile of fatty acids with approximately a 90% proportion of unsaturated fatty acids and a range of only 7.73% (for CRb) to 17.89% (for REp) of saturated acids. The main SFA was palmitic acid, and there were also stearic, arachidic, and decanoic acids present. In the group of unsaturated fatty acids, the dominant compound was oleic acid (C18:1). The total share of this fatty acid ranged from 37.14 to 42.79% for CRb and RBWg blends, respectively. The C18:2 n-6 α-linoleic acid dominated among the PUFAs, but the C18:3 n-3 (ALA) acid, which was also present in the analyzed blends, was more important from a nutritional point of view. ALA is not synthesized by the human body, it must be supplied by a person’s diet, and it is necessary for the proper functioning of the body [34,35]. Moreover, ALA is a precursor in the n-3 fatty acid metabolic pathway, allowing the synthesis of eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) in the organism [36,37]. The main sources of DHA and EPA are fish and seafood, which are eliminated in plant-based diets. However, ensuring an adequate level of DHA and EPA is crucial as the deficiency of these fatty acids can cause many adverse health effects [38,39,40].

3.2. Effect of Pretreatment on the Capacity of YBMCs

Both native S. cerevisiae yeast and autolyzed yeast were used in the present study to obtain YBMCs with oil blends with a nutritionally favorable fatty acid ratio. A significantly (p < 0.05) higher increase in EY was observed in YBMCs obtained from autolyzed yeast compared to capsules based on native yeast (Figure 1). The structure of the cell wall determines its permeability; in NY, the passive transport was much slower hence the observed lower oil loading yield [41]. The biggest difference was observed in the case of RWBg encapsulation, and EY increased in AY versus NY by about 35.46%. Bishop et al. [42] indicate in their work that the encapsulation of oils in yeast cells is a process consisting of two sub-processes: releasing the yeast cell components into the reaction environment and the simultaneous extraction of oil from the environment, immobilized inside the cell. Shi et al. [23] indicate that the pre-treatment of yeast biomass by means of plasmolysis may result in an increase in encapsulation yield by as much as 50%. However, other authors in the published report indicated that cell plasmolysis did not result in a significant increase in the oil loading into capsules [19]. The calculated EY ranged from 8.25 to 13.98%, depending on the oil blend used. This may result from the conditions of the encapsulation process carried out in the aquatic environment. The high water content causes the walls of yeast cells to become hydrophilic due to the polysaccharides present in them, which limits the process of diffusion of hydrophobic molecules into the cells [43]. Nevertheless, Shi et al. [44], in their studies, showed similar EY values using yeast as an encapsulation carrier. In other studies, however, Pham-hoang et al. [45] showed that EY can be as high as 55%, depending on the encapsulated material.

3.3. YBMCs Morphology

Figure 2 presents SEM pictures of the NY and AY microstructures, as well as YBMCs obtained from them from each of the 3 oil blends. Micrograph analysis showed significant morphological differences between the yeast cells without oil charged inside and the final microcapsules. Strong aggregation of NY cells was observed (Figure 2A), which was characterized by an oval, bubble-like shape. After the autolysis process, irregularly shaped collapsed bubbles were formed (Figure 2B). After the encapsulation process, the resulting capsules were mostly spherical, with the exception of RBWg/NY and REp/NY, whose shape was irregular. Literature data show that the aggregation of yeast cells is caused by a high content of β-glucans [46]. A strong agglomeration of microcapsules was also observed, especially in the case of NY capsules. Also, Czerniak et al. [24] observed similar agglomeration behavior for fish oil containing YBMCs. The reduction of YBMCs agglomeration due to cell autolysis increased the outer surface of the microcapsules and, consequently, the encapsulation efficiency. Therefore, preventing the aggregation of yeast cells in order to increase the diffusion rate of the encapsulated material into the YBMCs is recommended.

The size of yeast shells is important and determines their stability and microencapsulation efficiency. Small sized capsules are preferred as these make it easier to ensure uniformity and quality in food products [47]. The size distribution of YBMCs is presented in

Table 3. YBMCs size distribution in µm.

Oil Blends	NY			AY
	Dx (10)	Dx (50)	Dx (90)	Dx (10)	Dx (50)	Dx (90)
RBWg	170.98 ± 11.18	621.03 ± 55.68	1520.72 ± 184.96	77.70 ± 2.30	261.86 ± 22.27	1133.33 ± 207.75
CRb	197.34 ± 11.11	798.88 ± 69.90	1958.45 ± 126.62	77.56 ± 1.63	305.88 ± 22.25	1033.61 ± 127.78
REp	172.72 ± 17.01	780.38 ± 79.21	2134.76 ± 150.17	88.75 ± 4.03	409.83 ± 40.75	1743.94 ± 230.84

. The NY has uniform particle size distribution, as well as the obtained microcapsules (shown on Figure 3A). There is a noticeable shift towards higher values of the particle size distribution increasing after the absorption of oil during the encapsulation. In case of the AY, there is no uniformity in the particle size distribution. There is a wide range of different sizes of particles with local maxima (shown in Figure 3B). The autolysis process causes the removal of inside organelles from yeast cells and their partial disintegration, which can explain this characteristic of two sets of particle size distribution. Furthermore, autolysis does not impact every cell in the same way and usually leads to the limitation of cells aggregating and a more representative particle size distribution. On the other hand, the NY and NY-based microcapsules form aggregates (proven by SEM images), which can explain two separate maxima in the case of the AY samples and the uniform distribution of NY. Larger YBMCs provide better protection for the encapsulated material, but dissolve less well in the end products [48]. It has also been shown that the presence of large particles may be undesirable in most food products [49]. Capsules that are too small, however, can be empty containers that do not contain the encapsulated material inside [48].

3.4. FT-IR Analysis

The changes in the functional groups of the microcapsules were analyzed by FT-IR spectroscopy. It was previously proven that FT-IR microspectroscopy in the ATR mode can successfully study the composition of the global cell wall due to the reduced depth of penetration of the photon beam [50]. The spectra of the resulting changes are shown in Figure 4. The absorption spectra of the YBMCs showed no significant changes resulting from the content of various oil blends. A characteristic absorption band was observed at wave numbers 3400 cm⁻¹ and 2900 cm⁻¹, responsible for the OH vibration bands of yeast and the asymmetric CH₂ stretching vibrations of lipids [19,51]. Absorption maxima were recorded at the wave number 1600 cm⁻¹, which corresponds to amide I and amide II bands in proteins, and the 1050 cm⁻¹ β-1,3-glucans absorption band [52]. FT-IR analysis also showed a band related to the COO stretching vibration of triglycerides at 1742 cm⁻¹ [51].

3.5. Thermal Oxidative Stability of YBMCs

Oil blends with a ω3/ω6 ratio of 5:1 are characterized by a large amount of unsaturated fatty acids (the sum of MUFA and PUFA is about 90%) and thus are very susceptible to oxidation. Many different factors affect oxidative stability, including resistance to oxidation caused by atmospheric oxygen, temperature, light, metal ions, etc. In these tests, the oxidative stability of the oil blends and the plasmolyzed and non-plasmolyzed YBMCs within the oils were assessed by DSC and TG measurements.

The high content of unsaturated fatty acids determined by chromatography (Table 2) within the oil blends used for encapsulation was confirmed by the curves of the softening characteristics of oils, determined by the DSC method (Figure 5). According to the literature data, the low temperatures of endothermic peaks are responsible for PUFA, and their content is higher in relation to the lower value of the endothermic recorded during the analysis [53,54]. The DSC melting curves of edible fats have different peak temperatures. Triacylglycerols containing unsaturated fatty acids in their structure are characterized by negative maximum peak temperatures (Figure 5). It proves the content of MUFA and PUFA in triacylglycerols of the tested oils [55]. In Figure 5, no endothermic peaks were observed; the maximum temperature would have had positive temperature values.

The DSC curves of native and autolyzed yeast (Figure 6A) indicate that NY shows an endothermic peak at about 61 °C, corresponding to the phase transition temperature of yeast cell phospholipid bilayers. The phase transition temperature of yeast depends on many factors, such as the temperature during their breeding, the microbiological medium used, or the method of aeration of the culture, but it depends also on the growth phase in which the yeast was collected and in the published works of other authors, different values were assumed [19,43]. In contrast to NY, AY, due to the liquefaction of the cell membrane caused by the autolysis process, saw only a slight phase transition at a temperature of about 55 °C, which is consistent with the decrease in this temperature after yeast cell lysis, described by other authors [51]. The encapsulation of oils in yeast cells is evidenced by small peaks with maxima in negative temperatures (Figure 6B). In the yeast figures, small endothermic peaks are present only for NY yeast, with positive temperatures of about 50 °C (Figure 6A). The presence of this peak may indicate the degradation of proteins (peptides). The peaks of temperature, which are in the range of positive temperatures, indicate the melting of carbohydrates.

Changes in the course of the DSC curves for NY-based YBMCs are more pronounced. Endothermic peaks characterized by maximum temperatures at sub-zero temperatures indicate the presence of oils in the yeast capsules. In addition, the temperature values confirm the presence of triacylglycerol, the structure of which contains mono and polyunsaturated fatty acids. A decrease in the temperature of the endothermic reaction was observed, which may result from the integration of the encapsulated oil with the yeast bivalent lipid, which led to an easier transition from the gel to the liquid crystal state. van der Walls interactions between the oils and the cell membrane led to the reduction of its cohesiveness and, consequently, to its liquefaction [19]. At the same time, in the case of AY without and with encapsulated oils, an endothermic peak shift towards higher temperatures was demonstrated. As indicated by Kavosi et al. [51], such changes may indicate an increase in the thermal stability of the encapsulated oil.

3.6. Water Activity and LF NMR Relaxometry

The NY and AY trials showed a_w values of 0.2741 ± 0.0001 and 0.1660 ± 0.0001, respectively. Measurements of water activity carried out for the YBMCs showed different values of a_w in the measuring chamber, which made it impossible to unambiguously characterize the value of this parameter. This means that there is no water available for the YBMCs to show any possibility of evacuation. All the water is trapped in the YBMCs structure.

LF NMR is a method used in food analysis since the 1990s [56,57,58,59]. It is a fast and accurate alternative to the use of drying and solvent extraction for determining the fat and moisture content of a biological system and is particularly suitable for the analysis of complex systems such as food products [60,61]. It involves measurements of the relaxation time of spin-lattice T₁ and spin-spin T₂. Both parameters characterize the molecular dynamics of oil and water protons in the tested sample. Changes in their values reflect the relative changes in bulk water relative to bound water (T₁) or describe the molecular dynamics of protons fractions. The analysis of the molecular dynamics of water in the tests, carried out by LF NMR methodology, showed that in NY and AY, significant differences had occurred for the values of spin-lattice relaxation times T₁ (Figure 7). The spin-lattice T₁ relaxation times for the YBMCs obtained from NY are longer, which may indicate a greater ordering of molecules containing protons [62,63].

As in the case of changes in a_w, higher values were found in all NY samples, which means greater mobility of water molecules. Plasmolysis removed the internal yeast organelles and thus facilitated the encapsulation of the oil, hence the lower T₁ time value. Better encapsulation is also evidenced by the lower thermodynamic changes observed in the DSC analysis (see Section 3.5). Comparison of the T₁ values for the YBMCs showed that the largest (26%) difference between the NY and AY trials was recorded for CRb, and the lowest (5%) for RBWg.

The values of spin-spin T₂* times, presented in Figure 8, were also analyzed by using the shape of the free induction decay (FID) signals. YBMC samples containing oils are characterized by signals that are exponentially decaying. This exponentially decaying signal comes from the mobile liquid protons fraction. Thus, the results indicate that these relaxation time signals come only from the encapsulated oil, as confirmed by the results from the DSC.

Additionally, when analyzing the shape of the FID signals (Figure S1), it was found that only NY and AY were characterized by two times T₂*, described by the Gaussian curve and the exponential curve [64,65]. The Gaussian function comes from the solid proton fraction. In combination with the aw results, a very short T₂* time of 0.03 ms for NY and 0.05 ms for AY can be attributed to the unstructured water molecule. No water activity was recorded in the YBMCs; therefore, it should be concluded that the exponential components of the T₂* times are the result of the relaxation of the protons of the structural components of oil.

4. Conclusions

Microcapsules from new blends of oils with a favorable fatty acid profile were successfully prepared via a yeast-based microencapsulation process. The conducted research allows us to conclude that NY is a less efficient material for the oil encapsulation process. Earlier plasmolysis of yeast cells significantly improves the efficiency of the process. Moreover, importantly, YBMCs obtained from AY are characterized by lower aggregation, positively influencing their application value in food technology. The use of the encapsulation process resulted in the reduction of the oxidative changes of the encapsulated oils observed during thermal treatment, especially in YMBCs from autolyzed yeast; therefore, it is possible to effectively protect them in yeast capsules. More research on YBMC digestion, oil release, and in vitro and in vivo biological activity would be useful, which would show whether the encapsulation process also maintains the activity of the compounds with beneficial pro-health effects present in cold-pressed oils.

5. Patent

The results presented in this article were used to prepare a patent application to The Patent Office of the Republic of Poland (patent application No. P.440018, dated 2021-12-29).