Nanoencapsulation of Saccharomycopsis fibuligera VIT‐MN04 using electrospinning technique for easy gastrointestinal transit

Abstract

In this study, probiotic yeast Saccharomycopsis fibuligera (S. fibuligera) VIT‐MN04 was encapsulated with wheat bran fibre (WBF) and exopolysaccharide (EPS) along with 5% polyvinylpyrrolidone (PVP) using electrospinning technique for easy gastrointestinal transit (GIT). The electrospinning materials viz. WBF (10%), EPS (15%), PVP (5%) and electrospinning parameters viz. applied voltage (10 kV) and tip to collector distance (15 cm) were optimised using response surface methodology to produce fine nanofibres to achieve maximum encapsulation efficiency (100%) and GIT tolerance (97%). The probiotic yeast was successfully encapsulated in nanofibre and investigated for potential properties. The survival of encapsulated S. fibuligera VIT‐MN04 was increased compared to the free cells during in vitro digestion. In addition, encapsulated yeast cells retained their viability during storage at 4°C for 56 days. The nanofibres were characterised using scanning electron microscopy, atomic force microscopy, X‐ray diffraction, thermogravimetric analysis, zeta potential analysis and Fourier transform infrared spectroscopy followed by gas chromatography–mass spectrometry and nuclear magnetic resonance analysis. This work provides an efficient approach for encapsulation of probiotic yeast with the nanofibres which can also broaden the application of the prebiotic like WBF providing an idea for the efficient preparation of functional synbiotic supplements in the food industry.

1 Introduction

Recently, the consumption of probiotic functional foods has been increased in the market due to its wide range of health benefits [1]. Probiotics are affected by several factors like temperature, moisture, texture and aroma of the food product which disturbs their action in the gastrointestinal system during the consumption of the probiotic food. Therefore, it is necessary to protect probiotics using various materials viz. gums, proteins, polysaccharides and fibres through the encapsulation technique. Nanoencapsulation has many advantages like narrow size, controlled release and delivery in target sites [2]. The encapsulation process protects the functionality of the probiotics which attributes targeted delivery in the gut region [3].

The combination of probiotics and prebiotics are known as synbiotic and being used in food industries to explore their synergistic properties [4]. Probiotics and prebiotics are included in the diet to increase the beneficial gut microbes and their activities, thus generating benefits to human health. Several health benefits such as pathogen inhibition, lactose tolerance, cholesterol removal, vitamin production, the bioavailability of minerals, prevention of colon cancer, better digestion, gut function and immune regulation are reported after encapsulation of probiotics using prebiotics [5, 6].

Dietary fibre plays an important role in gut microbiota. Among the cereal fibres, wheat fibre has been reported as a dominant source of fibre in many western countries [7]. Wheat bran fibre (WBF) contains many bioactive compounds like arabinoxylans flavonoids, carotenoids, and sterols. Some of the food products are coated with WBFs viz. biscuits, bread, pasta, noodles and doughnut [8]. Also, it has health benefits such as reduction of cholesterol level in plasma and glucose level in blood [9]. Moreover, WBF as prebiotic has been reported to be used for the improved GIT tolerance compared to inulin [10].

Exopolysaccharides (EPSs) produced by probiotics showed beneficial physiological effects such as anti‐inflammatory, antitumour, antigastritis, antiulcer properties and cholesterol‐lowering effects [11] and used as a potential probiotic carrier. EPS played a crucial role in the development of fermented food products due to its high viscosity. Probiotic EPS exhibited potential properties such as cell adhesion, cholesterol‐lowering effects and immunomodulation [12].

Electrospinning is a fabrication technique which produces a large volume of nanofibres by applying a high voltage to a polymer solution. Encapsulation by electrospinning technique offered more advantages such as high immobilisation efficiency, robust protection and minimised sensorial effect [13]. The nanofibre fabrication was affected by electrospinning parameters (applied voltage, tip to collector distance flow rate) and solution parameters like polymer concentration and viscosity. The specific surface area, volume, and size of the pores of nanofibres showed considerable effects on cell adhesion, growth, and proliferation which affected the release of probiotics [14, 15].

A water‐soluble polymer, polyvinylpyrrolidone (PVP) can be used as an encapsulating agent for probiotics. There was a report on encapsulation of probiotic bacteria using synthetic polymer PVP which improved the nanofibre production through electrospinning process due to their hydrophilic nature [13].

This work deals with the production of nanofibres using WBF and EPS along with PVP by electrospinning technique. The probiotic yeast Saccharomycopsis fibuligera (S. fibuligera) VIT‐MN04 was encapsulated in the above‐mentioned nanofibres. To improve the encapsulation process, the parameters were optimised using Box Behnken design (BBD) by response surface methodology (RSM). The survival of encapsulated probiotic yeast was evaluated during in vitro digestion and the viability was noted at two different temperatures during storage. Further, encapsulated nanofibres were characterised using various instrumental analyses.

2 Materials and methods

2.1 Probiotic yeast and growth conditions

In our previous study, S. fibuligera VIT‐MN04 was reported as a potential probiotic yeast strain isolated from goat intestine [16, 17]. In 100 ml of YEPD media (Himedia, India) yeast cells were inoculated and incubated at 35 ± 2°C for 24–48 h with 200 rpm. The yeast culture was centrifuged at 5000 rpm for 15 min (Lab Tech, India) and lyophilised at −80°C.

2.2 Extraction of dietary fibre from wheat bran

The dietary fibres from wheat bran were extracted following the standard method [18]. Fat was extracted and removed from wheat bran by Soxhlet apparatus to get fat free powder. Then, starch was degraded in a phosphate buffer at pH 6.0 using heat‐stable α‐amylase (Himedia, India) and incubated for 30 min at 90°C. For degradation of protein, the residue was treated with protease after adjusting the pH at 7.5 and suspension was incubated at 60°C for 30 min. Then, the degradation of amyloglucosidase was done at pH 4.5 keeping the suspension at 60°C for 30 min. The suspension was centrifuged at 4000 rpm for 10 min after each treatment. Then the residue was washed with warm water (70°C) followed by 95% (v/v) ethanol and acetone wash and dried at 40°C in a vacuum oven for overnight. The dried residue was further extracted using three solvents viz. ethanol, dichloromethane, and n‐hexane for 8 h using a Soxhlet apparatus. After each treatment, centrifugation was done and the residue was dried at 50°C in a vacuum oven.

2.3 Extraction of exopolysaccharide from probiotic yeast

The EPS was extracted from probiotic yeast S. fibuligera VIT‐MN04 following the method of Li et al. [19]. The yeast was inoculated in 100 ml of basal medium and incubated on a rotary shaker (180 rpm) at 22°C for 7 days. Then, the culture was centrifuged at 10,000 rpm for 20 min at 4°C. To the supernatant, two volume of ice‐cold isopropanol was added to precipitate the EPS overnight. The precipitate was centrifuged at 10,000 rpm for 30 min. Then, 10 ml of supernatant was taken to dialysis through 10 kDa membrane against distilled water at 4°C for 72 h with 2–4 changes per day to remove low molecular weight impurities and the remaining were lyophilised overnight.

2.4 Production of nanofibre‐encapsulated S. fibuligera VIT‐MN04

Extracted WBF and EPS were mixed and amended with 3% (w/v) PVP (SRL, India) and dissolved at 60°C. The spinning solution was characterised by measuring viscosity and pH. Further, the solution was sterilised at 121°C for 15 min and fabricated at 25°C with 12 kV applied voltage in ESPIN‐NANO apparatus manufactured by Peco, Chennai. The collected nanofibres were dried at 80°C for 6 h [13]. The probiotic yeast S. fibuligera VIT‐MN04 was added to the spinning solution and electrospun at 25°C with a flow rate of 0.1 ml/h and 12 kV applied voltage. The probiotic yeast S. fibuligera VIT‐MN04 encapsulated in nanofibres was vacuum packed and stored at 4°C until further use.

2.5 Process optimisation using response surface methodology

The optimisation of electrospinning materials, as well as electrospinning parameters, was done using RSM. The five independent variables viz. WBF, EPS, PVP, applied voltage and tip to collector distance were selected for BBD and 54 experiments were conducted to investigate their interaction effects on encapsulation efficiency (EE) (%), and GIT tolerance (%). The quadratic model was used to analyse the data. Each factor in the design was studied at three different levels as shown in Table 1. The 3D contour plots were prepared to know the interactions of different factors and to evaluate the optimised conditions which influenced the responses. The results of the experimental design were analysed and interpreted using Design‐Expert 11 (Stat‐Ease Inc., Minneapolis, MN, USA) statistical software [20].

Table 1.

Levels of the variables used in the BBD design

Variables	Code	Level −1	Level 0	Level + 1
WBF, %	A	5	10	15
EPS, %	B	10	15	20
polyvinylpyrrolidone, %	C	3	5	7
applied voltage, kV	D	7	10	13
tip to collector distance, cm	E	10	15	20

2.6 Encapsulation efficiency

The encapsulation materials viz. WBF and EPS (2:6 ratio) were mixed and the suspension was autoclaved at 121°C for 15 min. Then, probiotic yeast S. fibuligera VIT‐MN04 (2%) was added to the suspension and stirred for 30 min at room temperature. The suspension was electrospun using ESPIN‐NANO apparatus and the nanofibres were produced. Then nanofibres encapsulated yeast cells were inoculated in YEPD medium again to enumerate the number of viable yeast cells. The EE of probiotic yeast cells was calculated (1) as follows:

$$\mathrm{EE}(\text{\%})=(X_t/X_i)\times 100$$ (1)

where X_t is the total amount of encapsulated probiotic yeast and X_i represents total amount of free cells used for encapsulation process [21].

2.7 Survival of probiotic yeast S. fibuligera VIT‐MN04 during in vitro digestion

The probiotic yeast cells (encapsulated and free cells) were evaluated for their GIT tolerance following the procedure of Wang et al. [22]. Initially, the cells were treated with simulated gastric juice (pepsin 3 mg/ml; pH‐2) and kept at 37°C for 3 h (100 rpm, Lab Tech, India). Further, the cells were treated with simulated intestinal juice (pancreatic 3 mg/ml; 1% bile salt; pH‐8) and incubated at 37°C for 4 h. After each treatment, the survival rate of probiotic yeast was calculated by pour plate method. The survival of free and encapsulated cells of S. fibuligera VIT‐MN04 was investigated.

2.8 Viability of probiotic yeast during storage

The encapsulated as well as free cells of S. fibuligera VIT‐MN04 were stored at 4 and 25°C for 56 days. The encapsulated and free yeast cells were inoculated on YEPD broth after storage period and the yeast colonies were counted.

2.9 Structural analysis of encapsulated S. fibuligera VIT‐MN04 nanofibres

The nanofibres were subjected to scanning electron microscopy (SEM) analysis (FEI Sirion, Eindhoven, the Netherlands). The known quantity of encapsulated nanofibres was dried for overnight and then fibres were mounted onto the cover slip at an acceleration voltage of 15 kV under different magnifications.

The surface topology of the nanofibre was predicted by atomic force microscopy (AFM) analysis. A thin probiotic fibre was prepared on the glass slide. The analysis was performed at 25°C with resonance frequencies of V250–300 kHz and typical scan speeds of 0.3–0.7 Hz.

2.10 Characterisation of encapsulated S. fibuligera VIT‐MN04 nanofibre

The crystalline nature of nanofibre was determined by X‐ray diffraction (XRD) analysis (Bruker D8 diffractometer). Ni‐filtered Cu K α radiation was employed to obtained XRD pattern in the 2θ range (20°–80°) at a scan rate of 1.0° min⁻¹ at 25°C. The stability of the nanofibre was analysed by thermogravimetric analysis (TGA) (SDTQ 600V20.9 Build 20) at a heating rate of 10°C/min in a nitrogen atmosphere. The zeta potential of the nanofibre suspensions (1%) was measured using Malvern Zetasizer Nano‐ZS90 (Malvern Instruments Ltd., UK). The samples were prepared by diluting the nanofibre suspensions (1%) into a concentration of 0.01% in purified water [23]. Further, the nanofibres were analysed by Fourier transform infrared spectroscopy (FT‐IR) analysis (Shimadzu, DR‐800). The nanofibres were mixed with KBr and pressed into transparent thin pellets. FT‐IR spectra of nanofibres were obtained in the range of 4000–400 cm⁻¹. Further, the nanofibres were analysed by gas chromatography–mass spectrometry (GC‐MS) spectrophotometer (JEOL GC MATEII). Nanofibres (2 mg) were dissolved in an equal amount of BSTFA and pyridine with double amount of acetone. The suspension was vortexed for 10 s and incubated at 70°C for 20 min and chloroform was added to this mixture. Helium (1 ml/min) was used as the carrier gas. The injector and detector were kept at 250 and 200°C, respectively [24]. Nuclear magnetic resonance (NMR) analysis was done using ¹³ C NMR spectrometer (Bruker Advance II 500 spectrometer, Bruker Co., MA) to predict their chemical structures. The known quantity of nanofibres was mixed with 99.96% D₂ O. ¹³ CNMR spectrum was obtained at a probe temperature of 25°C. The spectrum showed resonance signals (δ) which were reported in parts per million [25].

2.11 Statistical analysis

All experiments were conducted in triplicate and average values were calculated for individual studies. Analysis of variance (ANOVA) was used to compare the data using Design Expert 11 (Stat‐Ease Inc., USA) statistical software.

3 Results and discussion

The schematic representation of this study is shown in Fig. 1.

Fig. 1.

Nanoencapsulation of probiotic yeast S. fibuligera VIT‐MN04

3.1 Process optimisation for nanofibre production using RSM

The fine nanofibre production was achieved by the optimised parameters WBF (10%), EPS (15%), PVP (5%), applied voltage (10 kV) and tip to collector distance (15 cm). The experimental design and obtained results from design expert software are given in Table S1. The factors were optimised by BBD with five central points, for EE (Response 1) and GIT tolerance (Response 2) (see Fig. 2). A, B, C, D, and E are coded terms for the five test variables, i.e. WBF, EPS, PVP, applied voltage, and tip to collector distance, respectively. The responses of predicted and experimental values were computed by means of ANOVA to check whether the polynomial expression is able to predict the responses statistically.

Fig. 2.

Response surface plots showing the effects of variables on production of nanoencapsulated S. fibuligera VIT‐MN04

(a) Encapsulation efficiency (%), (b) GIT tolerance (%)

The ANOVA for the quadratic model of response 1 (encapsulation efficiency) was studied and the second‐order polynomial equation (2) is given as follows:

$$\begin{array}{rl}& \mathrm{Response}1-\mathrm{Encapsulation}\mathrm{efficiency}(\text{\%})\\ & =99.87+0.6212\times A+0.8281\times B-0.8619\times C\\ & -1.01\times D+0.3206\times E+0.5225\times AB+0.465\times AC-0.0325\times AD\\ & +0.78\times AE+1.53\times BC-0.1925\times BD-1.74\times BE\\ & +1.44\times CD+1\times CE+0.305\times DE-2.45\times A^2\\ & -1.06\times B^2-1.68\times C^2-2.9\times D^2-2.19\times E^2\end{array},$$ (2)

where R 1 is the response 1 representing the EE (%) and 3D plots for most significant interactions were illustrated in Fig. 2 a. The results indicated a positive effect on S. fibuligera VIT‐MN04 encapsulated in nanofibre either with independent factor or combination of two factors. In this case, A, B, C, D, E, AB, AC, AE, BC, BE, CD, CE, A ², B ², C ², D ², E ² are significant model terms. All the variables exhibited significant influence on EE (%) Interactive effect of variables, BC (EPS and PVP), BE (EPS and tip to collector distance), CD (PVP and Applied voltage) and CE (PVP and tip to collector distance) showed a significant impact on EE (%) compared to other interactions. The model was statistically fit and significant data is given in Table 2.

Table 2.

ANOVA analysis for encapsulation efficiency (%) – Response 1

Source	Sum of squares	df	Mean square	F ‐value	p ‐value
model	198.00	20	9.90	52.66	0.0001a
A ‐WBF	6.18	1	6.18	32.85	0.0001a
B ‐EPS	10.97	1	10.97	58.36	0.0001a
C ‐PVP	11.89	1	11.89	63.22	0.0001a
D ‐applied voltage	16.18	1	16.18	86.07	0.0001a
E ‐tip to collector distance	1.64	1	1.64	8.75	0.0067a

^a Significant value.

The ANOVA for the quadratic model of response 2 (GIT tolerance) was studied and the second‐order polynomial equation (3) is given as follows:

$$\begin{array}{rl}& \mathrm{Response}2-\mathrm{GIT}\mathrm{tolerance}(\text{\%})\\ & =93.86+1.12\times A+077531\times B-1.05\times C-1.22\times D+0.4531\times E\\ & +0.4675\times AB-0.9425\times AC-0.3425\times AD+0.6675\times AE\\ & +1.5\times BC+0\times BD-1.66\times BE+1.25\times CD+0.885\times CE+0.305\times DE\\ & -2.85\times A^2-2.04\times B^2-2.9\times C^2-3.78\times D^2-3.27\times E^2\end{array},$$ (3)

where R 2 is the response 2 representing the GIT tolerance (%) and three dimensional plots indicated positive effect on S. fibuligera VIT‐MN04 encapsulated in nanofibre either with independent factor or combination of two factors (see Fig. 2 b). In this case, A, B, C, D, E, AB, AC, AE, BC, BE, CD, CE, A ², B ², C ², D ², E ² are significant model terms. The significant influence on GIT tolerance (%) was noted in the case of all the variables. The interactions AC (WBF and PVP), BC (EPS and PVP), BE (EPS and tip to collector distance), CD (PVP and Applied voltage) and CE (PVP and tip to collector distance) were found to show the significant impact on GIT tolerance (%) compared to other interactions. The model was statistically fit and significant data is given in Table 3. The actual and predicted values were highly comparable for all the responses and the normal plots of residuals compared with predicted versus actual values for each response were illustrated in Fig. 3.

Table 3.

ANOVA analysis for GIT tolerance (%) – Response 2

Source	Sum of Squares	df	Mean Square	F‐value	p‐value
model	313.75	20	15.69	119.75	0.0001a
A ‐WBF	19.98	1	19.98	152.53	0.0001a
B ‐EPS	9.08	1	9.08	69.28	0.0001a
C ‐PVP	17.58	1	17.58	134.18	0.0001a
D ‐applied voltage	23.99	1	23.99	183.10	0.0001a
E ‐tip to collector distance	3.29	1	3.29	25.08	0.0001a

^a Significant value.

Fig. 3.

Normal residual plots and predicted versus actual plots for responses

(a) , (c) Encapsulation efficiency (%), (b) , (d) GIT tolerance (%)

Optimisation results indicated that interactions of BC, BE, CD, and CE were found to be significant in both the responses (R 1 and R 2) which indicate that EPS along with PVP at optimised voltage and tip to collector distance could improve the fibre EE to protect the nanoencapsulated cells. These results suggested that change in fibre diameter occurred due to applied voltage because the higher voltage increased the fibre diameter [26].

The interaction of AC (WBF and PVP) was found to be significant only in the case of R 2 (GIT tolerance %). This result indicates that the interaction of WBF along with PVP at the optimised applied voltage and tip to collector distance could protect the probiotic yeast cells effectively. The hydrogen bonding in PVP allowed gentle cell encapsulation and increased the cell viability (79%) which was already reported in encapsulated Saccharomyces. cerevisiae yeast cells. The carbonyl groups of PVP could preserve the cell integrity and functional properties of encapsulated yeast cells [27].

The uniform nanofibres were obtained at the optimised tip to collector distance because the morphology of nanofibres was affected by the distance due to deposition and evaporation time of polymer solution [28]. The increased applied voltage and the tip to collector distance yielded nanofibres with a different mean diameter ranging from 250 to 300 nm. These results indicate a non‐linear relationship between the applied voltage and fibre diameter which was influenced by tip to collector distance [29].

3.2 Encapsulation efficiency

The composition of spinning solutions significantly affected the EE. Probiotic yeast S. fibuligera VIT‐MN04 encapsulated in nanofibres revealed that EPS and PVP showed a significant effect on EE. Moreover, applied voltage highly influenced the encapsulation process resulting enhanced nanofibre production showing 99–100% yeast cell viability ensuring the protection of probiotic yeast S. fibuligera VIT‐MN04 encapsulated in nanofibres. Whereas microencapsulated S. fibuligera VIT‐MN04 exhibited 92% cell viability using oat bran gum was reported in our previous study [30]. Therefore, the nanoencapsulation of probiotic yeast showed greater protection to the yeast cells compared to microencapsulation. There was a report on probiotic bacteria Lactobacillus rhamnosus encapsulated in nanofibres using sodium alginate and poly vinyl alcohol (PVA) which exhibited 83% viability after electrospinning [31].

3.3 Survival of probiotic yeast during in vitro digestion

The probiotic yeast cells (free and nanoencapsulated) were tested for their tolerance under simulated gastrointestinal conditions (in vitro digestion). The viable yeast cell counts before and after gastric juice digestion followed by intestinal juice digestion was illustrated in Fig. 4 a. The loss of viability in non‐encapsulated (free cells) was found to be more in intestinal phase due to cell disruption by digestive enzymes compared to gastric phase due to the acidic condition (pH‐2). The survival of S. fibuligera VIT‐MN04 encapsulated in nanofibres was greater compared to non‐encapsulated cells. The survival of encapsulated probiotic yeast was found to be 6.23 CFU/ml after simulated gastric condition and 6.15 CFU/ml after simulated intestinal condition. In case of free cells, the viable count was 5.96 CFU/ml after simulated gastric condition and 5.22 CFU/ml after simulated intestinal condition. The viability of nanoencapsulated cells was found to be 97% whereas free cells showed 82% viability during in vitro digestion.

Fig. 4.

Evaluation of nanoencapsulated S. fibuligera VIT‐MN04 nanofibre

(a) Survival during in vitro digestion, (b) Viability during 56 days of storage

Fung et al. [13] reported that the probiotic bacteria Lactobacillus acidophilus encapsulated in nanofibres obtained from agro‐waste materials showed improved GIT tolerance (90%). Other workers also reported that probiotic bacteria Lactobacillus plantarum (L. plantarum) encapsulated with fructooligosaccharides (FOS) showed improved viability [32]. Mathew et al. [33] reported a decrease in viability in the case of L. plantarum 423 encapsulated in nanofibres using PVA. Therefore, these findings indicate that nanoencapsulated S. fibuligera VIT‐MN04 got better protection compared to the nanoencapsulated bacterial cells reported so far.

3.4 Viability of nanoencapsulated probiotic yeast during storage

The viability of nanoencapsulated yeast cells along with free cells was investigated at 4 and 25°C (see Fig. 4 b). The loss of viability was found to be minimum at 4°C compared to 25°C in case of nanoencapsulated cells. The free cells exhibited significant loss of viability compared to the nanoencapsulated cells at both the temperatures. Thus, the probiotic yeast S. fibuligera VIT‐MN04 encapsulated in nanofibres could retain the viability of better protection after 56 days of storage at 4°C compared to free cells.

Similar results were reported by other workers. The probiotic bacteria Bifidobacterium animalis ssp. lactis Bb12 was microencapsulated with inulin which showed better protection at 4°C compared to 25°C [34]. The Saccharomyces cerevisiae encapsulated in microfibres with polyethylene glycol showed stable viability up to 7 days at room temperature [35]. The probiotic bacteria Lactobacillus plantarum encapsulated with pectin and rice bran was reported to retain improved viability after freeze drying [36]. Therefore, the results of this work confirmed that encapsulating materials viz. WBF, EPS along with PVP served as efficient matrices for effective nanoencapsulation of probiotic yeast which could be stored for longer period of time.

3.5 Structural characterisation

Nanofibres produced from the blends of WBF, EPS and PVP containing probiotic yeast S. fibuligera VIT‐MN04 were found to have a regular texture, smooth surface with uniform size and consistent diameters. The size of the nanofibres ranged from 200 to 400 nm which was determined by SEM analysis (see Fig. 5 a).

Fig. 5.

Structural analysis of nanoencapsulated S. fibuligera VIT‐MN04 nanofibre

(a) SEM, (b) AFM analysis

The surface topology of encapsulated probiotic yeast S. fibuligera VIT‐MN04 was investigated by AFM analysis with nanoscale resolution. The surface of encapsulated cells showed smooth inclusions as illustrated in Fig. 5 b. This result suggested that electrospinning at 10 kV considerably improved the cohesive interactions between encapsulating materials and probiotic yeast.

3.6 Characterisation of nanoencapsulated probiotic yeast

XRD analysis was done to predict the crystallinity of nanofibre (see Fig. 6 a). The nanofibre exhibited two peaks at 2θ  = 13° and 30°, which indicate that nanofibre was partially crystalline and crystallinity was found to be 50.23%. The average crystalline size was calculated using the Scherer equation which was found to be 16.94 nm.

Fig. 6.

Characterisation of nanoencapsulated S. fibuligera VIT‐MN04 nanofibre

(a) XRD, (b) TGA, (c) Zeta potential, (d) FTIR analysis

TGA analysis was done and the thermogram revealed that the nanofibres were degraded mainly in two step processes (see Fig. 6 b). The results showed degradation at 150°C (8.36%) and 200°C (71.17%) which indicated that nanofibres were considerably thermostable.

The zeta potential of nanofibres was found to be negative −98 mV (see Fig. 6 c). The negative charge represented the presence of carboxyl anion (–COO–) groups on nanofibres, which could improve mucus permeation [23]. FT‐IR analysis revealed the presence of the following functional groups on nanofibres (see Fig. 6 d). The broad absorption at 3315 cm⁻¹ corresponds to –OH stretching indicated the presence of polysaccharide compounds [37] which revealed the presence of EPS. Another band at 2972 cm⁻¹ representing C–H stretching confirmed the presence of PVP. Five bands at 1660, 1440, 1290, 1087 and 1045 cm⁻¹ confirmed the presence of cellulose material due to the presence of WBF. The β‐glycosidic bond observed at 879 cm⁻¹ indicated the presence of cellulose [38].

The chromatogram of GC‐MS analysis showed four significant peaks which represent polycosanols at a retention time of 12.43 min (docosanol), 19.15 min (tetracosanol), 21.03 min (pentacosanol), and 25.84 min (heptacosanol) on nanofibres as shown in Fig. 7. The results confirmed the presence of WBF in the nanofibre composition, which could help in blood circulation and cholesterol‐lowering in the host. Similar retention time and ion fragment patterns of the peaks were reported by Irmak et al. [39].

Fig. 7.

GC‐MS analysis for nanoencapsulated S. fibuligera VIT‐MN04 nanofibre

NMR analysis was done and the monomeric composition of nanofibres was determined by ¹³ C spectrum (see Fig. 8). The anomeric region 16–113 ppm showed six signals which confirmed the presence of aliphatic groups (C–H). The ¹³ C spectrum of nanofibre showed four signals at δ 77 ppm, which indicated the presence of the cellulose skeleton. The side chain of methylene carbons was observed at δ 18 ppm, δ 31 ppm and δ 175 ppm indicating the presence of PVP and carbonyl (CO) groups. A similar pattern was noted by Stephen et al. [40].

Fig. 8.

NMR analysis for nanoencapsulated S. fibuligera VIT‐MN04 nanofibre

4 Conclusion

The probiotic yeast was successfully encapsulated in nanofibres obtained from the blending of WBF, EPS and PVP. The RSM was used to study the individual and interactive effects of encapsulation materials and electrospinning parameters on EE (%) and GIT tolerance (%). The nanoencapsulated S. fibuligera VIT‐MN04 showed 15% increased survival compared to free yeast cells after in vitro digestion and retained viability for 56 days of storage at 4°C. The presence of polysaccharides and cellulose in nanofibres could serve as prebiotic to improve the efficiency of nanoencapsulated probiotic yeast in the gut region.