1. Introduction
Probiotics are living microorganisms that provide health benefits to their host (or consumer) by maintaining the gut microbiome balance. Their benefits may include intestinal health improvement, immune response enhancement, and better serum cholesterol control [
1]. Nowadays, probiotics are commercially available as dietary supplements (e.g., probiotic capsules and tablets [
2]) and functional foods (e.g., yogurt [
3], cheese [
4], cereal [
5], chocolate [
6], and fruit juice [
7]). However, to deliver their health benefits, a sufficient cell concentration must be administered in the food products. In general, it is recommended to incorporate 6–7 log probiotic cells per mL or gram product [
1,
8,
9,
10], and a total of 9–10 log probiotic cells should be consumed daily [
1,
11].
Pediococcus acidilactici are homofermentative gram-positive lactic acid bacteria known to have a great capacity to survive the harsh environment in the animal and human digestive systems (e.g., acidic pH, pepsin, and bile salt), enabling gut colonization [
12,
13]. Over the past decades, it has considerably gained research interest due to its potent probiotic characteristics, such as good antimicrobial activity [
14,
15], good adherence to intestinal cells [
14,
15], and the capability to produce useful metabolites such as bacteriocin and gamma-aminobutyric acid [
16,
17]. In addition, it has wide applications in the food industry, ranging from fish feed supplementation [
18,
19], starter culture for traditional sausage production [
20], orange juice supplementation [
21,
22], and bio-preservative agents in food products [
23]. However, during processing, storage, and consumption in the gastrointestinal tract (GIT), the probiotics are exposed to several environmental stresses, including heat, desiccation, and low pH, which reduce their viability [
21,
24,
25,
26,
27]. The probiotics’ survival against such environmental stress could be improved by encapsulation [
24,
25].
Spray drying is a commonly used encapsulation technique [
28,
29,
30], as it is simple, fast, cost-effective, and scalable compared to other drying encapsulation methods [
31,
32]. In spray drying encapsulation, probiotics are added to the encapsulation material solution, known as feed solution, then atomized into small particles, exposed to hot air (150 °C to 250 °C), and transformed into powders [
33,
34]. The resulting powder will contain probiotics encapsulated within the coating matrix that protects the cells from upcoming environmental stress [
35]. However, spray drying exposes the probiotics to heat and desiccation stress, which could reduce the viable cell number [
31,
36,
37]. Hence, selecting appropriate parameters used in spray drying is important to maintain a high encapsulation efficiency [
38,
39,
40].
One of the critical parameters in spray drying is inlet air temperature, which refers to the pre-heated drying air entering the drying chamber [
41]. Choosing a suitable inlet air temperature is critical in obtaining good quality powder, such as low moisture content and water activity, which is required to prevent microbial growth or contamination and maintain product stability during storage [
38,
40]. Studies by Flores et al. [
39] and Ortega and Vandeker [
40] have shown that high inlet air temperature is favourable as it results in low moisture content and water activity. However, such high temperature also induces viable cell loss as it gives relatively high stress that could damage the cell wall, DNA, and RNA and disrupt their metabolic activity [
8,
40,
42].
Besides the inlet temperature, selecting an appropriate encapsulation material is also crucial to obtain a powder with high viable cell concentration. No single biopolymer can provide all the ideal criteria for encapsulation materials (e.g., edible, low-cost, idle in nature, and good physicochemical properties) [
43]. Hence, two or three materials are often used to obtain synergic properties in maintaining a high viable probiotic cell count [
44]. Whey protein isolate (WPI) and gum arabic (GA) have been shown to have good properties as spray drying encapsulation materials, as they are able to construct physically strong and stable matrices [
45]. Furthermore, their interactions demonstrate excellent interfacial activity and emulsifying properties that can protect the probiotic cells during spray drying [
46]. Another study demonstrates that WPI, in combination with GA, exhibited the highest probiotic survival during GIT simulation compared to WPI combined with other materials, such as locust bean gum and maltodextrin [
47]. Moreover, using response surface methodology, the use of WPI and GA was predicted to encapsulate
Lactobacillus acidophilus with a high encapsulation efficiency of 93.95% [
44].
As of now, the effect of inlet air temperature and the use of WPI and GA as spray drying encapsulation material for P. acidilactici has never been studied. Hence, this study investigated the effect of varying inlet temperature (120 °C, 150 °C and 170 °C) and WPI:GA ratio (1:1, 3:1, and 1:3) on P. acidilactici survivability during spray drying, storage, and GIT simulation. Additionally, the production yield and physicochemical properties of all samples were analysed, including moisture content, water activity, Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM).
2. Materials and Methods
2.1. Materials and Culture
Encapsulation materials were food grade, and the other materials for survival and physicochemical analysis were analytical grades. Whey protein isolate 90 (WPI) (JFO store, Jakarta, Indonesia) and gum arabic (GA) (ALMA Chemical, Demak, Indonesia) were purchased using local e-commerce in Indonesia. Microbiological growth media used were de Man-Rogosa-Sharpe (MRS) broth (MERCK, Darmstadt, Germany) and MRS agar (MERCK, Darmstadt, Germany). For GIT simulation, the MRS broth was supplemented with glucose (MERCK, Darmstadt, Germany), KH2PO4 (MERCK, Darmstadt, Germany), CaCl2 (MERCK, Darmstadt, Germany), and KCl (MERCK, Darmstadt, Germany). The rehydration media for spray-dried probiotic and an additional supplement to gastrointestinal simulation media was NaCl (Himedia, Mumbai, India). The pH of gastrointestinal simulation media was maintained by adding HCl (MERCK, Darmstadt, Germany) and NaOH (ROFA, Bandung, Indonesia). P. acidilactici culture was obtained from Universitas Gadjah Mada (UGM), Food and Nutrition Culture Collection. The bacterial identity was confirmed through gram-staining and 16S rRNA sequencing.
2.2. Culture Preparation for Spray Drying
P. acidilactici from MRS agar was subcultured twice in fresh 50 mL MRS broth, followed by incubation at 30 °C for 18 h until it entered the late log phase. Then, 50 mL culture (OD600 0.7 equal to 7–8 log CFU/mL) was harvested and washed twice with 25 mL 0.9% NaCl solution. Cell harvest and wash were done by centrifugation at 2438× g, 25 °C, for 15 min. Lastly, the culture was concentrated by removing the supernatant, and the pellet was resuspended in 10 mL 0.9% NaCl solution to get a higher cell concentration (9–10 log CFU/mL).
2.3. Viable Cell Counting
The viable cell counting was performed using the Miles and Misra method with adjustments [
48]. First, 100 µL culture or 1 g of spray-dried sample was transferred to 900 µL or 9 mL 0.9% NaCl solution, respectively, followed by serial dilution. Then three drops of 10 µL culture from each dilution were dropped onto the MRS agar and allowed to set. The agar plates were incubated at 30 °C for 36–48 h, and the number of colonies was calculated.
2.4. Spray Drying
Feed solution containing probiotic culture (1% v/v) and 20% w/v WPI-GA was prepared to be subjected to spray drying. The effect of inlet temperature was investigated by keeping the WPI-GA ratio fixed at 1:1, while varying the inlet temperature at 120 °C, 150 °C and 170 °C. To evaluate the effect of WPI:GA ratio, the inlet temperature was fixed at 150 °C, while WPI:GA ratio was varied to 1:3, 1:1, and 3:1.
After the culture was prepared (described in
Section 2.2), feed solutions containing encapsulation material were prepared. A total of 200 g WPI and GA were dissolved in 1 L mineral water and the solution was homogenized using a hand blender (Bamix Deluxe hand blender, Mettlen, Switzerland) for 5 min at maximum speed. Once homogenized, the 10 mL culture prepared in
Section 2.2 was added and the feed solution was homogenized for another 2 min. The spray drying was carried out using a pilot-scale spray dryer (LPG 5, Changzhou Huaihai Drying Equipment Co., Ltd., Changzhou, China). The solutions were fed into the chamber through a peristaltic pump at a constant flow rate of 15 rpm/min. Other parameters were fan speed at 45 Hz, atomisation at 250 Hz, and air hammer within 1 s every 20 s. Spray-dried samples were cooled down and further stored in a Ziplock plastic bag. The Ziplock bag was kept inside an aluminum bag, added with silica gel, and sealed with a heat sealer until further analysis.
2.5. Viability Loss
The viability loss was defined as a log reduction of viable cell concentration as described in Equation (1) [
37,
49,
50]. N0 is the viable cell concentration (log CFU/g) before spray drying and N
t is the viable cell concentration (log CFU/g) after spray drying. For gastrointestinal simulation, N
0 is the initial viable cell concentration (log CFU/g) before the simulation, and N
t is the viable concentration (log CFU/g) after the simulation.
2.6. Gastrointestinal Simulation
The survival during GIT simulation was studied to monitor viability loss during consumption. The method was adapted from a previous study with modifications [
44]. In general, spray-dried and free cells of
P. acidilactici (control) were sequentially exposed to simulated gastric juice (SGJ) for 2 h and simulated intestinal juice (SIJ) for 4 h. Firstly, the formulation for SGJ was prepared according to a study by Kulkarni et al. [
51]. Briefly, MRS broth were added with glucose (3.5 g/L), NaCl (2.05 g/L), KH
2PO
4 (0.60 g/L), CaCl
2 (0.11 g/L), and KCl (0.37 g/L), and then adjusted to pH 2.0 using 1 M HCl. Then, pepsin (from porcine stomach mucosa, Sigma Chemical Co., St. Louis, MO, USA) was added to the sterile SGJ stock by 13.3 mg/L. Sterile 25-mL Erlenmeyer flasks were prepared and filled with 9 mL of sterile SGJ. Into each 9 mL sterile SGJ solution, 1 g of spray-dried samples or 1 mL of free cells (control) were added, producing suspension that was homogenized using a vortex for 2 min. Viable cell counting was performed to obtain initial cell concentration before GIT simulation. Next, the suspensions were incubated in a shaker incubator (TOU-50N Orbital Shaker Incubator, MRC, Holon, Israel) for 2 h at 37 °C and 150 rpm for gastric juice simulation. Once the incubation in SGJ was done, the samples were neutralised with 1 M NaOH to pH 7, quenching the pepsin enzymatic reaction. Viable cell counting was performed to obtain viable cell concentration after SGJ simulation. Then, porcine bile (0.7%
w/
v; Sigma Chemical Co., St. Louis, MO, USA) was added to each flask and incubated at 37 °C and 150 rpm for 4 h for SIJ simulation. Once the incubation was complete, the last viable cell counting was conducted to obtain viable cell concentration after sequential SGJ and SIJ simulation.
2.7. Storage Test
P. acidilactici viability during storage was investigated to observe the viable cell reduction during storage at room temperature for 4 months. During the 1st month, the viable cell count of spray-dried
P. acidilactici was measured every week, and then the frequency was changed to once every month. Viable cell counting was done as described in
Section 2.3.
2.8. Physicochemical Analysis
The production yield was expressed in percentage, calculated using Equation (2). The mass of encapsulation material used in all samples was 200 g. After weighing, the powder was stored in a sealed aluminium foil bag until further analysis.
The moisture content analysis was adapted from a previous study [
52]. A gram of sample was heated at 105 °C for 5 min and the average moisture content was measured using a moisture analyser (Ohaus
® MB-45 moisture balances, Parsippany, NJ, USA). The water activity (a
w) was determined by Pawkit Water Activity Meter (Decagon, Pullman, WA, USA). Samples were poured into the water activity measurement cup, and the water activity meter analysed the moisture content for 5 min [
52,
53].
2.9. Data Analysis
All the experiments were performed in triplicates. Data from the experiment were analysed by GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego. Ca, USA,
www.graphad.com, accessed on 19 November 2022. The effects were considered significant at
p < 0.05. Results were presented as the mean ± standard deviation. Statistical analysis on encapsulation efficiency and physicochemical properties (excluding FT-IR and powder morphology) was performed by one-way analysis of variance (ANOVA). Meanwhile, statistical analysis on gastrointestinal simulation and storage test was performed using a two-way analysis of variance (ANOVA). Tukey’s honestly significant difference test (
p < 0.05) was conducted as a posteriori contrast after rejecting the null hypothesis.
4. Conclusions
This study investigates the effect of inlet air temperature (120 °C, 150 °C and 170 °C) and WPI:GA ratio (1:1, 3:1, 1:3) on spray-dried P. acidilactici survivability and physicochemical properties. The results show that increasing the inlet air temperature was favourable in terms of production yield, moisture content, and aw. However, increasing the inlet temperature to 170 °C could lead to a significant viability loss. Unlike the inlet air temperature, the effect of WPI:GA ratio on the survival of spray-dried cells, yield, moisture content, and aw was not apparent. Furthermore, increasing the inlet air temperature to 150 °C resulted in the lowest viable cell reduction (0.5 log cycle) after the GIT simulation. For samples containing various WPI:GA concentrations, the viable cell reduction after simulated GIT was significantly lower when a higher WPI proportion (WG11 and WG31) was used. According to the SEM analysis, samples with higher WPI content exhibited a smoother spherical surface, while those containing a higher GA content demonstrated more dents, wrinkled surfaces, and blow-holes. During storage at 25 °C, viable cell counts decreased to below 7 log CFU/g more rapidly as the inlet temperature increased, which correlates with the encapsulation efficiency. Meanwhile, varying WPI:GA ratios did not influence the survivability of spray-dried P. acidilactici during storage. FT-IR spectra indicated no chemical interaction between wall materials and P. acidilactici. This study confirms that selecting the appropriate inlet air temperature and wall material ratio is vital in obtaining P. acidilactici powder with good physicochemical properties, high encapsulation efficiency, and survivability against gastrointestinal and storage conditions. Future investigations could focus on the effect of spray drying parameters on P. acidilactici functionalities.