1. Introduction
Probiotic bacteria (defined as: “living microorganisms which exert beneficial effect on the host health when consumed in adequate amounts”) are intensively studied worldwide [
1].
Lacticaseibacillus rhamnosus GG (LGG) is a Gram-positive Lactic Acid Bacterium (LAB), and one of the best-studied probiotics in clinical trials. This strain exhibits most of the features required for probiotics, and has been shown to be safe and non-pathogenic [
1,
2,
3]. It has been previously reported that LGG exerts beneficial effects in treating and/or preventing several disorders, including ulcerative colitis, diarrhea, atopic dermatitis, rotavirus infections, sepsis and meningitis [
2,
3,
4,
5]. The competition with pathogens for binding sites and production of antimicrobial compounds are the main mechanisms suggested to contribute to the probiotic action of LGG in the gastrointestinal tract (GIT) [
2,
3,
4,
6]. One of the LGG characteristics responsible for its health-benefit properties is resisting low pH levels, the ability to adhere mucus and epithelial cells as well as prolonged residence in GIT [
1,
3,
4]. Probiotic microorganisms should be present in the product at minimum numbers of 10
6–10
7 CFU (Colony Forming Units) per mL or g of product [
7,
8]. The technological issues related to the development of products containing beneficial microflora at recommended levels, maintaining their viability during shelf life and stabilization throughout the GIT are a challenge [
9].
Spray drying is a fast and cost-effective technique to produce powders (solid microparticles) from starting liquid raw materials, being one of the promising processes to produce dry, probiotic formulations, as well as a strategy to protect and improve their viability within the GIT [
9,
10,
11,
12,
13,
14]. Moreover, spray drying is one of the encapsulation methods, which refers to a process where the active ingredients or cells are surrounded (encapsulated) by a protective continuous film of polymeric materials [
15]. Polysaccharides and proteins are widely used to prepare carriers/delivery systems, playing a pivotal role in their structure and stability [
16,
17,
18]. Many natural-based wall/carrier materials have been proposed for improving LAB (including probiotic strains) survivability during spray drying and passage through the GIT [
18,
19,
20]. Various carriers such as skim milk and calcium-fortified skim milk [
11,
21], whey proteins [
22], maltodextrin [
1], native rice starch and inulin [
23], fructooligosaccharides [
14], agava fructans and buttermilk proteins [
24], trehalose [
25], polysaccharides (alginate, carrageenan, pectin, xantan, gellan) [
1], vegetable juices [
26] as well as almond milk [
10] can be considered a promising strategy to improve stability and viability of probiotics. However, there are very limited information about maintaining probiotic properties of LAB after spray drying [
27]. An innovative idea is to obtain reconstituted plant water extracts (also known as plant milks) and was already reported in for spray-dried reconstituted soymilk and almond-based milk [
10,
28]. Reconstitution (as a result of rehydration by adding water) has been known for decades in the case of spray-dried animal (bovine, camel) milks [
29]. Moreover, due to the increase in vegan, vegetarian and allergic consumers, food and pharmaceutical industries are currently searching for alternatives in plant-based carriers. Indeed, plant-based products are raising interest as innovative potential carriers ensuring probiotics for vegan consumers [
8,
30,
31,
32]. Therefore, sourcing biopolymers such as plant-derived proteins and polysaccharides is of great importance for the development of innovative functional products, such as new probiotic carrier matrices [
16,
18,
33].
Plant by-products (such as pomace, seeds, kernels, stalks, oilseed cakes and meals) are produced in large quantities worldwide, and have great potential to be used to provide phytochemicals and high value-added biopolymers [
18]. The use of new types of biopolymers is important to the green label trend—the term green refers to origin, which involves waste and by-products valorization [
12,
18]. Oil cake/oil meal is a by-product of the extraction of oil from seeds [
34,
35]. These residues are an abundant source of compounds (antioxidants and biopolymers such as proteins, polysaccharides, fibers) with beneficial properties that can be used in many fields. Another advantage is the economic aspect: oil cakes are a cheap, safe material available all year round. The use of oil cakes can be a sustainable alternative to reduce waste and also contributes to the development of new low-cost products [
34]. Flaxseed oil cake (FOC) is an inexpensive by-product of pressing flaxseed (
Linum usitatissimum L.) oil and is a source of many bioactive substances such as proteins, polysaccharides, fiber and polyphenols. Flaxseed global production is estimated to be more than 1.2 million tons [
36], thus generating large amounts of FOC. The valorization of FOCE (flaxseed oil cake extract) is a relatively new issue. FOCE is a liquid mixture obtained by hot extraction from FOC, the main components of which are primarily protein (FP—flaxseed protein) and mucilage (also known, as flaxseed gum—FG), abundant also in antioxidants [
13,
35,
37]. Some several technological applications of FOC and FOCE are reported, including spray-dried emulsions and powders with emulsifying activity [
12,
13,
37], and as dairy alternatives [
38,
39,
40] which demonstrates the potential of this valuable agro-industrial by-product in the concept of circular economy and sustainable development. A feature of FG is the formation of a multiform structure and increase the resistance of multiphase systems to environmental stresses, thus FG can be used as a thickener, stabilizer, gelling agent and emulsifier [
17,
35,
41]. Moreover, flaxseed mucilage showed good prebiotic potential, that can protect LAB cells from the adverse gastric environment and digestion [
15,
42]. In fact, the application of prebiotics (that could be used by bacteria for survival through the GIT) for encapsulation purposes has shown many advantages [
23,
43]. On the other hand, FP has good film-forming properties that can participate in encapsulation processes [
44,
45]. Previous works have shown that FOCE (used as liquid matrix or spray-dried powders) can be used as a stabilizing agent due to the synergistic effect of FP and FG [
13,
35,
37]. Moreover, flaxseed polysaccharides have mucoadherent properties, and could be used in therapeutic or cosmetic applications [
46]. Mucoadhesive polymers have been found to facilitate probiotic adhesion in the GIT, therefore are good candidates as encapsulating polymers for probiotic delivery systems [
47]. Several authors reported the application of flaxseed mucilage as encapsulating material for LAB and their good survivability after spray drying and during incubation in simulated gastrointestinal conditions [
42,
48,
49]. There are some reports indicating that the high efficiency of microencapsulation could be achieved by synergistic effect of the wall polymers (charged polysaccharides and amphiphilic proteins) through electrostatic interactions [
15,
17,
20,
21,
41]. Therefore, FOCE presumably could act as an effective encapsulating agent and carrier for LAB, due to its biopolymers (proteins and polysaccharides) content.
The objective of this research was to valorize spray-dried FOCE as a natural carrier for probiotic LGG. Particularly, this study was designed to evaluate the influence of the spray drying process on powders properties as well as viability and probiotic properties of LGG in initial and reconstituted FOCE under simulated conditions of the GIT.
2. Materials and Methods
2.1. Materials and Chemicals
Flaxseed oil cake (FOC) was purchased from ACS Sp. z o.o. (Bydgoszcz, Poland). The proximate composition of FOC (based on supplier information) was: solids—80.50%, including: proteins—41.97%; carbohydrates—27.99%; fiber—6.29%; fat—6.11%; ash—4.50%. Lacticaseibacillus rhamnosus GG (ATCC53103), was procured from ATCC (Manassas, VA, USA). Buffered peptone water, microbiological agar, MRS agar and broth were purchased from Oxoid (Basingstoke, UK). Potassium chloride, sodium chloride, potassium thiocyanate, disodium hydrogen phosphate, monosodium dihydrogen orthophosphate, calcium chloride, sodium hydrogen carbonate, hydrochloric acid, ammonium chloride, sodium peroxide, urea, α-amylase, uric acid, mucin from porcine stomach (type II), glucose, glucuronic acid, glucosamine hydrochloride, bovine serum albumin (BSA), pepsin, pancreatin, oxgall, Triton X-100, cholesterol, ethanol, ninhydrin, glacial acetic acid, cadmium chloride and hexadecane were purchased from Merck Chemical (Saint Louis, MI, USA). All reagents were of analytical grade.
2.2. Preparation, Fermentation and Spray Drying of Flaxseed Oil Cake Extract with LGG (FOCE-LGG)
The preparation and fermentation of FOCE-LGG was carried out as described in previous study [
40]. Briefly, FOC was extracted with hot distilled water (1:10
w/
w, 90 °C, 1 h, 250 rpm), then cooled down to 20 °C, centrifuged (4000 rpm, 30 min) to obtain FOCE. Subsequently FOCE was filtered, homogenized (12000 rpm, SilentCrusherM, Heidolph, Germany), and fermented by LGG (42 °C, 24 h). Powdered FOCE-LGG samples were obtained using a laboratory scale spray dryer (Büchi B-290, Büchi Labortechnik AGT, Flawill, Switzerland). Three inlet temperatures were used: 110 °C, 140 °C and 170 °C. The outlet temperature was maintained at 55 ± 5 °C, and the air flow was 40 m
3/h. The powders were collected in a sterile glass collection vessel. Total solids content (TSC) of non-dried FOCE and powders (denoted as FOCE-LGG-110, FOCE-LGG-140 and FOCE-LGG-170) was evaluated following the standard method (no. 968.11) of AOAC (Association of Official Agricultural Chemists) [
50].
2.3. Powders Characterization
The powders were characterized for water activity (a
w, MS1 Set-aw, Novasina, Lachen, Switzerland), morphology (SEM microscopy, Vega 3 LMU, Tescan, Brno, Czech Republic), chemical composition (FTIR spectroscopy, Perkin Elmer Spectrophotometer 100, Waltham, MA, USA), and particles size distribution (Mastersizer 2000 with a Scirocco 2000 dry sampling system, Malvern Instrument Ltd., Worcestershire, UK) as described in previous studies [
12,
13,
37]. Moreover, bulk (ρ
b) and tapped (ρ
t) densities were determined as described elsewhere [
13], using the following formulas:
The Carr’s index and Hausner ratio were used to express flowability and cohesiveness of the powders [
13]. The indexes were calculated from the ρ
b and ρ
t values, based on the following equations:
Whereas for powders evaluation a scale based on European Pharmacopoeia standards (
Table 1) was used [
51].
2.4. FOCE-LGG Reconstitution, Determination of pH, Free Amino Acids, Sulfhydryl Groups (-SH) and Disulfide Bonds (-S-S-) Contents
The reconstitution of the FOCE-LGG samples was carried out by adding the individual powders to distilled water to obtain the initial dry matter content of the starting samples (taking into account the total solids content of the powders), and then stirred until a homogeneous (37 °C, 20 min, 50 rpm) [
13]. pH measurements of the samples were carried out directly at 25 °C using a pH-meter (CP-411, Elmetron, Zabrze, Poland). Free amino acids were determined using ninhydrin-Cd reagent as described elsewhere [
39]. The sulfydryl groups (-SH) and the disulfide bonds (-S-S-) contents were analyzed according to the protocol described by Gong et al. [
52].
2.5. Enumeration of LGG Counts
The LGG counts in FOCE-LGG, reconstitution and at each stage of the GIT (mouth, stomach and small intestine) was determined in compliance with ISO 6887-1:2017. The samples (1 mL) were diluted with 9 mL of sterile buffered peptone water, and further ten-fold serial dilutions were prepared. LGG counts were determined on MRS agar after incubation at 37 °C under anaerobic conditions for 72 h. The enumeration of all microorganisms was performed in triplicate (by counting plates with 30–300 colonies) and the viable cell counts were expressed as log CFU/mL of the samples [
40].
2.6. Gastrointestinal Tract Simulator (GITS)
The Simulator of Human Intestinal Microbial Ecosystem (SHIME) was adopted to assess the behavior of selected features of the LGG administered in the tested products. The model consisted of three bioreactors simulating the processes taking place in selected sections of the digestive system—mouth (Reactor 1), stomach (Reactor 2) and small intestine (Reactor 3), completed with the mucin adherence test (ascending colon). Control of filling and intake of ingredients at specified times was carried out using peristaltic pumps with a total retention time of 6 h. The dwell time in individual reactors did not exceed 2.5 h and was dependent on the volume [
53,
54]. The content of each bioreactor was magnetically stirred (33 rpm, IKA, Staufen, Germany) and maintained at a temperature of 37 °C ± 1 °C. Cultures were incubated in anaerobic conditions modified with the composition of N
2 and CO
2 [
55]. The pH and temperature values were monitored electronically (Adwa, Szeged, Hungary).
2.6.1. Simulated Digestion
Saliva Preparation
The saliva medium was prepared according to Oomen et al. [
56] and consisted of: 10 mL of 8.96% KCl, 10 mL of 2.0% KSCN, 10 mL of 5.7% Na
2PO
4, 1.7 mL of 17.53% NaCl, 1.8 mL of 4.0% NaOH, 8 mL of 2.5% urea, 145 mg α-amylase, 15 mg uric acid and 50 mg mucin filled up to 500 mL with distilled water. The pH was adjusted to 7.0 ± 0.2 with 0.1 M NaOH.
Gastric Medium Preparation
Gastric medium was prepared based on protocol of Oomen et al. [
56] with some modifications. The electrolyte solution consisted of: 15.7 mL of 17.53% NaCl, 3.0 mL 8.88% NaH
2PO
4, 9.2 mL of 8.96% KCl, 18 mL of 2.22% CaCl
2, 10 mL of 3.06% NH
4Cl, 10 mL of 6.5% glucose, 10 mL of 0.2% glucuronic acid, 3.4 mL of 2.5% urea, 10 mL of 3.3% glucosamine hydrochloride and organic solution: 1.0 g BSA, 1.0 g pepsin, 3.0 g mucin. The mixture was filled up with 500 mL distilled water. The pH value of the gastric juice was adjusted to 3.0 ± 0.2 by 0.1 M HCl.
Intestinal Juice Preparation
Intestinal juice was prepared as described by Bondue et al. [
57] by mixing in 0.9 g of pancreatin, 4.0 g of ox-gall, 2.5 g NaHCO
3 and filled the mixture to 1000 mL with distilled water. Sodium hydroxide solution (0.1 M) was used to adjust to pH 6.2 ± 0.5.
Mucin Agar Preparation
Mucin agar was prepared by boiling distilled water and 1% agar, when the mixture was cooled to 65 °C, 5% mucin from porcine stomach (type II) was added. The pH was adjusted to 6.8 with 0.1 M NaOH [
58,
59].
2.6.2. Characterization of Survival in GITS
The samples were mixed with saliva solution in a ratio of 10:1 (v/v). Incubation was carried out for 10 min with constant stirring (50 rpm) (Reactor 1). Subsequently, 50 mL of mixtures (2 mL/min) were transferred to the prepared gastric medium (Reactor 2), to obtain the final ratio 5:8 (v/v), and pH 3.0 ± 0.2 (maintained by using 0.1 M HCl). This mixture was incubated for 2 h, then transferred (2.0 mL/min) to the next Reactor 3 (intestinal juice). The volume transfer was calculated to give a final ratio of 5:6 (v/v). The pH was adjusted with 0.1 M NaOH, followed by incubation for another 2 h. The final step in controlling LGG in GITS was to evaluate the ability to colonize mucin as the active layer of the gut. For this purpose, 1 mL of the mixture was taken from Reactor 3 and added to a 12-well plate covered with 1.2 mL of mucin agar. Incubation was carried out at 37 °C with constant agitation (30 rpm). After 80 min, unadhered bacteria were removed by rinsing three times with PBS. LGG cells remaining on mucin agar were separated from the medium using 0.5% Triton X-100 in PBS. The total number of adhered cells (in triplicate) was evaluated by the plate method described above. The obtained bacterial counts were converted in accordance to counts resulted from Reactor 3.
The survival of bacteria (SUR) LGG in GITS (for saliva, gastric juice and intestinal juice) was calculated according to Sumeri et al. [
54]:
where: CFU
t stands for instantaneous concentration of bacteria (CFU/mL), V
t is the volume of culture (mL) in the bioreactor vessel at time point t, CFU
F is the concentration of bacteria in FOCE-LGG variants (CFU/mL), and V
F is the volume of FOCE-LGG variants (mL) injected into the vessel.
2.7. Determination of Probiotic Properties of LGG on GITS Stages
2.7.1. Cholesterol Binding Activity
Cholesterol binding (CH
b) activity was determined on the basis of its loss after incubation with FOCE-LGG and FOCE-LGG-110, FOCE-LGG -140 and FOCE-LGG -170 samples obtained after passage through SHIME. Samples taken from Reactor 3 as well as from “fresh” FOCE-LGG were loaded into 12-well plates with 1% cholesterol (dissolved in 96% ethanol) to obtain a final cholesterol concentration of 1.66 g/L. The samples were incubated for 18 h (30 °C), then centrifuged (Centrifuge MPW 351R, 7500 rpm, 10 min, 4 °C) to separate the culture fluid. In order to determine the cholesterol residues in the samples, the procedure of Cholesterol RTU kit (BioMerieux, Marcy l’Etoile, France) was followed. The absorbance measurement was performed using the NanoDrop ND 1000 spectrophotometer at 500 nm. The percentage of bound cholesterol from the environment was calculated according to the formula:
where: A is the initial absorbance of mixture, B is the absorbance of mixture after inxubation.
2.7.2. Surface Hydrophobicity Assay
The BATH (Bacterial Adhesion to Hydrocarbons) method with hexadecane was used to assess hydrophobicity of LGG cells following procedure of Rahman et al. [
60] with some changes. Five milliliters of bacterial suspensions were mixed with 2 mL of hexadecane by vortexing for 60 s, then incubated for 2 h (37 °C). Changes of absorbance were measured at 620 nm using a spectrophotometer (BioPhotometer D30, Eppendorf). The hydrophobicity (SH%) was expressed according to formula:
where: A
0 is the initial OD at 620 nm and A is the final OD at 620 nm.
It was assumed that result: SH > 70% indicates high hydrophobicity; SH 20–70% indicates medium hydrophobicity, whereas SH < 20% indicates low hydrophobicity.
2.8. Statistical Analysis
All experiments were carried out three times. Results are expressed as mean ± standard deviation. One-way and two-way ANOVA with Tukey’s tests were conducted using the Statistica 13.0 software (StatSoft, Kraków, Poland), and p values < 0.05 are considered to be statistically significant.