Egészségügy | Gyógynövények, gombák » Co-encapsulation of probiotic Lactobacillus acidophilus and Reishi medicinal mushroom (Ganoderma lingzhi) extract in moist calcium alginate beads

International Journal of Biological Macromolecules 192 (2021) 461–470 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elseviercom/locate/ijbiomac Co-encapsulation of probiotic Lactobacillus acidophilus and Reishi medicinal mushroom (Ganoderma lingzhi) extract in moist calcium alginate beads Iman Mirmazloum a, *, Márta Ladányi b, Mohammad Omran a, Viktor Papp c, Veli-Pekka Ronkainen d, Zsolt Pónya e, István Papp a, Erzsébet Némedi f, Attila Kiss g a Department of Plant Physiology and Plant Ecology, Institute of Agronomy, Hungarian University of Agriculture and Life Sciences, Budapest, Hungary Department of Applied Statistics, Institute of Mathematics and Basic Science, Hungarian University of Agriculture and Life Sciences, Budapest, Hungary Department of Botany, Institute of Agronomy, Hungarian University of Agriculture and Life Sciences, Budapest, Hungary d Biocenter Oulu, University of Oulu, Oulu,

Finland e Division of Applied Food Crop Production, Department of Agronomy, Institute of Agronomy, Kaposvár Campus, Hungarian University of Agricultural and Life Sciences, Kaposvár, Hungary f Expedit Nodum Ltd., Budapesti Str 106, Budapest, Hungary g Agro-Food Science Techtransfer and Innovation Centre, Faculty for Agro-, Food- and Environmental Science, Debrecen University, Debrecen, Hungary b c A R T I C L E I N F O A B S T R A C T Keywords: Prebiotic β-Glucan Calcium lactate CLMS Double layer coat Probiotic protection Probiotic L. acidophilus La-14 cells were co-encapsulated with Ganoderma lingzhi extract to prolong the viability of the cells under simulated gastrointestinal (SGI) condition and to protect the active ingredients of Reishi mushroom during the storage period. Combinations of distinctive reagents (sodium alginate, chitosan, maltose, Hydroxyethyl-cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and calcium lactate) were tested. Optimal double layer

Ca-alginate hydrogel beads were fabricated with significantly improved characteristics. The incorporation of maltose significantly decreases the release rate of mushrooms phenolics, antioxidants, and β-glucan during the storage time. Significant improvement in probiotic cells viability under SGI condition has been found and confirmed by confocal laser microscopy in maltose containing double layer coated calcium alginate beads variants. The encapsulation of newly formulated prebiotic Reishi extract and probiotic L. acidophilus is creating a new potential food application for such medicinal mushrooms and natural products with unpleasant taste upon oral consumption. 1. Introduction Synbiotic or functional food ingredients with proven health proper­ ties are gaining more and more attention in food industry. Synbiotic is a term referred to the food products to be comprised of both probiotics and prebiotics [1]. The global probiotic market is predicted to experi­ ence a considerable 7%

compound annual growth rate (CAGR) by 2023 (https://www.industryarccom/Report/18555/ (accessed 27 July 2020)). Probiotics are able to colonize, influence, and enhance the gut microflora by outcompeting the pathogenic bacteria throughout the intestinal tract [2]. In sight of several studies, the health promoting properties of probiotics such as enhanced lactose utilization and im­ mune system modulation have been revealed [3,4]. The probiotics are also proven to be able to facilitate the bioavailability of metal ions and to contribute in gastrointestinal health [5]. The optimum colonization of orally delivered probiotic bacteria such as Lactobacillus acidophilus relies on their viability and adequate quantity upon arrival to the large in­ testine and the availability of adjacent carbon and energy sources [2]. Prebiotics on the other hand, are the natural ingredients that can survive the gastric condition and selectively serve distinct probiotic bacteria to enhance their growth and

activity in the colon [6]. The combination of probiotics and prebiotics in close physical proximity and coating (encapsulating) them with protective biocompatible materials to enhance their stability during storage or upper gastrointestinal condi­ tion have been the subject of several studies with promising results [7,8]. Encapsulation is a modern solution to preserve the probiotics viability by incorporation of bacterial cells or metabolites into a specific biocompatible matrix or membrane to reduce the effect of * Corresponding author at: Department of Plant Physiology and Plant Ecology, Institute of Agronomy, Hungarian University of Agriculture and Life Sciences, Ménesi Str. 44, H-1118 Budapest, Hungary E-mail address: Mirmazloum.Seyediman@uni-matehu (I Mirmazloum) https://doi.org/101016/jijbiomac202109177 Received 30 May 2021; Received in revised form 3 September 2021; Accepted 25 September 2021 Available online 1 October 2021 0141-8130/ 2021 The Authors. Published by Elsevier

BV This is an open access article under the CC BY license (http://creativecommonsorg/licenses/by/40/) I. Mirmazloum et al International Journal of Biological Macromolecules 192 (2021) 461–470 environmental factors and cell loss rate under the gastrointestinal con­ dition [9,10]. The extent of permeability in coating technology is a major parameter to consider when active ingredients are the subject of encapsulation. Multilayer coating approach is recommended to mini­ mize the diffusion rate and to sustain the permeation of active molecules [10]. The low cost, biocompatible and biodegradable sodium alginate polymer has been extensively applied for encapsulation and protected delivery of cells and macromolecules [11,12]. Several ingredients with thickening and gelling properties have been used in encapsulation and hydrogel beads formulation of which the chitosan and sodium alginate can be referred to as the most commonly used ingredients. The appli­ cation of other

biodegradables, biocompatible and mucoadhesives polymers such as HEC and HPMC were also reported to enhance the encapsulation of active ingredients [13–16]. The HPMC in particular is used for encapsulation of chemically incompatible or hygroscopic in­ gredients [17]. Ganoderma (Polyporales, Basidiomycota) is a large, economically important cosmopolitan polypore genus comprises of about 180 whiterot fungi species [18,19]. Due to the woody fruiting bodies which often has a bitter taste, Ganoderma species are not classified as edible mushrooms [20], but several Ganoderma species (e.g, G applanatum, G. lingzhi, G lucidum, G sinense and G tsugae) have been used as a functional food to prevent and treat a variety of immunological diseases [21,22]. The health benefits of the various Ganoderma species and their compounds responsible for beneficial effects are intensely studied worldwide [23–26]. The most studied Ganoderma species, which has been described in traditional Asian medicine

under several popular names (such as “Lingzhi” in China or “Reishi” in Japan) is taxonomically identical with G. lingzhi [27–29] However, G lingzhi is commercially sold under the name of G. lucidum [30] and this scientific binomial has widely been used incorrectly for the commercially cultivated East Asian medicinal mushroom [31]. The prebiotic properties of G lingzhi espe­ cially for Lactobacillus genus has been also reported which had made it an excellent candidate for our synbiotic encapsulation strategy [32,33] which can be further be considered as nutraceutical [34]. In the current work, three different molecules (D-(+)-maltose monohydrate, (hydrox­ ypropyl) methyl cellulose and hydroxyethyl-cellulose with texture and taste modifying properties were investigated for their potential contri­ bution in reducing the diffusion rate and enhancing the stability of encapsulated Reishi mushroom active ingredients. The viability of a coencapsulated Lactobacillus strain with

Reishi mushroom extract in the selected formulation during the GIS was also studied. 2.2 DNA molecular marker and identification of Reishi mushroom The applied basidiocarp of Reishi mushroom (Fig. 1A) has grown from commercially produced mushroom spawn bag (Oriens Gomba Ltd., Hungary). The specimen (Oriens-1) was systematically identified by means of genetic marker sequencing. A Phire® Plant Direct PCR Kit (Thermo Scientific, USA) was used to generate molecular data. Using the primers ITS1F (5′ -CTTGGTCATTTAGAGGAAGTAA-3′ ) and ITS4 (5′ TCCTCCGCTTATTGATATGC-3′ ) [35,36], and the PCR protocol [37], the ITS (internal transcribed spacer) regions of the nuclear ribosomal DNA has been amplified. The PCR products were visualized and checked by gel electrophoresis and the purified PCR products were sequenced using the same primers at the Biological Research Centre (Szeged, Hungary). The nucleotides chromatogram was checked, assembled and edited with CodonCodeAligner 7.01 The obtained

nucleotide sequence amplified from the applied Reishi mushroom in this study has been named (Oriens1) and deposited in NCBI GenBank (www.ncbinlmnihgov/genbank/) under the accession number of MW139644. The phylogenetic analysis based on the ITS sequences of Oriens-1 and of a selection of reference sequences from other laccate Ganoderma species was also done to show the position of the studied mushroom strain (Fig. 1B) 2.3 Mushroom cultivation and harvesting Basidiocarps of Oriens Reishi mushroom were grown in glass bottles. The spawn substrate consisted of 39% sawdust, 39% wood chips, 19% wheat straw, 3% gypsum, and mixed with 28 L water. The medium was put into 5 L bottles for the cultivation. The medium was sterilized at 120 ◦ C and cooled under aseptic conditions. The inoculated bottles were closed with a sterilized cotton wool and a paper layer. Thereafter, the bottles were incubated in the dark at 23 to 25 ◦ C for 11–13 days when they have been transferred to the fruiting

room, with the cotton wool, and the paper removed to enhance aeration and stimulate pinning when the mycelia fully colonized the medium. The cultivation room was maintained under constant conditions of temperature (25 ◦ C), and nat­ ural ventilation. Mature basidiomata were harvested, cut into slices and dried at 40 ◦ C for 72 h. 2.4 Extraction and samples preparation Polysaccharides of Reishi mushroom were isolated by following the method of Yin and Dang [38] and Pillai, Nair, and Janardhanan [39], with modifications. The dried basidiocarp of mushroom were vigorously grinded and defatted with petroleum ether, re-dried at 40 ◦ C for 6 h 2. Materials and methods 2.1 Materials and instruments Calcium L-lactate hydrate, polyoxyethylene sorbitan monolaurate (Tween 80), D-(+)-maltose monohydrate, (Hydroxypropyl) methyl cel­ lulose (HPMC), Hydroxyethyl-cellulose (HEC), Folin–Ciocalteus phenol reagent, Gallic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Bile extract porcine, sodium

citrate tribasic dehydrate, low molecular weight chi­ tosan and sodium alginate (from Macrocystis pyrifera, MW: 80,000–120,000. M/G ratio: 156) were purchased from Sigma Chem­ icals (St. Louis, Missouri, USA) Petroleum ether was purchased from Promochem (LGC Standards GmbH, Wesel, Germany). Sodium chloride puriss was purchased from Lachner (Czech Republic). Mushroom β-glucan quantification kit was obtained from Megazyme Int. (Wicklow, Ireland). All of the chemicals and solvents were of analytical grade A commercial Lactobacillus acidophilus (100 billion CFU/g) was used in this research. A Christ ALPHA 2–4 LSC freeze drier (MARTIN CHRIST Gef, GmbH, Germany), a 1800 UV–VIS spectrophotometer (Shimadzu Inc., Kyoto, Japan), a microplate spectrophotometer (PowerWave XS2, Bio­ Tek, USA) and a FluoView FV1000 confocal laser scanning microscope (Olympus, Tokyo) with UplanSApo 10×/0.40, 20×/075, 40×/130, and 60×/1.30 objectives (Olympus) were used in our studies Fig. 1 The applied

Reishi mushroom (G lingzhi) in this study (A) and the phylogenetic analysis of the ITS nuclear ribosomal DNA of the applied Oriens Reishi mushroom (B). 462 International Journal of Biological Macromolecules 192 (2021) 461–470 I. Mirmazloum et al before extraction with 75% ethanol in Erlenmeyer flasks (1:10 w/w). The flasks were put in an ultrasonic bath for 15 min at 70 ◦ C and then the content was transferred to Duran bottles glass with airtight caps and the extraction was carried out at 70 ◦ C for 8 h in an orbitally shaking water bath. The solution was cleaned by filtration (Whatman number 4 paper filter) and the filtrate was concentrated to the one fourth of the original volume in a vacuum rotary evaporator (at gradually increased temper­ ature of 55–75 ◦ C). Ice-cold 95% ethanol (five times the remaining volume) was added to the round bottom flask and kept at 4 ◦ C for 48 h. The precipitate (in 20% of total volume) was collected after centrifu­ gation (8000 rpm

for 20 min), vacuum rotary evaporated (at gradually increased temperature of 55–75 ◦ C) to a gel like texture, frozen at − 80 ◦ C for 2 h and lyophilized in a Christ ALPHA 2–4 LSC freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) at − 40 ◦ C to − 45 ◦ C for 48 h at 5 Pa, to get a light brown extract that was stored at room temperature until use. Extraction yield (%) was determined as: Table 1 The applied ingredients of single layer coating formulation of Reishi extract and L. acidophilus containing hydrogel beads Ingredients Control S Var. 1S Var. 2S Var. 3S Sodium alginate (MW: 80000–120,000) Reishi extract 2% (w/ w) 5% (w/ w) 5% (w/ w) – 2% (w/ w) 5% (w/ w) 5% (w/ w) – 2% (w/ w) 5% (w/ w) 5% (w/ w) – – 2% w) 5% w) 5% w) 2% w) – – – – 0.5% (w/ w) – L. acidophilus (108–9 cells ml− 1) D-(+)-maltose monohydrate (Hydroxypropyl) methyl cellulose Hydroxyethyl-cellulose Yield (%) = (weight of the

dried extract × 100) (w/ (w/ (w/ (w/ 0.5% (w/ w) Crosslinking solution Concentration Reaction time Temperature Stirring Calcium lactate 6% 30 min 25 ◦ C 50 rpm /(weight of the initial mushroom sample) subjected for the second layer of coating. Equal weight of deionized water (used as storage solution) was added to the beads containing each formula and stored at 4 ◦ C for further analysis (the first measurements were conducted immediately after this step when referred to 0 h in this work). 2.5 Preparation of bacterial cell culture Liquid anaerobic culture was initiated from L. acidophilus La-14 in sterile MRS (Sigma) broth at 37 ◦ C for 36 h. An aliquot of this culture was transferred to MRS broth supplemented with 0.1% of TWEEN 80 at 37 ◦ C for 36 h under anaerobic condition using CO2 generating sachets (Microbiology Anaerocult A, Merck KGaA, Germany) in an anaerobic culture jar system. Optical density at 600 nm (OD600) was measured to ensure the cell density

reached about 1.0–20, representing the nominal cell populations of 108–9 cells ml− 1. Cells (20 ml) were centrifuged (1500 rpm, 5 min, 4 ◦ C) and then washed twice with sterile peptone water. The washed cells were re-suspended in 10 ml sterile molecular biology grade water and stored at 4 ◦ C until being added to the beads core preparation (5% w/w). 2.62 Double-layer coated beads To enhance the stability and to more protect the encapsulated active ingredients, the beads were coated with a second polyelectrolyte layer. The concentration of gelling/thickening agents was reduced to half strength for ease of immersion and more uniform spherical coating formation around the initial beads. No bacterial cells have been added into the second matrix to be able to monitor the movement of stained bacteria through the first coating layer. Single-layered beads from each variation (control. S and Var 1S–3S) were submerged in the same in­ gredients solution with half strength (except

for Reishi extract) and denoted as control D, and Var. 1D–3D (Table 2) The beads were picked out from the new matrix using bacterial inoculating loops with 2.2 mm in diameter. The beads were then pushed out from the loop and dropped into the 2ed crosslinking solution (6% calcium lactate + 0.4% chitosan) by a burst of air generated by a manual air compressor. The double layered beads “(average size 2.3 ± 08 mm in diameter) then were filtered out, rinsed with distilled water (5 s); surface dried on sterile paper towel, weighted and placed in sterile glass Petri dishes. Equal weight of deionized water was added to the beads containing each 2.6 Encapsulation of Reishi mushroom extract and probiotic L. acidophilus The basic ingredients of the liquid core consisted of freeze-dried Reishi extract (5% w/w), L. acidophilus (5% w/w) and sodium alginate (2% w/w) that were sequentially dissolved in distilled water at room temperature using a magnetic stirrer for 4 h for complete dissolution.

The solution was then kept at fridge at 4 ◦ C overnight to eliminate the trapped air bubbles. 2.61 Single-layer coated beads Different variations of core liquid content were prepared by sup­ plementing the basic preparation with D-(+)-maltose monohydrate, hypromellose ((Hydroxypropyl) methyl cellulose) and hydroxyethylcellulose (HEC) at 2.0, 05 and 05% w/w concentration, respectively The denoted names and supplemented ingredients for different varia­ tions (control S and Var. 1S–3S) are listed in Table 1 The prepared formulations (3 × 25.0 ml) were transferred to calcium lactate (6%) solution (with mild stirring at 50 rpm) dropwise using a hypodermic syringe and needle with the inner size diameter of 200 μm (with a cut-off angle of 45 degrees) from 3 cm distance above the crosslinking solution surface. By tapping the syringe needle to the edge of a 200-ml beaker, the droplets were going deep into the calcium lactate solution by about 2 cm where the round shape liquid-core beads

(average size 1.5 ± 03 mm in diameter) with alginate-gel membrane were forming immedi­ ately as a result of crosslinking reaction between sodium alginate and calcium ions. The beads were filtered out after 30 min using a stainlesssteel sieve with wire mesh size of 96 μm and rinsed with distilled water for 15 s. Half of the beads were surface dried on sterile paper towel, weighted and placed in sterile glass Petri dishes while the other half was Table 2 The applied ingredients of double layer coating formulation of Reishi extract and L. acidophilus containing hydrogel beads Ingredients (bead content) Control D Var. 1D Var. 2D Var. 3D Sodium alginate (MW: 80000–120,000) Reishi extract 1% (w/ w) 5% (w/ w) – 1% (w/w) 1% (w/w) 5% (w/w) 5% (w/w) – – – 1% (w/ w) 5% (w/ w) 1% (w/ w) – – – – 0.25% (w/ w) – D-(+)-maltose monohydrate (Hydroxypropyl) methyl cellulose Hydroxyethyl-cellulose 463 0.25% (w/ w) Crosslinking solution Concentration Reaction

time Temperature Stirring Calcium lactate chitosan 6%–0.4% 60 min 25 ◦ C 20 rpm I. Mirmazloum et al International Journal of Biological Macromolecules 192 (2021) 461–470 formula and stored at 4 ◦ C for further analysis (the first measurements were conducted immediately after this step when referred to 0 h in this work). mixed vigorously. After 20 min of incubation and regular vortexing in an ice-water bath, 7.0 ml of 12 M sodium acetate buffer (pH 38) was added to each tube and mixed well. A total of 02 ml of a amylogluco­ sidase (1630 U/ml) plus invertase (500 U/ml) was mixed with the tubes content and incubated at 40 ◦ C for 30 min in a water bath with inter­ mittent mixing on a vortex stirrer. Aliquot of 10 ml of the solution (from 9.3 ml final volume) was taken and centrifuged at 1500 rpm for 10 min from which eventually, 0.1 ml of the supernatant was transferred to glass test tubes (16 × 100 mm) and incubated at 40 ◦ C for 20 min with the addition of

0.1 ml of sodium acetate buffer (200 mM, pH 50) plus 3.0 ml of GOPOD Reagent The absorbance of these solutions was measured at 510 nm against the blank reagent made of 0.2 ml of sodium acetate buffer (200 mM, pH 5.0) plus 30 ml of GOPOD Reagent in a microplate spectrophotometer (PowerWave XS2, BioTek, USA). The glucose content (total glucan plus free glucose and glucose from sucrose) in the solutions was analysed by measuring the absorbance at 510 nm against the blank reagent made of 0.2 ml of sodium acetate buffer (200 mM, pH 5.0) plus 30 ml of glucose-oxidase-peroxidasereagent (GOPOD) 2.7 Determination of total phenolics content (TPC) and encapsulation efficiency A modified spectrophotometric method of Folin-Ciocalteu was applied to determine the total phenolics content of the Reishi mushroom basidiocarp or extract-containing formulations and the storage aqueous solutions of encapsulated Reishi mushroom extract to compare the release rate of phenolic compounds from the beads [40].

From the above samples, solutions of 1:1 ratio in deionized water (except from the storage solutions) was prepared and centrifuged at 6000 rpm for 10 min. Of the supernatant, 2 ml (instead of 100 μl of applied Gallic acid stan­ dard dilution to correspond to dry weight of Reishi basidiocarp or extract) was mixed with 500 μl of deionized water and 100 μl of FolinCiocalteu reagent (10% v/v) in 5 ml Eppendorf tubes, and gently mixed. After 6 min of incubation at 25 ◦ C in dark, 1 ml of freshly prepared 7.5% (w/v) Na2CO3 was added to the reaction mix. The final volume was raised to 4 ml with deionized water, incubated in dark for 60 min at 25 ◦ C, and the absorbance was recorded at 760 nm. Gallic acid (0156–5 mg ml− 1) was used to construct a calibration curve (R2 = 0.9934), and the total phenolic contents of the samples were calculated as Gallic acid equivalents (mg GAE g− 1 DW of applied Reishi basidiocarp or extract in beads core formulation). The results were reported as

the mean ± standard deviations of 3 spectrophotometric measurements. To deter­ mine the encapsulation efficiency 200 mg of surface dried differentially formulated beads or each initial preparation (without encapsulation) were dissolved in 5 mL of sodium citrate (100 mM), sonicated for 10 min, and centrifuged for 10 min at 3000 rpm. The encapsulation effi­ ciency was calculated according to following equation: 2.92 Measurement of total and β-glucan content The β-(1,3)-(1,6) d-glucan content of the samples was detected by the Megazyme test kit in triplicates with modifications of the method described by McCleary and Draga [42]. The same samples (Section 2.91) were subjected for total glucan content measurement The dissolution and dispersion of the samples was achieved by adding icecold sulfuric acid (12 M) to the samples and vigorous mixing (10–15 s) every 15 min in an ice-water bath for 2 h. Deionized water was added sequentially to each tube and mixed by vortexing (10–15 s).

The tubes with loosened caps were placed in a hot water bath (~100 ◦ C) and after 5 min; the caps were tightened for 2 h of incubation at 100 ◦ C. To neutralize the solution, 6.0 ml of KOH (10 M) was then added, and mixed well with the tubes contents. The contents of each tube were transferred quantitatively to volumetric flasks and the volume was adjusted to 100 ml with 200 mM sodium acetate buffer (pH 5). Aliquots (2 ml) of the solution was centrifuged at 4000 rpm for 10 min and 0.1 ml of the supernatant was incubated with 0.1 ml of a mixture of exo-1,3beta-glucanase (20 U/ml) and beta-glucosidase (4 U/ml) in 200 mM sodium acetate buffer (pH 5) at 40 ◦ C for 60 min. The glucose-oxidaseperoxidase-reagent (30 ml) was then added to each reaction tube and incubated at 40 ◦ C for another 20 min. The β-glucan content was determined by subtracting the α-glucan content from the total glucan content. EE (%) = TPCe /TPCi × 100 where TPCe was the total phenolics content encapsulated

in beads, while TPCi was the total phenolics content in the initial preparations used for the encapsulation. 2.8 Free radical-scavenging activity (DPPH assay) Method of Brand-Williams, Cuvelier, and Berset [41] were applied with small modifications. The reaction consisted of 18 ml of 130 μM DPPH (completely dissolved in absolute ethanol), 100 μl supernatant of each preparation formula plus 100 μl of deionized water or 200 μl of the beads storage aqueous solution (more details in Section 2.6) The mix­ tures were incubated at 23 ◦ C for 20 min in dark. The radical-scavenging activity was expressed as the percentage of DPPH discoloration (deter­ mined at 517 nm) using the following equation: 2.10 Cell viability tests The viability of applied L. acidophilus was constantly monitored during the 48 h of storage period in each differentially formulated bead matrix before the encapsulation to monitor the effect of supplemented reagents. Both microscopy and plate count method have been

used to follow the cell viability of encapsulated probiotic bacteria in the selected formulation before and after simulated gastric and intestinal digestion. %Radical Scavenging Activity = ([ADPPH − AS ]/ADPPH ) × 100 where ADPPH is the absorbance of the reaction mixture containing 0.1 ml of H2O and AS is the absorbance of the solution containing 01 ml of each sample after 20 min. The radical scavenging activity was expressed as % of DPPH inhibition and the results are presented as the mean ± standard deviations. 2.101 Confocal laser scanning microscopy (CLSM) The bacterial cell viability and their distribution throughout the beads were studied with confocal laser scanning microscopy (Olympus FV 1000, and FV10-ASW (Ver1.4) Japan) The anaerobically grown cells were stained with fluorescent molecular probes using LIVE/DEAD Bac­ Light Bacterial Viability Kit (Thermo Fisher Scientific, USA) containing SYTO 9 dye as green fluorescent and propidium iodide (PI) as a red fluorescent

nucleic acid stain before encapsulation and SGI treatment. The PI can only inter the cells with damaged/inactive membranes, and quenches the emission of SYTO 9 fluorescence signal. Hence, the live cells emit the green fluorescence signal, while the dead cells fluoresce red. For CLSM application, 5 μL of both SYTO 9 and PI dyes were mixed together (1:1) of which 1 μL was added to 0.5 ml of each probiotic- 2.9 Determination of glucans content 2.91 Measurement of α-glucan and D-glucose To determine the α-glucan contents of the preparations, 2 ml of each formula containing 5% Reishi extract or 2 g of surface dried single and double-layer coated beads (different variants containing 5% G. lingzhi extract) corresponding to 100 mg of original basidiocarp or extract was added to Fisher Brand glass culture tubes. To dissolve the starch/ glycogen, 2.0 ml of ice-cold KOH (2 M) was added to each tube and 464 I. Mirmazloum et al International Journal of Biological Macromolecules 192 (2021)

461–470 containing matrix before and after the GIS. The solutions were incubated for 15 min in dark at room temperature prior to visualization with CLSM. Images were obtained with UplanSApo 60×/13 (Oil) and with 10×/0.4 (Air) objective lenses (Olympus, Japan) from free cells and the beads, respectively. The excitation/emission wavelength of 473 nm and 485–530 nm was used for SYTO 9 and 543 nm and 550–560 nm for PI, respectively in a dual-channel sequential imaging mode. The images were analysed using image analysis software (FV10-ASW (Ver1.4)) for CFU counts. Microbiological assays were conducted in triplicates and the results were expressed as mean values with standard deviations. Prior to statistical analyses, colony forming unit (CFU) of L. acidophilus data was normalized by log10 transformation. Assumptions normality and homogeneity of variances were accepted by Shapiro-Wilks and Levenes tests (p > 0.05) Homogeneous subsets were separated by Tukeys post hoc test (p

> 0.05) Statistical analysis was performed using IBM SPSS v25 (Armonk, NY: IBM Corp. [45]) while the charts are pro­ duced in Excel except bubble chart that was generated in R 4.03, using the package ggpubr [46]. 2.102 Simulated gastrointestinal fate of beads Simulated gastrointestinal model for the stomach and small intestine were used to investigate the viability of free and encapsulated probiotics [43]. Simulated gastric solution consisted of sodium chloride (2 g) and 6 M hydrochloric acid (7 ml) in 1.0 liter ddH2O The solution (pH 25) was prepared 30 min before the treatment and filter sterilized using 0.22 μm pore size Millex GP filter unit (Millipore, Bedford, USA). Aliquots (20 g) from the freshly prepared beads core preparations (Var. 1S and Var 1D) containing equal total cell concentrations (5% w/w of 108–9 cells ml− 1) in free and encapsulated form were mixed with gastric juice (2 ml) in separate autoclaved test tubes in duplicates [44]. The tubes were then

incubated at 37 ◦ C for 2 h in anaerobic culture jar system in an orbital shaking incubator. The simulated small intestinal fluid was prepared in 5.0 mM phosphate buffer (pH 70) containing calcium chloride (025 M), sodium chloride (3.75 M) and 5% bile salts solution that was prepared by dissolving porcine bile extract (4 g in 80 ml phosphate buffer, pH 7.0) From this solution, 10 ml was added to each simulating intestinal phase test tubes containing 2.0 g of treated samples (free and encapsu­ lated) with gastric solution and incubated for another 2 h. The beads and free cells were then separated from the supernatant by centrifugation at 1500 rpm (10 min, at 4 ◦ C). The samples of free cells after simulated gastric and intestinal treatment were subjected for direct staining and confocal laser microscopy and also for serial delusion (10− 0–10− 5) and colony forming unit (CFU) count analysis on MRS broth plates. Whereas, the samples of encapsulated cells (not stained) were

subjected for disintegration by submerging 2 g of surface dried beads in 10 ml of a calcium chelator sodium citrate dehydrate solution (100 mM) for 10 min with gentle shaking at room temperature for plate count analysis. Di­ lutions of dissociated cells from the dissolved beads before gastro in­ testinal treatment, after 2 h gastric phase treatment, and/or after 4 h intestinal phases treatment were plated on MRS agar plates and incu­ bated anaerobically for 48 h at 37 ◦ C for viable cell counting. Blank MRS agar plate was employed as blank control. 3. Results and discussion 3.1 Molecular identification and extraction yield The applied Reishi mushroom (Fig. 1A) specimens sequence of ITS nuclear ribosomal DNA was clustered with G. lingzhi sequences in our phylogenetic analysis (Fig. 1B) including the holotype (GenBank NO JQ781877), in a well-supported clade (MLBS = 100%). The interspecific and intraspecific phytochemical profile of Reishi mushrooms can vary significantly among

differentially originated specimens [47]. Therefore, the molecular identification of the applied mushroom was important to be able to compare the biological activity of our cultivated mushroom extract (e.g β-glucan and antioxidants) with other published reports The 75% ethanolic crude extraction resulted in 8.1 g dry Reishi extract from 100 g of dried basidiomata. The content and composition of active molecules in Reishi mushroom can also change upon different drying and extraction temperatures [48]. Thus, the proper identification of the species, the cultivation system, and finally the extraction methods should be outlined and be considered for different research and indus­ trial objectives. 3.2 The TPC, antioxidant capacity and β-glucan content of G lingzhi There has been significant increase in the content of studied active ingredients in the obtained extract when compared with raw mushroom materials in the applied basic formulations (Control Fr.Bo (fruiting body) and Control

Ext.) as presented in Table 3 A significant and more than 6 fold increase in TPC were recorded in Reishi extract-containing basic formulation (17.9 mg GAE g− 1 DW) in comparison with TPC of Reishi basidiocarp-containing (Fr.Bo) basic formulation (≈26 mg GAE g− 1 DW). The TPC content of the two basic preparations did not change significantly during the 48 h of storage. The free radical scavenging activity of Reishi extract (23%) was significantly higher than the basi­ diocarp (14%) at 25 mg ml− 1 (Table 4). Our results were showing relatively lower values than earlier reports on Reishi extracts obtained from ultrasound assisted extraction [49,50]. Though, the aim of measuring this parameter in our study was to monitor the release rate of antioxidant molecules after encapsulation using different formulations. The antioxidant capacity in the applied basic ingredients did not change significantly during the storage period (Table 4). The addition of maltose, HPMC and HEC did not

alter the antioxidant properties of the applied Reishi extract (Data are not shown). The β-glucan content of applied Reishi mushroom was also following the same trend when almost a 50% higher content was detected in the extract-containing formulation (≈2%) when compared to basidiocarp-containing formu­ lation (≈1%) as shown in Table 5. The β-glucan content in basidiocarpcontaining basic formulation did not change during the 48 h of storage but in case of β-glucan in Reishi extract-containing basic formulation, a slightly but significantly higher content was detected after 48 h than the first measurement. This may be due to the slower solubility rate of β-glucans in this formulation during the storage time. Different values for β-glucans content in Reishi or Ganoderma mushroom have been re­ ported ranging from as little as 4 g 100 g− 1 to as high as 55.9 g 100 g− 1 of dried materials [51,52]. The remarkable deviation in the reported values can be due to the taxonomical

confusion, improper identification, 2.11 Statistics The release rate of G. lingzhis total phenolic compounds, radical scavenger compounds and β-glucan from single- and double-layered calcium alginate beads during 48 h in aqueous solution were compared by Repeated Measures ANOVA with two independent factors (of between-subjects effects) as ‘treatments’ (Control Fr. Bo, Control Ext., Control, Var1, Var2, Var3) and ‘layer’ (single or double) while ‘elapsed time’ (0, 1, 6,12, 24, and 48 h) was considered as withinsubjects effect factor. The overall effect was tested by Wilks lambda that expresses the unexplained variance rate. Normality of the residuals was proven with Shapiro-Wilks test (p > 0.05) Sphericity assumption was satisfied in all the three cases (with Greenhouse-Geissers epsilon values 0.71, 077, and 086, respectively) According to Levenes test, in some cases homogeneity of variances was violated (p < 0.05), therefore in case of significant results, we used

Games-Howells post hoc tests to separate significant groups of between-subject effects. Significant within-subject effects were further analysed in pairwise testing with estimated marginal means by Bonferronis adjustment. Encapsulation efficiency and CFU data were tested by two-way ANOVA with factors ‘treatments’ (Control, Var.1, Var2, Var3) and ‘coating’ (single or double) for efficiency and ‘digestion’ (initial prepa­ ration, gastric and intestinal) with ‘coating’ (free cells, single, double) 465 I. Mirmazloum et al International Journal of Biological Macromolecules 192 (2021) 461–470 Table 3 The release rate of G. lingzhis total phenolic compounds from single and doublelayered calcium alginate beads during 48 h in aqueous solution Sample name 0h 1h 6h 12 h 24 h 48 h Single layer coating Control 2.55 ± Fr.Bo 0.012 Ba Control 17.92 ± Ext. 0.017 Ea Control 3.94 ± S. 0.017 Da Var.S1 0.78 ± 0.037 Aa Var.S2 3.71 ± 0.043 Ca Var.S3 3.76 ± 0.037 Ca 2.59

± 0.005 Ba 17.94 ± 0.037 Fa 5.08 ± 0.005 Eb 1.96 ± 0.024 Ab 4.66 ± 0.029 Cb 4.87 ± 0.016 Db 2.63 ± 0.017 Aa 17.90 ± 0.023 Fa 9.40 ± 0.012 Ec 3.82 ± 0.021 Bc 8.33 ± 0.021 Dc 7.52 ± 0.014 Cc 2.65 ± 0.020 Aa 17.93 ± 0.009 Fa 10.38 ± 0.014 Ed 7.58 ± 0.021 Bd 9.82 ± 0.024 Dd 9.42 ± 0.028 Cd 2.70 ± 0.005 Aa 17.95 ± 0.024 Ea 11.15 ± 0.020 De 8.22 ± 0.035 Be 11.10 ± 0.028 De 10.05 ± 0.045 Ce 2.72 ± 0.008 Aa 17.88 ± 0.021 Fa 11.32 ± 0.012 Ee 8.51 ± 0.043 Be 11.09 ± 0.045 De 10.16 ± 0.035 Ce Double layer coating Control 2.51 ± Fr.Bo 0.019 Ea Control 17.74 ± Ext. 0.026 Fa Control 0.55 ± D. 0.012 Da Var.D1 0.08 ± 0.008 Aa Var.D2 0.25 ± 0.037 Ba Var.D3 0.41 ± 0.021 Ca 2.51 ± 0.021 Ea 17.90 ± 0.038 F ab 1.56 ± 0.026 Db 0.14 ± 0.037 Aa 1.20 ± 0.021 Bb 1.04 ± 0.029 Cb 2.63 ± 0.066 BC a 17.91 ± 0.020 E ab 3.54 ± 0.005 Dc 2.57 ± 0.008 Cb 2.27 ± 0.016 Bc 2.00 ± 0.028 Ac 2.65 ± 0.020 Aa 18.00 ± 0.026 Eb 6.24 ± 0.031 Dd 4.31 ± 0.032 Bc 5.22 ±

0.045 Cd 5.11 ± 0.021 Cd 2.70 ± 0.012 Aa 17.95 ± 0.029 F ab 8.29 ± 0.014 Ee 5.49 ± 0.021 Bd 7.52 ± 0.042 Ce 7.75 ± 0.024 De 2.71 ± 0.017 Aa 17.86 ± 0.020 F ab 8.56 ± 0.020 Ee 5.75 ± 0.049 Bd 8.17 ± 0.016 Df 8.05 ± 0.021 Cf Table 4 The release rate of G. lingzhis radical scavenger compounds from single and double-layered calcium alginate beads during 48 h in aqueous solution (DPPH assay: % of inhibition). Sample name Upper case letters: comparison of treatments within time elapsed in single or double layer coating (Games-Howells, p < 0.05) – read vertically Lowercase letters: comparison of time effects within single or double layer coating treatments (Bonferronis, p < 0.05) - read horizontally In single layer coated beads: Var.S1: Plus 2% D-(+)-maltose; VarS2: Plus 05% (Hydroxypropyl) methyl cellulose; Var.S3: Plus 05% Hydroxyethyl-cellulose In double layer coated beads: Var.D1: Plus 1% D-(+)-maltose; VarD2: Plus 025% (Hydroxypropyl) methyl cellulose; Var.D3:

Plus 025% Hydroxyethyl-cellulose Values are in mg GAE g− 1 dry weight of applied Reishi basidiocarp or extract. Control Fr.Bo: fruiting body of Reishi mushroom 0h 1h 6h 12 h 24 h 48 h Single layer coating Control 14.11 ± Fr.Bo 0.086 Ca Control 23.28 ± Ext. 0.032 Da Control 7.65 ± S. 0.032 Ba Var.S1 7.03 ± 0.032 Aa Var.S2 7.59 ± 0.086 Ba Var.S3 7.55 ± 0.065 Ba 14.19 ± 0.112 Ca 23.29 ± 0.019 Da 8.79 ± 0.086 Bb 8.06 ± 0.032 Ab 8.81 ± 0.056 Bb 8.84 ± 0.032 Bb 14.28 ± 0.086 Ca 23.37 ± 0.037 Da 9.16 ± 0.065 B bc 8.77 ± 0.086 A bc 9.14 ± 0.056 B bc 9.07 ± 0.032 AB b 14.43 ± 0.032 C ab 23.41 ± 0.019 Da 10.02 ± 0.086 Bc 8.99 ± 0.032 Ac 9.96 ± 0.032 Bc 10.11 ± 0.086 Bc 14.71 ± 0.086 Cb 23.37 ± 0.037 Da 14.02 ± 0.097 Bd 9.25 ± 0.056 Ac 14.26 ± 0.032 Bd 14.38 ± 0.086 Bd 14.83 ± 0.086 C ab 23.29 ± 0.019 Da 14.06 ± 0.086 Bd 9.35 ± 0.065 Ac 14.34 ± 0.032 Bd 14.40 ± 0.141 BC d Double layer coating Control 14.11 ± Fr.Bo 0.086 Da Control 23.28 ± Ext.

0.032 Ea Control 5.38 ± D. 0.000 Ca Var.D1 3.78 ± 0.065 Aa Var.D2 5.27 ± 0.112 BC a Var.D3 5.07 ± 0.065 Ba 14.19 ± 0.112 Da 23.29 ± 0.019 Ea 6.04 ± 0.037 Cb 4.77 ± 0.097 Ab 5.80 ± 0.032 Ba 5.68 ± 0.032 Ba 14.28 ± 0.086 Da 23.37 ± 0.037 Ea 6.49 ± 0.049 Cb 5.29 ± 0.117 Ab 6.06 ± 0.056 B ab 6.15 ± 0.086 B ab 14.43 ± 0.032 C ab 23.41 ± 0.019 Da 7.09 ± 0.131 B abc 5.95 ± 0.112 Ab 6.82 ± 0.086 Bb 7.01 ± 0.056 Bb 14.71 ± 0.086 Cb 23.37 ± 0.037 Da 8.28 ± 0.067 Bd 7.20 ± 0.065 Ac 8.08 ± 0.056 Bc 8.23 ± 0.117 Bb 14.83 ± 0.086 C ab 23.29 ± 0.019 Da 9.29 ± 0.049 B cd 7.68 ± 0.056 Ac 9.27 ± 0.086 Bd 9.50 ± 0.117 Bc Upper case letters: comparison of treatments within time elapsed in single or double layer coating (Games-Howells, p < 0.05) – read vertically Lowercase letters: comparison of time effects within single or double layer coating treatments (Bonferronis, p < 0.05) - read horizontally In single layer coated beads: Var.S1: Plus 2% D-(+)-maltose;

VarS2: Plus 05% (Hydroxypropyl) methyl cellulose; Var.S3: Plus 05% Hydroxyethyl-cellulose In double layer coated beads: Var.D1: Plus 1% D-(+)-maltose; VarD2: Plus 025% (Hydroxypropyl) methyl cellulose; Var.D3: Plus 025% Hydroxyethyl-cellulose Control Fr.Bo: fruiting body of Reishi mushroom growth habitat or cultivation condition and different extraction methods and quantification. The partial degradation of β-glucan under in vitro gastric condition has been reported [53]. The protected quantity of β-glucan by encapsulation, can therefore contribute to a sustainable delivery of more intact β-glucan to pass through the gastrointestinal tract. similar tendency after double layer coating (Var. D1) In case of phenolic compounds, the between-subjects effects were all significant (treatment: F(4;26) = 656,941.13, layer: F(2;26) = 812,41841, interaction: F(3;26) = 3937.33, all with p < 0001) and so were the within-subjects effect (elapsed time: F(3.53;9172) = 107,40306, p < 0001)

Unlike maltose, the addition of HPMC and HEC did not show any protective effect on the release rate of Reishi extract active substances from the beads. This diminished release rate was consistent during the sampling time intervals up to 48 h. There are only a few reports on the application of maltose or its derivatives in controlled release of active ingredients encapsulated in biopolymers matrixes or its ability to con­ jugate and make cross linking network with other natural polymers [54]. The attachment of maltose to other Reishi polysaccharides that may have been occurred during the overnight storage before encapsu­ lation may have resulted in some conjugated polymers with enhanced hydrophobicity and consequently decreased the release of active mole­ cules from the alginate and chitosan semipermeable membrane. Another potential explanation can be on role of the maltose to physically 3.3 Protective effect of encapsulation on active molecules release rate The released quantity of

the total phenolics, antioxidants and the β-glucan from the beads with single and double layer coatings during the 48 h of storage was considered for statistical analysis of the data. Comparing the phenolic and radical scavenger compounds as well as β-glucan, we detected significant overall multivariate effects (Wilks lambda < 0.01 for the elapsed time effect and for all the two-way and the three-way interactions with p < 0.001) As it can be seen in Table 3, a significantly lower release rate was recorded when maltose has been applied in the formulation (Var. S1) after single layer coating with 466 I. Mirmazloum et al International Journal of Biological Macromolecules 192 (2021) 461–470 and double layer coated beads are presented in Table 5. In case of β-glucan, the between-subjects effects were all significant (treatment: F (4;26) = 15,863.05, layer: F(2;26) = 25,88302, interaction: F(3;26) = 15.06, all with p < 0001) and so were the within-subjects effect (elapsed

time: F(4.3;11185) = 206335, p < 0001) The overall released content of β-glucan was significantly decreased as a result double layer coating of the basic formulation. Except the consistent and significant effect of the maltose addition, the HPMC and HEC did not show a considerable protective effect on the release rate of both single and double-layer coated beads. Hydroxypropyl methylcellulose (HPMC); a cellulose derivative that has been utilised in film forming material and drug delivery systems for sustained controlled release [55,56], did not show a convincing role in reducing the release rate of Reishis active molecules from the beads at the form and applied concentration in this study. The same results were found in case of supplemented hydroxyethyl-cellulose in the core formulation in our samples. There are reports however indicating the successful applicability of HEC for the pH sensitive controlled release of some drugs [57,58]. To determine the encapsulation efficiency, the

same TPC assay was used for the samples of non-capsulated and encapsulated formulations and the results are presented in Fig. 2 The layer and treatment effects as well as their interaction were proved to be significant on encapsulation efficiency (F(1;16) = 6245.68, F(3;16) = 1133, F(2;16) = 1256, respectively, all with p < 0.001) Significantly higher encapsulation efficiency was recorded in single-layer coated (≈83%) that the doublelayer coated beads (≈74%). That makes sense when considering the washing step of single-layer coated beads before the second layer coating step during which a portion of phenolic compounds could be lost. The application of reagents (maltose, HPMC and HEC) had no significant effect on the encapsulation efficiency when compared with the control. Considering the larger molecular sizes of β-glucan and the bacterial cells (1–10 μm) comparing to phenolic compounds, higher encapsulation efficiency can be anticipated for these compartments of the beads.

A selected set of obtained data on release rate of studied molecules from the beads (at 0,12, 24 and 48 h of the storage time) have been converted to the percentage of their initial detected quantity in perpe­ tration formulations and the results are presented in a constructed bubble chart (Fig. 3) The sizes of the spheres are representing the saved quantity of the different active molecules at measurement intervals. This figure is clearly showing a summarized overview of protective effect of maltose supplementation on the release rate of different beads. It became evident that the double layer coating and perhaps the presence of chitosan in the second coating layer remarkably saved more antiox­ idant molecules comparing to the single layer coated beads with no Table 5 The release rate of G. lingzhis β-glucan from single and double-layered calcium alginate beads during 48 h in aqueous solution (g 100 g− 1 of different formu­ lations and in the beads storage solution). Sample

name 0h 1h 6h 12 h 24 h 48 h Single layer coating Control 0.93 ± Fr.Bo 0.019 Ca Control 1.97 ± Ext. 0.016 Da Control S. 0.83 ± 0.015 B ca Var.S1 0.63 ± 0.003 Aa Var.S2 0.81 ± 0.009 Ba Var.S3 0.82 ± 0.009 BC a 0.95 ± 0.015 Ba 2.01 ± 0.009 C ab 0.89 ± 0.011 B ab 0.87 ± 0.015 AB bc 0.82 ± 0.007 Aa 0.84 ± 0.003 AB a 0.95 ± 0.001 Ba 2.01 ± 0.014 D ab 0.99 ± 0.012 B bcd 0.84 ± 0.009 Ab 1.00 ± 0.013 B bcd 1.09 ± 0.006 Cb 0.97 ± 0.006 Ba 2.00 ± 0.014 D ab 1.03 ± 0.012 B abc 0.86 ± 0.009 A bc 1.04 ± 0.004 Bc 1.16 ± 0.010 Cb 0.96 ± 0.008 Aa 2.02 ± 0.019 D ab 1.25 ± 0.012 B cd 0.95 ± 0.010 Ac 1.31 ± 0.007 C de 1.30 ± 0.004 BC c 0.95 ± 0.011 Aa 2.02 ± 0.016 Cb 1.36 ± 0.012 Bd 0.93 ± 0.006 A bc 1.34 ± 0.009 Be 1.33 ± 0.016 B bc Double layer coating Control 0.92 ± Fr.Bo 0.020 Ca Control 1.95 ± Ext. 0.003 Da Control D. 0.81 ± 0.007 BC a Var.D1 0.51 ± 0.004 Aa Var.D2 0.77 ± 0.006 Ba Var.D3 0.81 ± 0.006 BC ac 0.96 ± 0.003 Da 1.99 ± 0.017 E ab

0.83 ± 0.007 Ca 0.54 ± 0.004 Aa 0.73 ± 0.009 Ba 0.81 ± 0.009 C ab 0.95 ± 0.001 Ca 2.01 ± 0.011 D ab 0.92 ± 0.007 Ca 0.61 ± 0.007 Aa 0.85 ± 0.008 Ba 0.90 ± 0.007 C bc 0.97 ± 0.005 Ba 2.03 ± 0.019 C ab 1.00 ± 0.012 B abc 0.82 ± 0.007 Ab 0.99 ± 0.003 Bb 0.99 ± 0.006 B cd 0.95 ± 0.008 Aa 2.02 ± 0.001 Db 1.24 ± 0.011 Cb 1.01 ± 0.007 Bb 1.10 ± 0.005 Cb 1.10 ± 0.009 C bde 0.95 ± 0.013 Aa 2.03 ± 0.003 D ab 1.29 ± 0.011 Cc 1.01 ± 0.013 Ab 1.22 ± 0.010 Bb 1.23 ± 0.003 BC e Upper case letters: comparison of treatments within time elapsed in single or double layer coating (Games-Howells, p < 0.05) – read vertically Lowercase letters: comparison of time effects within single or double layer coating treatments (Bonferronis, p < 0.05) - read horizontally In single layer coated beads: Var.S1: Plus 2% D-(+)-maltose; VarS2: Plus 05% (Hydroxypropyl) methyl cellulose; Var.S3: Plus 05% Hydroxyethyl-cellulose In double layer coated beads: Var.D1: Plus 1%

D-(+)-maltose; VarD2: Plus 025% (Hydroxypropyl) methyl cellulose; Var.D3: Plus 025% Hydroxyethyl-cellulose Control Fr.Bo: fruiting body of Reishi mushroom smoothing surface of the microparticles and imparting a smaller contact area with the core medium and consequently slowing the release of encapsulated phenolic compounds. The effect of different formulation in single and double-layer coted beads release rate of antioxidants is presented in Table 4. In case of radical scavenger compounds, the between-subjects effects were all significant (treatment: F(4;20) = 18,471.26, layer: F(2;20) = 138,411.49, interaction: F(3;20) = 186712, all with p < 0001) and so were the within-subjects effect (elapsed time: F(3.85;7694) = 978311, p < 0.001) Similar to TPC, the addition of maltose resulted in the highest protective effect in both single and double layer coated beads during the 48 h of storage. When compared with the basic formulations (Control S. and Control D), there were significant

and positive effects when HPMC (Var. S2 and D2) and HEC (Var S3 and Var D3) were applied in the formulations in some of the sampling times but not as pronounced and consistent as maltose-containing formulations (Table 4). The results on release rate of Reishi extracts β-glucan from the single Fig. 2 The encapsulation efficiency of Reishis total phenolic compounds (TPC) in single and double-layer coated beads with supplementation of maltose, HPMC and HEC. Different letters are for significantly different groups (Tukeys, p < 0.05) Upper case: comparison of layers by treatments; lower case: com­ parisons of treatments by layers. 467 I. Mirmazloum et al International Journal of Biological Macromolecules 192 (2021) 461–470 strain to the acidic pH. The number of viable bacteria has significantly influenced when single and double-layer coatings were applied. Signif­ icantly higher colonies were recovered from disintegrated beads with bauble-layer coating after the both gastric

and intestinal treatment whereas the number of viable bacteria in single-layer coated beads declined significantly between the gastric and intestinally digested samples. These results were further validated by CLSM imagining. As pre­ sented in Fig. 5, the cells that were damaged and lost their membrane integrity emitted the red signal while the green florescence signal was captured from the viable cells. The images are also showing that the alginate-based encapsulation can successfully retain and protect the cells physically within the beads. It was also observed that the cells tended to be accumulated around the coating shell or the beads walls as lower signal intensity were observed among most of the studied beads. There are already several reports on enhanced cell viability of multilayer-encapsulated cells during the storage and upon oral ingestion [61–63]. Our contribution was intended to better visualize the process and show the approximate physical position of the cells inside

the beads. The prebiotic properties of the Reishi mushroom extract has been re­ ported in our earlier research [64,65], supporting its ability to enhance the growth of adjacent co-encapsulated cells and the gut microbiota when disintegrated from the beads. The positive effect of coencapsulated prebiotics such as inulin on the growth of probiotic bac­ teria has been reported earlier [66,67]. The combination of plate count method and the microscopy proved to be adequate for evaluation and analysis of the cells viability and their status under different treatments. Fig. 3 Representation of the saved quantity of β-glucan, antioxidants and total phenolics from single and double-layer coated beads influenced by supple­ mentation of maltose, HPMC and HEC during the storage period. The smaller the sphere, the higher the diffused quantity of encapsulated molecules. incorporated chitosan. Our results showed a relatively lower release rate of phenolic compounds during the storage in

biological pH than the other studies reported on around 50% release of active molecules during the first 2–3 h after encapsulation [59,60]. The release rate of active ingredients can vary when assuming the applied quantity and the ratio of the beads being supplemented in functional food product such as dairy product or beverages. Therefore, this parameter should be studied and evaluated in the final prototypes. 4. Conclusion The medicinal Reishi mushroom with its evident health promoting properties has been considered for functional food application recently. The identity of the applied strain was confirmed and cultivation and extraction of polysaccharide-reach fraction was established. To mask the bitter taste of the mushroom extract and to protect the biologically active molecules upon oral administration, biopolymer encapsulation strategy was adopted. Using the food friendlier ingredients (eg food grade calcium lactate instead of calcium chloride and the application of a

commercial lactobacillus stain), hydrogel beads were produced with 3.4 Viability of free and encapsulated L acidophilus during simulated gastrointestinal treatment Based on the results of active molecules release rate, the Var. S1 and Var. S2 formulations containing maltose were selected for the cell viability test upon gastrointestinal simulation. The free and encapsu­ lated cells in single and double layer coating systems were subjected for plate count of colony forming units (CFU) and CLS microscopy. Based on the statistical analysis, the layer and digestion effects as well as their interaction were all significant on CFU counts (F(2;14) = 307.21, F (2;14) = 1146.63, F(2;14) = 23515, respectively, all with p < 0001) As presented in Fig. 4, the non-capsulated cells lost their viability under the simulated gastric condition indicating the sensitivity of the applied Fig. 4 Logarithmic CFU values of free and encapsulated L acidophilus in single and double-layer coated maltose

containing alginate beads (kept in storage solution for 48 h) after simulated gastrointestinal digestion. Different letters are for significantly different groups (Tukeys, p < 0.05) Upper case: comparison of layers by digestion; lower case: comparisons of digestions by layers. Fig. 5 CLSM images of viable encapsulated bacteria (in green) in maltosecontaining calcium alginate moist beads after exposure to simulated gastroin­ testinal condition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 468 International Journal of Biological Macromolecules 192 (2021) 461–470 I. Mirmazloum et al single and double layer coatings. To increase the functionality of the potent Reishi ingredient, probiotic lactobacillus was also incorporated in this study. The role of different taste and texture modifiers (maltose, HEC and MPMC) on hydrogels antioxidants and glucans diffusion rate were investigated during the 48

h of storage time. The selected formu­ lations were subjected for GIS and microscopy and microbiology eval­ uation. Remarkable β-glucan content on the obtained Reishi mushroom extract was documented. The significantly positive effect of maltose supplementation and the double-layer coating on reducing the release rate of the active molecules (phenolic compounds, antioxidants and β-glucan) were confirmed. The double-layer coated moist alginate-based beads could protect the viability of the incorporated probiotic cells as documented by plate count and microscopy. The applied method has a potential to conveniently imply the extracts of medicinally important species with previously limited food industrial use due to their bitterness or unpleasing taste or aroma. The combination of prebiotic substances with probiotic bacteria using the presented approach can bring countless opportunities to produce novel functional food and beverage variants with enhanced stability of the bioactive

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