2.1. Seaweeds used in the study
The brown seaweeds used in this study, Himanthalia elongata (Linnaeus) S.F. Gray (common name Sea Spaghetti) and Ascophyllum nodosum (Linnaeus) Le Jolis were harvested from the Northwest coast of Ireland in November 2020 and were supplied, dried and milled (size of 250-1mm) by the Irish company SeaLac Limited (Sligo, Ireland). A low temperature drying method described previously [16] where conditions do not exceed 28°C was used to dry harvested biomass.
2.2. Generation of Ascophyllum nodosum fermentate
Ascophyllum nodosum was fermented using a commercially available LAB preparation (Ecosyl TM, Volac, UK) containing the bacterium Lactobacillus plantarum MDT/1. Fermentation was carried out by re-suspending dried, milled (250-1 mm size) A. nodosum supplied by SeaLac Ltd. at a ratio of substrate: ddH2O of 1:2 (w:v). To this mixture, the commercial bacterium mixture was added at a ratio of 24:1 (w:v, seaweed mixture to bacterium). This mixture was maintained at 25°C for 5 days. The LAB strain used for fermentation was heat deactivated after fermentation at 95°C for 15 mins in a water bath and the fermentate was freeze-dried using an industrial scale freeze dryer, the FD 80 model (Cuddon Engineering, Marl-borough, New Zealand) following in-house drying protocols.
2.3 Generation of Himanthalia elongata hydrolysate
The Himanthalia elongata hydrolysate was generated using Xylanase powder (from Aspergillus oryzae > 2500 units/g, Merck, Dublin, Ireland). 100 g of dried and milled H. elongata was placed in 1000 mL ddH2O in a bioreactor. The pH of the reaction was maintained at 3.8 and temperature was maintained at 70°C using 1M HCl and the temperature probe controller of the bioreactor. The hydrolysates were generated using a New Brunswick (Mason Technologies, Dublin, Ireland) 1.5 L bioreactor with temperature and pH control. Prior to initiating hydrolysis, the temperature of the mixture was adjusted to 70°C. The reaction was maintained at these conditions for 3 hr and subsequently the hydrolysate was heat deactivated by heating the mixture in a water bath to 95°C for 15 mins to deactivate the enzyme. The mixture was cooled to room temperature, frozen at -20°C and subsequently freeze-dried as described earlier.
2.4 Nutritional and bioactive composition – Analytical procedures
Elemental analysis was conducted using a LECO CNS928 Macro Analyzer (USA) to determine the carbon, nitrogen, and sulphur content of the seaweeds used in the study. Likewise, a TGM 800 Thermogravimetric Moisture Analyser (LECO Corporation, USA) was used to determine the moisture content following the AOAC Official Methods of Analysis 950.46. The ash content was measured by charring the sample using a Carbolite Muffle Furnace at 550°C following the AOAC method 942.05.
The Oracle NMR Smart TRAC rapid Fat Analyser (CEM Corporation USA) measured fat content following the AOAC moisture and fat in meat and poultry products, Official Methods 985.14 and 985.26 as described previously [17]. Prior to the fat content analysis, the samples were pre dried using a TGM 800 Thermogravimetric Moisture Analyser automated for oven drying.
Protein content was determined following the AOAC nitrogen-based combustion technique (Dumas method), Official method 992.15 (1990) [18]. Briefly, the protein concentration was measured using a LECO FP628 Protein analyser (LECO Corp., MI, USA) and a nitrogen factor of 5.13 was used to calculate the percentage protein of each sample [19].
Total dietary fibre was determined using the Ankom (US) Dietary Fiber Automatic Analyser following the AOAC Total, Soluble, and Insoluble Dietary Fiber in Foods, Official methods 991.43 (1990) [20].
Iodine content was measured using a spectrophotometric method and an Iodide colorimetric assay using a kit supplied by Biovision (Biovision, USA) as described previously [17]. A specific optical density at 330 nm was recorded during the study, wherein the principal relied on the iodide ion as a catalyst converting the yellow coloured substrate to a colourless solution.
Total phenolic content was determined using methods based on a spectrophotomet ric method using a Folin Ciocalteu’s reagent (purchased from Merck, Arklow, Ireland). The polyphenols were extracted following the method by Stankovic (2011) [21] or Kupina et al., (2018) [22]. Briefly, phloroglucinol or Gallic acid obtained from Merck, Ireland was dissolved in methanol at different concentrations ~ 1-250 µg/mL to generate a standard plot. The test samples at a concentration of 1 mg/mL, 2.5 mL of 10% Folin-Ciocalteu’s reagent, and 2.5 mL of 7.5% Na2CO3 purchased from Merck, UK were used for the analysis. After incubation for 45 minutes at 45°C, the absorbance was recorded at 765 nm and the results obtained were expressed as phloroglucinol equivalents (PGE eq. per mg of sample) or Gallic acid equivalents (mg GAE/100 g of sample) respectively.
The phlorotannin content of the hydrolysate and fermentate was determined by a method developed by Lopes et al. (2012) [23]. Briefly, the phlorotannins were extracted using 100 mL of acetone: water (70:30, v/v) added to 5 g of test sample. The sample mixture was vortexed at 400 rpm for 1 hour. After extraction, acetone remaining in the samples were separated using a rotary vacuum evaporator (Model no. 1000243991; Mason Technology, Buchi, Switzerland). Subsequently, the organic fractions were pooled and dried under nitrogen at 30°C. A mixture of cellulose and methanol was used to purify phlorotannins. Pigments were removed using toluene. Phlorotannins were released from cellulose using acetone: water (70:30, v/v). The phlorotannin fractions obtained were dried under nitrogen and subsequently freeze-dried. Following hydrolysis and fermentation, different 10-kDa, 50-kDa, 100-kDa seaweed permeate fractions were generated using molecular weight cut-off (MWCO) filters (Merck Millipore, Carrigaline, Co. Cork, Ireland) using a centrifugation procedure described previously [24]. Permeates were frozen and subsequently freeze-dried as described previously.
2.5. Preparation of medium for cultivation of methanogens
The methanogens Methanococcus maripaludis (MM) and Methanobrevibacter ruminantium (MR) were purchased from The Leibniz Institute DSMZ - German culture collection of Microorganisms (Leibniz, Germany). Both the anaerobic bacterial cultures were grown in DSMZ 141 medium but lacked Wolin’s vitamin solution. Weimar and colleagues [15] previously made the media for cultivation of methanogens. Briefly, the medium composition was as follows: KCl (0.34 g/L); MgCl2.6H2O (4 g/L); NH4Cl (0.25 g/L); MgSO4.7H2O (3.45 g/L); CaCl2.2H2O (0.14 g/L); K2HPO4 (0.14 g/L); NaCl (18 g/L); Fe(NH4)2(SO4)2.6H2O solution (0.1% w/v) (2 mL/L); CH3COONa (1 g/L); yeast extract (2 g/L); Trypticase peptone (2 g/L); Na Resazurin solution (0.1% w/v) (0.5 mL/L); NaHCO3 (5 g/L); L-Cysteine-HCl.H2O (0.5 g/L); Na2S.9H2O (0.5 g/L); and Modified Wolin’s mineral solution ~ 10 mL/L [Nitrilotriacetic acid (1.5 g/L); MgSO4.7H2O (3 g/L); MnSO4.7H2O (0.5 g/L); NaCl (1 g/L); FeSO4.7H2O (0.1 g/L); CoSO4.7H2O (0.18 g/L); CaCl2.2H2O (0.10 g/L); ZnSO4.7H2O (0.18 g/L); CuSO4.5H2O (0.01 g/L); KAl(SO4)2.12H2O (0.02 g/L); H3BO3 (0.01 g/L); Na2MoO4.2H2O (0.01 g/L); NiCl2.6H2O (0.03 g/L); Na2SeO3.5H2O (0.30 mg/L); Na2WO4.2H2O (0.04 mg/L)]. To prepare the Modified Wolin’s mineral solution, Nitrilotriacetic acid was dissolved first, and the pH was adjusted to 6.5 with KOH. Subsequently, the remaining minerals were added, and the final pH was adjusted to 7.0 with KOH. Once the Modified Wolin’s mineral solution was prepared, the mineral solution was added to the main medium, except for the bicarbonate, L-Cysteine-HCl and sodium sulphide. In order to make the medium anoxic, Nitrogen (N2) gas was used to sparge the mixture for 30–45 min. After the partial removal of dissolved oxygen from the medium, the bicarbonate was added, the final pH was adjusted to 7.0 and the medium were autoclaved. Following sterilization, L-Cysteine-HCl and sodium sulphide from sterile anoxic stock solution was added to the medium inside the anaerobic chamber (gas mix, 4% H2, 5% CO2, and 91% N2) with < 8 ppm O2. Note: L-Cysteine-HCl was filter sterilized using a syringe micro filter (0.2 mm).
For inoculum development both bacteria were transferred to the anoxic freshly prepared medium in plastic cell culture tubes and were incubated inside the an-aerobic chamber (Whitley A35 anaerobic workstation, model number: NF237032833, PL ref: 799009 purchased from the UK) at 37°C. Intermittently, the Optical Density (OD) was measured at 595 nm using a spectrophotometer purchased from Thermofisher scientific (Multiskan FC model number: NF980033586) provided with an inbuilt software (SKANIT software 4.1) package for determining the growth of bacterial cultures.
2.6. Microplate assay and seeding techniques (agar solid plate) for screening the methane inhibiting effect of seaweed hydrolysate and fermentate and MWCO fractions
A 96 well microplate assay was used to screen the effect of different seaweed treatments and fractions generated from the same on the ruminant specific methanogenic bacteria ~ Methanococcus maripaludis and Methanobrevibacter ruminantium following the method described by Weimar et al. (2017) [15]. 2-Bromoethanesulfonic acid (BES) was used as the positive control inhibitor and all the experiments were carried out inside an anaerobic chamber maintained at 37°C. Prior to the experiment, the sample seaweed extracts or fractions were dissolved in distilled water (1 mg/mL). BES was used at a concentration of 1 mg/mL. The microplates and all other required equipment and materials were pre-incubated in the anaerobic chamber to remove traces of oxygen for at least 2–3 days prior to the experiment. Briefly, 100 µL of sample seaweed extracts or fraction was added to the 96 well plate. To this, 100 µL of bacterial cultures (in their exponential phase) were added. The final culture volume in the assay was 200 µL. Inoculum OD values were recorded at 595 nm. For the positive control, 100 µL of BES was added to 100 µL of bacterial cultures. Plates were incubated at 37°C for 11 days. Similarly, for the negative control, 100 µL of only medium and 100 µL of each bacterial culture was added to individual wells on the plate. After addition of sample extracts and bacterial cultures, a thin plastic film was placed on top of the microplate to avoid spilling of the cultures during OD measurements. Subsequently, the OD values for each well were recorded at 405 nm using a spectrophotometer for time zero (T = 0, day 1) and on the 4th, 6th, 8th and 11th day post plate set up. The plate was always kept in anaerobic condition and an anoxic atmosphere was maintained using portable boxes preincubated with anaerobic gas packs (AGS AnaeroGen compact bag, Oxoid). The seaweed extracts and controls were tested in triplicate.
Additionally, a spread plate technique was carried out. Briefly, the bacterial cultures were seeded independently into the prepared medium and were examined for zones of inhibition in accordance with previously published antimicrobial screening assay procedures [25]. For seeding, 10 mL of bacterial cultures were added to 100 mL agar broth, and this was poured into petri-dishes. Following solidification, small holes were made in the agar plates using sterile borers, approximately 50 µL of each seaweed extracts including the hydrolysate, and fermentate fractions were added independently to the wells. Upon absorption of the extracts, the plates were inverted and incubated inside the same anaerobic chamber at 37°C for 2–3 days. Following incubation, the appearance of zones of inhibition were observed. BES was used as a positive control.
2.7 Identification of secondary metabolites in the seaweed samples using HPLC-Q-TOF mass spectrometry
The Q-TOF Premier mass spectrometer connected to an Alliance 2695 HPLC system (Waters Corporation, Milford, MA, USA) was used for untargeted analysis of metabolites of the seaweed hydrolysate and fermentate following the method of Hossain et al. 2010 [26] with slight modifications. Briefly, the separation of analytes was performed on an Atlantis T3 C18 column (100 x 2.1 mm, 3 µm) using a binary mobile phase of 0.1% aqueous formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The temperature of the column was set at 40°C. A stepwise gradient of solvent B was administered from 10–90% at a flow rate of 0.3 mL/min for 25 min. The capillary and cone voltages for the mass spectrometry were set at 3.0 kV and 50V, respectively, whilst the source temperature and desolvation temperatures were set at 120°C and 350°C, respectively. Electrospray ionisation (ESI) mass spectra were recorded for mass scan range m/z 100–2,000 in negative ion mode. Waters untargeted (MSe) mode was applied with the low collision energy set at 5 eV, and the high collision energy ramped between 15–40 eV. Accurate masses of the recorded m/z values were achieved through the internal reference compound (i.e. leucine-enkephalin) delivered through lock spray.
2.7.1 Phlorotannin oligomer characterisation
Phlorotannin oligomers were detected by direct infusion of samples into the electrospray ionisation source of the quadrupole time of flight mass spectrometry (Q-ToF-MS) (Waters Corp. Milford, USA) as described previously by Kirke et al., (2019) [27]. This was a non-bias screening method. Mass spectral data were obtained in negative electrospray ion mode (ES−) with mass scan ranging from 300 to 3000 m/z. The capillary and cone voltages were set at 2.6 kV and 35 V, respectively. The desolvation gas was set at 800 L h− 1 and the cone gas at 50 L h− 1. The samples were infused at 1 µL min− 1 for a total run time of 2 min.
The oligomer profile of phlorotannins for each extract was mapped using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (Waters Corp. Mil-ford, USA) following the method of Tierney et al. [28]. The system consisted of a binary pump solvent manager coupled with an Acquity™ TQD-MS (Waters Corp., Milford, USA). It was operated in negative electrospray ion (ES−) mode with multiple reaction monitoring (MRM) method. The MRM method was developed and optimized with a phlorotannin-enriched sample using Intellistart™ software (Waters Corporation) according to molecular masses of phlorotannins containing 3–16 phloroglucinol units (maximum permitted by TQD). A Waters Acquity ™ UPLC HSS PFP column was used (100 Å, 1.8 µm particle size, 2.1 mm × 100 mm) at 40°C. Mobile phases consisted of distilled water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The flow rate was 0.5 mL min− 1 and the injection volume set at 1 µL. The elution gradient was set as follows: 0.5% B from zero to 10 min, 0.5–30% from 10 to 26 min, 30–90% B from 26 to 28 min, and 0.5% B from 28 to 30 min. The capillary voltage was set at 2.8 kV. The desolvation gas and cone gas were set at 1200 L h− 1 and 50 L h− 1, respectively. The source temperature and desolvation temperature were set at 450°C and 150°C, respectively, for maximum sensitivity. Methanol blanks were injected between samples to ensure that the peaks observed were in fact those of the sample and not resultant from the retention of compounds on the column and subsequent carry-over from previously analysed samples. Due to the lack of commercial phlorotannin standards, individual molecular weights detected (374–1987 Da) could not be quantified and only the relative abundance was expressed.
2.7.2 Peptide analysis
The hydrolysate and fermentate 10-kDa permeate fraction were processed for MS analysis using the Preomics Phoenix Clean-up Kit (96×), (Preomics, D-82152 Planegg/Martinsried, Germany) in accordance with the manufacturers’ instructions and as described previously [29]. The sample was acidified and hydrophobic and hydrophilic contaminants removed using a series of wash steps and peptides eluted from the cartridge and prepared in loading buffer for LC-MS analysis. Peptides were identified using a mass spectrometer nano ESI-QqTOF (6600 plus TripleTOF, AB SCIEX, Framingham, MA, USA) using liquid chromatography and tandem mass spectrometry (LC–MS/MS). A total of 1 µL of microalgal permeate was loaded onto a trap column (3 µ C18-CL 120 Ᾰ, 350 µM × 0.5 mm; Eksigent, Redwood City, CA, USA) and desalted with 0.1% TFA (trifluoroacetic acid) at 5 µL/min during 5 min. The peptides were then loaded onto an analytical column (3 µ C18-CL 120 Ᾰ, 0.075 × 150 mm) equilibrated in 5% acetonitrile 0.1% FA (formic acid). Elution was carried out with a linear gradient from 7 to 45% B in A for 20 min, where solvent A was 0.1% FA and solvent B was ACN (acetonitrile) with 0.1% FA) at a flow rate of 300 nL/min. The sample was ionized in an electrospray source Optiflow < 1 µL Nano applying 3.0 kV to the spray emitter at 200°C. Analysis was carried out in a data dependent mode. Survey MS1 scans were acquired from 350 to 1400 m/z for 250 ms. The quadrupole resolution was set to ‘LOW’ for MS2 experiments, which were acquired from 100 to 1500 m/z for 25 ms in ‘high sensitivity’ mode. The following switch criteria were used: charge: 1 + to 4+; minimum intensity; 100 counts per second (cps). Up to 50 ions were selected for fragmentation after each survey scan. Dynamic exclusion was set to 15 s. The system sensitivity was controlled by analysing 500 ng of K562 protein extract digest (SCIEX); in these conditions, 2260 proteins were identified (FDR < 1%) in a 45 min gradient. Protein Pilot v 5.0. (SCIEX) default parameters were used to generate peak list directly from 6600 plus Triple TOF wiff files. The Paragon algorithm of ProteinPilot v 5.0 was used to search different databases. Peptides were identified with a confidence of ≥ 95%.
2.7.3 Volatile analysis
2.7.3.1 SPME Analysis - Sample preparation
0.5g of the fermentate was added to a 20 mL screw capped SPME vial and equilibrated to 40°C for 10 mins with pulsed agitation of 5 sec at 500 rpm. Sample introduction was accomplished using a Gerstel MPS auto-sampler.
2.7.3.2 GC-MS analysis for volatiles
GC-MS analysis was carried out as described previously [30]. Briefly, a single 50/30 µm CarboxenTM/divinylbenzene/polydimethylsiloxane (23Ga DVB/CAR/PDMS, Agilent Technologies Ireland Limited) fibre was used. The SPME fibre was exposed to the headspace above the samples for 20 min at depth of 1 cm at 40°C. The fibre was retracted and injected into the GC inlet and desorbed for 2 min at 250°C. Injections were made on a Shimadzu 2010 Plus GC with an Agilent DB-624 UI (60m x 0.32mm x 1.8µm) column using a split/splitless injector in a splitless mode. A merlin microseal was used as the septum. The temperature of the column oven was set at 40°C, held for 5 min, increased at 5°C/min to 230°C, held for 15 min, then increased at 15°C/min to 260°C, held for 5 min yielding at total GC run time of 65 min. The carrier gas was helium held at a constant flow of 1.2ml/min. The detector was a Shimadzu TQ8030 mass spectrometer detector, ran in single quad mode. The ion source temperature was 220°C and the interface temperature was set at 260°C. The MS mode was electronic ionization (70v) with the mass range scanned between 35 and 250 amu. Compounds were identified using mass spectra comparisons to the NIST 2014 mass spectral library, a commercial flavour and fragrance library (FFNSC 2, Shimadzu Corporation, Japan) and an in-house library created using authentic compounds with target and qualifier ions and linear retention indices for each compound using Kovats index. Retention indices were matched against peer reviewed publications where possible to confirm compound identification. Spectral deconvolution was also performed to confirm identification of compounds using AMDIS. Batch processing of samples was carried out using MetaMS [31]. MetaMS is an open-source pipeline for GC-MS-based untargeted metabolomics. An auto tune of the GCMS was carried out prior to the analysis to ensure optimal GCMS performance. A set of external standards: 1-butanol, dimethyl disulphide, butyl acetate and cyclohexanone (Sigma Aldrich Ltd, Arklow, Ireland) at 10ppm concentration were run at the start and end of the sample set and abundances were compared to known amounts to ensure that both the SPME extraction and MS detection was performing within specification.
2.8 FAME analysis
Lipids from the fermentate and hydrolysate were extracted using an ethanol: ethyl acetate mixture (1:1, w/w), as described by Lin et al. [32]. Extracted lipids were then directly converted to fatty acid methyl esters (FAMEs) using boron trichloride in methanol (14%, w/v) without previous derivatization, with slight modifications of the method previously described by Araujo et al. [33]. Separation and analysis of the FAMEs was done using an Agilent 7890A/5975C GC-MSD system (Agilent Technologies, Santa Clara, CA, USA) equipped with Agilent J&W DB-Fast FAME column (30 m × 0.25 mm, 0.25 µm). A modified method from Agilent Application note 5991-8706EN was used for analysis [34]. Hydrogen was used as carrier gas in constant pressure mode at 8 PSI and sample injection volume was 1 µL in inlet split mode (25:1). The temperature program of the oven was as follows: 50°C (0.5 minutes), then 15°C/min to 194°C (4 minutes) and finally 4°C/min to 240°C (1 minute). Mass spectra were acquired in scan mode in the 40–550 AMU mass range.
Chromatographic peaks (total ion chromatogram, TIC) were identified by comparison of retention times (RT) and mass spectra with peaks of analysed FAMEs in the standard mixture. Identification was confirmed by searching the generated MS spectra within available spectral database (NIST11) using Agilent ChemStation software. Supelco Fame 37 mix (Sigma Aldrich, Darmstadt, Germany) was utilized for external calibration by making series of appropriate dilutions with hexane, with individual compound peaks used to construct a five-point calibration curve. Each individual FAME was quantified by measuring response (peak area after integration) of a selected quantifier ion from the compound spectrum using ChemStation software. 1 mL of glyceryl-tri-heptadecanoate (Sigma Aldrich, Darmstadt, Germany) in hexane (1 mg/mL, w/v) was added to lipid samples prior to derivatization, to correct for transesterification efficiency and procedural losses. All samples were analysed in triplicates and results expressed as concentration (mg/g) of each fatty acid in extracted fat.
2.9 TGA analysis
The A. nodosum fermentate was analysed thermogravimetrically to determine the cellulosic material present in the samples using a Mettler-Toledo GmbH TGA analyser instrument no. B848840113; serial no. 1630/1 (Mason Technology, Switzerland). Approximately 10–15 mg of the sample was placed in a special aluminium crucible and subsequently in the sample holder. The temperature range selected for the assay was 25 to 500°C with a constant heat rate of 10°C min− 1 and a Nitrogen gas flow rate of 30 mL min− 1. Cellulose (Merck, Dublin, Ireland) was used as a standard. Mathematical derivations were calculated using the Star e software (version: V16.00).
2.10 Protein digestibility-corrected amino acid score (PDCAAS) analysis
The protein quality of the developed animal feeds was determined by assessing the amino acid composition of each feed samples by using the in vitro Protein Digestibility corrected amino acid score (PDCAAS) enzyme digestion method, a Megazyme assay kit (Megazyme, Wicklow, Ireland). Detailed method for PDCAAS evaluation was conducted following the steps outlined in the study by De Bhowmick and Hayes (2022) [35]. Briefly, protein samples were digested sequentially as previously described using pepsin, trypsin, and chymotrypsin. The undigested proteins were removed by trichloroacetic acid (TCA) precipitation technique. α-Amino acid concentration was determined by Ninhydrin based optical density (recorded at 570 nm) method using L-Glycine Standards. Separate reagent solutions were prepared as follows: (i) Hydrochloric acid (0.06 N, pH 2.0); (ii) Trichloroacetic acid (40% w/v); (iii) Hydrochloric acid (0.001 N, pH 3.0); (iv) Tris Buffer (1.0 M, pH 7.4); (v) Sodium acetate buffer (50 mM, pH 5.5); (vi) Sodium acetate buffer (50 mM, pH 5.5); and (vii) Ninhydrin Reagent (2% solution). Subsequently, 500 mg of the three milled (ball mill - Mixer mill MM 400; Retsch; Ireland) samples were used for the assay. Following the step-by-step method15 enzymes pepsin and trypsin/chymotrypsin were added to the sample mixture with in between incubation at 37°C in a shaking incubator (Maxq 8000; model no: 444; serial no: 185550-31; Thermo Fisher Scientific; made in USA) set at 150 rpm. At the end of enzyme digestion, the samples were placed in a boiling water bath for 10 min for enzyme deactivation. Following incubation, the samples were removed from the water bath and vortexed. Finally, after completely cooling down the samples, 1 mL of 40% TCA solution was added to the mixture and incubated overnight at 4°C. Furthermore, acetate buffer (50 mM, pH 5.5) was used to make 10 to 20 fold dilutions for each of the samples. Subsequently, the diluted samples were further tested following the Ninhydrin assay.
2.11 Confocal microscopy
A small spatula (~ 10 mg) of the A. nodosum fermentate was placed onto a slide. 100 µL of a stain mixture comprising Nile Red (0.10 g/L in polyethylene glycol 200), Fast green (0.01 g/L in water) and Fluorescent Brightener 28 (0.125 g/L in water) mixed together in a ratio of 3:1:3 respectively, was added to the surface and a coverslip placed on top. The sample was examined with a Leica SP5 confocal scanning laser microscope (Leica Microsystems, Mannheim, Germany) using blue diode (405 nm), argon (488 nm) and HeNe (633 nm) lasers in three channels simultaneously. Cellulosic material stained by the fluorescent brightener is shown in blue. Material within the cell wall is indicated by the red colour (stained by Fast green) and the Nile Red labels trace amounts of lipid a green colour. Sequential images were acquired using triple channel imaging. Emission signals for FB28, Nile Red and Fast Green were sequentially collected using band pass filters 450–490 nm; 510–550 nm and 650–700 nm, respectively. Digital 8-bit images (512 x 512 pixels) were obtained for each separate excitation wavelength using 10x air and 63x oil immersion lenses and combined images were collected.
2.12 Statistical analysis
All the experiments were performed in triplicates and the results of each study were expressed as the mean ± standard deviation (SD) of the three independent analysis. Additionally, one way ANOVA and p- values were determined and excel ANOVA packages were used for the analysis.