Wild yeasts isolation
Using a sterile swab soaked in YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% bacteriological peptone and 1% glucose) there was a friction on surface of the fruits strawberry (Fragaria x ananassa), blackberry (Morus nigra), cherry (Eugenia uniflora), orange (Citrus x sinensis) and butiá (Butia capitata), being subsequently inoculated in a tube containing YM added with ampicillin (100 µg/µL). Cultures were incubated for 48 h at 28 °C with constant agitation of 150 rpm. For flowers and leaves of orchid (Aspasia lunata), pitaya (Hylocereus undatus), vine (Vitis vinífera), and Red Flanders and Old Ale beers, the same procedure was performed. All samples collected for yeasts isolation were obtained in the city of Pelotas (Latitude -31.776, Longitude -52.3594 31° 46’ 34” South, 52° 21’ 34” West), throughout spring/summer period, with daily temperatures around 25 to 30 °C.
Yeasts were isolated by streaking samples from cultures on YM agar + ampicillin. Plates were kept at 28 °C for 72 h for yeasts growth. The yeast morphology was observed using optical microscope BLUE1600BA-L-BT (Biofocus, Brazil) at 1000x magnification. Isolates were stored by freezing (- 80°C) using glycerol 30%, with identification codes being given to each isolate.
Fermentation of standard beer wort
In order to evaluate fermentation ability in standard beer worts, 400 mL of malt extract (Dry Brew, Liotecnica, Brazil) with a density of 11 °P, pH 5.0, sterilized by autoclaving for 15 min at 121 °C was used. Iso-α-acid from hops (HOPSTEINER, Germany) was added to final 15 IBU (International Bitterness Units). All isolates were cultivated in YM medium over 48 h, 28 °C and 150 rpm condition prior fermentation test. Yeast cells concentration was obtained by counting in a Neubauer chamber, then inoculum for fermentation was standardized at a concentration of 109 cells, which were obtained by centrifugation at 1.500 × g for 5 min in DTR-16000 centrifuge (DAIKI, Korea). Cells were suspended with 15 mL of malt extract 11 °P, subsequently inoculated in the total volume for fermentation.
Fermentation was conducted during 14 days, in an incubator at 22 °C, with no agitation. Production and release of CO2 was monitored through bubble formation in an air-lock piece, while biofilm formation, on medium surface, was visually observed. After this period, final density and pH were evaluated using a pHmeter (KASVI, Brazil), a densimeter (INCOTERM, Brazil) for density in g/cm³ measure and a refractometer (AKSO, Brazil) for measure in Brix degrees, data used to calculate percentage of attenuation by each isolate. Aroma resulting from malt extract fermentation was evaluated by simple sensory analysis, as conducted in Osburn et al. (2016) study. Saccharomyces cerevisiae YT001 (YEASTECH, Brasil) was used in this experiment as a reference yeast.
DNA extraction, PCR and sequencing
Total DNA from each isolate was extracted following the protocol described by Preiss et al. (2018)and its concentration quantified in Nanovue™ (Biochrom). Identification at species level was performed using ITS1 and ITS4 primers, which are responsible for amplifying the repetitive region of the 5.8S rRNA gene and ITS flanking regions (Internal Transcribed Spacer). PCR reaction was performed using 0,7 uL of previously extracted DNAs (1 ug/uL), 22 uL of Master Mix (Ludwig Biotecnology, Brazil), 1 uL (0.5 uM) of ITS1 primer (5′TCCGTAGGTGAACCTTGCGG) and 1 uL (0,5 uM) of ITS4 primer (5′TCCTCCGCTTATTGATATGC). Following incubation conditions for PCR were used: initial denaturation at 95 °C for 10 min, 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min and extension at 72 °C for 2 min, and then final extension at 72 °C for 8 min. PCR products sizes were analyzed by electrophoresis of 10 uL in 1% agarose gel (w/v) in TBE buffer (Tris 0.89 M, EDTA 0.02 M, Boric Acid 0.89 M), under 100 V, 500 mA over 2 h. Agarose gels were stained with 5 uL of ethidium bromide and bands sizes were predicted by comparison with 1 kb Plus DNA Ladder (ThermoFischer Scientific, United States).
The PCR product of each isolate was purified using GFX PCR DNA and Gel Band Purification (GE Healthcare, United States), quantified with NanovueTM and then submitted at an approximate concentration of 50 ng/l uL for sequencing by ACTGene Análises Moleculares company (Brazil) through Applied Biosystems AB-3500 platform.
Characterization of yeasts isolates
Evaluation of yeast growth in different stress conditions
Wild yeasts Hanseniaspora uvarum (PIT001), Pichia kluyveri (LAR001) and Candida intermedia (ORQ001) were selected in addition to S. boulardii, a yeast which was already part of microorganism bank of Microbiology Laboratory in the Technological Development Center of the Federal University of Pelotas.
Cultivation tests were performed in 10 mL of YPD medium (1% yeast extract, 2% bacteriological peptone, 2% glucose), following a protocol adapted from Zeng et al. (2019). Analysis of resistance to different conditions were tested: lactic acid 1%, acetic acid 0.2% and pHs 2.5, 6.0 and 8.0. Yeast cultures were maintained for 72 h, with samples being collected at 0 h, 4 h, 24 h, 48 h and 72 h, analyzing their biomasses by absorbance reads at 600nm in Biochrom Ultrospec-10 spectrophotometer (Amersham Biosciences, United States) and CFU/mL (Colony Forming Units). Incubation temperature was maintained constant at 28 °C, in an orbital shaker at 150 rpm. In addition, yeast growth was evaluated in YPD pH 6.0 at 37 °C, following same procedures for samples collection and incubation of previous tests. Growth index (GI) was calculated based in Zeng et al. (2019) and Bevilacqua et al. (2009) method, following the equation: GI= AbSs/AbSc × 100, where AbSs is the absorbance of the samples in different pH, temperature and acid type presence, and AbSc is the absorbance of the control samples. Values < 25% were considered inhibitory for microorganisms’ growth, between 25% - 75% partially inhibitory and > 75% growth was similar to respective yeast culture in optimal conditions (YPD pH 6.0, incubated at 28 °C).
Selected yeasts were also cultivated in YPD with different concentrations of Sodium Chloride (NaCl) salt, ranging from 0.5%, 1%, 5% and 10% (p/v), to assess the ability to tolerate ionic stress by halotolerance. Yeast growth was evaluated after 48 h at 28 °C by optical density, and if OD600nm ≥ 1.0, it was considered that yeast isolate was able to survive and grow in this condition.
Sensitivity to α-acids (spot test)
The analysis of yeast tolerance to α-acids from hops was performed based on adapted protocol from (Samanfar et al., 2017). Aiming the evaluation of yeast sensitivity to α-acids, it was added to YM agar medium 200 ppm of isomerized hop extract 30% (HOPSTEINER, Germany), and into plates containing this medium were applied 10 µL of 10X dilutions of yeast cultures in initial concentration of 108 CFU/mL. Plates were incubated for 48 h at 28 °C.
Determination of proteolytic activity
Protease activity was evaluated following protocol established by Zeng et al. (2019). Briefly, skim milk agar medium was used (1% skim milk, 0.1% glucose, 0.5% bacteriological peptone, 0.25% yeast extract, 1.5% agar), in which 10 µL from yeast cultures at 108 CFU/mL were applied. Positive result should be observed with formation of a halo around the drop, after an incubation during 48 h at 30 °C.
Gelatinase activity was evaluated according to Pereira et al. (2009) protocol. Culture medium was prepared with 1% yeast extract, 1.5% bacteriological peptone and 12% gelatin. Isolates were cultured for 24 h at 28 °C, then transferred to the medium containing gelatin with a platinum loop. Tubes were incubated at 30 °C for 7 days, and after that were maintained under refrigeration (4 °C – 10 °C) for 30 minutes. Positivity (i.e. enzymatic activity) in the test was based on the conversion of semi-solid medium into liquid. As a positive control, Staphylococcus aureus ATCC 25923 was used.
Auto-aggregation and co-aggregation
Auto-aggregation and co-aggregation tests were performed following Collado et al. (2008) protocols. Briefly, yeasts were cultivated for 24 h at 30 °C in YM medium, centrifuged for 5 min at 2.000 × g and absorbance OD600nm adjusted to 0.25 ± 0.02 using Phosphate-Buffered Saline (PBS). Auto-aggregation test was conducted under 18 °C, 30 °c and 37 °C. Absorbance at 600nmwas read at 2h and 20 h and these values were used in the equation [1-A600nm of final suspension/A600nm of initial suspension) x 100] for results as percentage.
In the co-aggregation test, pathogens Escherichia coli ATCC8739 (Gram-negative bacterium) and Listeria monocytogenes ATCC 7644 (Gram-positive bacterium) were cultivated for 24 h at 37 °C in Brain Heart Infusion (BHI) medium, centrifuged during 5 min at 8.000 × g, then their absorbances at 600nm were adjusted to 0.25 ± 0.02 using PBS solution. Equal volumes of pathogen and yeast were mixed (1:1) and incubated under the same conditions as performed in auto-aggregation test, as well as absorbance reads times. Results for co-aggregation were expressed as percentage, originated from the equation [(Apat + Aisol) – (Amix)/(Apat + Aisol)] x 100, in which “Apat + Aisol” represents the absorbance value for pathogen + isolate in time 0 h and “Amix” is the absorbance of microorganisms’ mixed suspensions in different periods of time.
Antimicrobial activity test
Bacteria related to foodborne diseases (FBDs) Pseudomonas aeruginosa ATCC27853, L. monocytogenes ATCC 7644, S. aureus ATCC25923, E. coli ATCC8739 and a wild isolate of Klebsiella sp. were used to test the capacity of isolated yeasts to inhibit growth of bacterial pathogens. Antimicrobial activity test was carried out according to the double-layer technique protocol presented by Amorim et al. (2018), with values of inhibition halos being expressed in millimeters (halo + drop). Following identifications were designated for inhibition capacity: + + + (inhibition zone diameter ≥ 24 mm), + + (16 – 23 mm), + (8 – 16 mm) and – (≤ 7mm); so, the larger the halo, greater the inhibitory power by yeast.
Gastrointestinal tract in vitro simulation
Gastrointestinal tract simulation tests were performed based on protocol described by Bonatsou et al. (2015), with some adaptations. Simulation of GI tract conditions was performed using two solutions: Gastric Digestion (GD) and Pancreatic Digestion (PD). Gastric digestion solution was prepared with NaCl (2.05 g/L), KH2PO4 (0.60 g/L), CaCl2 (0.11 g/L), KCl (0.37 g/L), pH 2.0 adjusted with HCl 1 M and autoclaved for 15 min at 121 °C; after sterilization, pepsin (0.0133 g/L) and lysozyme (0.01 g/L) were added. Pancreatic digestion solution was prepared with bile salts (3.0 g/L), Na2HPO4 (26,9 g/L), NaCl (8,5 g/L), pH 8.0 adjusted with HCl 1 M; after sterilization, pancreatin enzyme (0.1 g/L) was added. Yeasts were submitted to simulations of GD and PD, with solutions applied separately and in sequence.
Initially, 1 colony was selected from fresh cultures streaked on YPD agar to be inoculated in 10 mL of YPD medium, with incubation at 28 °C until reaching 108 CFU/mL concentration. After collecting a sample, yeast cultures were centrifuged at 1.500 × g for 10 min, washed with GD solution, and then suspended in 10 mL of the same solution. Incubation was carried out at 37 °C for 2.5 h, under agitation at 200 rpm in order to simulate peristaltic movements, and at test ending a sample was collected. Then cultures were centrifuged again at 1.500 × g for 10 min, washed with PD solution, and suspended in 10 mL of the same solution. Yeast cultures remained at 37 °C for 3.5 h, under the same agitation condition as described in previous test. Viable yeast cells were detected according to yeast counts before and after each treatment.
Concentrations of viable yeast cells were used to determine viability index (VI) during the test, based on the equation proposed by Zeng et al. (2019): VI=log Nt/log N0 x 100, in which Nt is related to yeast concentration at specific time and N0 refers to the initial cell concentration. The test was also performed in an isolated manner, in which it was split in two independent tests, one with GD solution and another one with PD solution, aiming to obtain specific VI for PD step. Again, yeasts started these tests with a concentration of 108 CFU/mL.
Data were analyzed by analysis of variance (ANOVA) and Student’s t test to determine significant difference (p<0.05) between means. All statistical analyzes were performed using GraphPad Prism 7 software.