Yeast strains and sorghum wort

The wild yeasts (traditional inoculum) and sorghum wort used in this study have been collected from randomly selected commercial traditional sorghum beer brewers at Williamsville-Macaci in the District of Abidjan (Southern Côte d’Ivoire). The slurry of wild yeasts was been sun dried into brown powder and lumps.

Fermentation conditions

Fermentation was carried out under agitation in rotary shaker 1-L sterile flasks fitted with a shaker. The flasks were filled with 500 mL sorghum wort which was previously pasteurized at 100 °C for 10 min and cooled to ambient temperature and closed with dense cotton plugs. Five (05) grams of wild yeasts which correspond to an inoculation rate of 1% (2.107 CFU/mL) were added to the wort. Fermentation was carried out during 24 h at 30 °C with agitator speed of 120 rpm. During latter, samples were collected to 0, 4, 8 and 24 h for physicochemical, microbiology and biology molecular analyses. Three independent experiments were carried out.

Analytical determination

pH, titratable acidity, density and total soluble solids

pH values were determined using a pH-meter (Hanna Instruments; HI 8010) and titratable acidity was measured by titration with 0.1 N NaOH and expressed as % meq of lactic acid in 10 mL of sample respectively. A densimeter (Mettler Toledo) was used to determine the density of each beer in 100 mL of sample and hand refractometer was used to determine total soluble solids (TSS) content expressed as °Brix from two or three drops of sample. For each parameter, three independent measurements were made.

Alcohol level determination by specific gravity method

The alcohol level was determined from original gravity (OG) (Density of sorghum wort) and final gravity (FG) (Density of beer) measured using a densimeter as previously described and expressed as follows:

$$Alcohol rate (mathrm{% }v/v)=frac{OG-OF}{0.0075} (1)$$

Microbiological analysis

Isolation and enumeration of yeasts

After decimal dilutions in the YPD medium (10 g/L yeasts extract (Difco)); (10 g/L bactopeptone (Becton Dickinson)); (10 g/L D-glucose (Sordalab)), counts were carried out in duplicate on YPDA (10 g/L yeasts extract (Difco)); 10 g/L bactopeptone (Becton Dickinson)); 10 g/L D-glucose (Sordalab)); Agar 10 g/L (Oxford) medium. The incubation period was two days at 30 °C. The results of the counts were expressed as log (CFU/mL) of fermenting wort and beer.

Isolation of non-Saccharomyces yeast strains

After enumeration on YPDA, 30 colonies were selected at each sampling time on the basis of size and appearance of colony outline out of a total of 300 colonies. This selection had been repeated two times (biological repetition). The selected colonies were plated on lysine agar medium (Oxoid LTD., Hampshire, England), a selective medium used for to differentiate between non-Saccharomyces and S. cerevisiae strains. The Saccharomyces cerevisiae NRRL Y-12632 strain has been used as negative control. This medium does not allow the growth of S. cerevisiae because these species are unable to use lysine for their growth. Isolates where growth was observed were retained for identification by PCR-RLFP followed sequencing.

Molecular identification of non-Saccharomyces yeast isolates

Amplification of the ITS region

PCR reaction method performed in this work has been described by Jeyaram et al. [41] on colonies with some modifications. Fresh yeast colonies (less than 5 days old) grown on Sabouraud Chloramphenicol agar were transferred in to 100 mL of molecular biology water under sterility conditions. For the amplification of the 5.8S rDNA-ITS region, the primers ITS1 (5′ TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATA-3′) were used as described by White et al. [42]. Amplification reactions of the 5.8S rDNA-ITS region were performed using a thermal cycler (Techne Prime). Thermocycler (Techne Prime, UK) according to the program described by Aa Kühle et al. [29] with some modifications:

-First step: Initial denaturation at 95 °C for 10 min.

-Second step: denaturation at 94 °C for 2 min followed by primers annealing at 55.8 °C for 1 min and extension at 72 °C for 2 min (30 cycles).

-Third step: final extension at 72 °C for 7 min marking the end of the amplification reaction. The size of the amplicons were determined by electrophoresis on a 1.5% agarose gel containing ethidium bromide (BET). Amplicons were stored at -20 °C for further use.

Restriction analysis

Digestion of the amplicons was performed using the restriction enzymes Hae III (Haemophilus aegyptius) and Hinf I (Haemophilus influenzae) reported in the literature [43, 44]. It was performed in Eppendorf tubes containing 20 µL of the reaction mixture. The Eppendorf tubes with amplicons were incubated in a dry water bath (Biobase, China) at 37 °C for 6 h. The sizes of digested fragments were analyzed on 3% agarose gel containing ethidium bromide (BET).

Yeast identification by sequencing and sequence analysis

The method used for sequencing was the same that was described by N’guessan et al. [14]. All amplicons digested used to carry out the sequencing. For sequencing, the primers used (NL1 5’-GCATATCAATAAGCGGAGGAAAAG-3’ and NL4 5’-GGTCCGTGTTTCAAGACGG 3’) have been described by Kurtzman and Robnett [45]. The PCR mix was prepared in the same conditions as for amplification of the ITS region. PCR was carried out in 5 steps: firstly an initial denaturation at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 30 s, then primers annealing at 54 °C for 40 s and DNA extension at 72 °C for 1 min 30 s. The last step which was the final extension was completed at 72 °C for 7 min.

PCR amplification and sequencing of the ACT1 gene and the ACT1 gene intron was carried out according to the method described by Jacques et al. [46]. The sequencing of amplified fragments was performed by the company Eurofins MWG Operon (Ebersberg, Germany). The sequences obtained were compared by using basic local alignment search tool (BLASTN) to provide sequence similarity with the sequences available in the National Center for Biotechnology Information (NCBI) GenBank databases (http://www.ncbi.nlm.nih.gov). A multiple sequence alignment was performed, and the phylogenetic tree was constructed by the neighbor-joining method [32] using MEGA (Molecular Evolution Genetic Analysis) software, version X [33].

Fermentation characteristics of identified yeasts species: Issatchenkia orientalis and Pichia kudriavzevii

Growth at different pH

Antunovics et al. [47] developed a modified approach to assess the growth of the identified species at different pH (3, 4, 5, 6). YPD broth (10 g/L yeast extract, 10 g/L bactopeptone, 10 g/L D-glucose) was previously used to culture the strains at 25 °C. Each specie was then inoculated into 50 mL of YPD broth at 0.2 OD. The culture flasks were then incubated for 24 h with agitation at 150 rpm. After 24 h, the optical densities (OD) were measured at 600 nm with a clear spectrophotometer view UV translluminator/visible spectrophotometer, and the growth of each species was assessed. The experiments were replicated three times.

Growth at different temperatures

The growth at different temperatures was carried out according the method described by Antunovics et al. [47]. Samples of overnight cultures (grown at 25 °C) were inoculated into broth YPD broth at O.D. 0.2 and incubated in a water-bath shaker at 30, 40, and 50 °C for 24 h for comparison of growth at different temperatures. The optical density at 600 nm was used to monitor the increase in cell population. The experiments were replicated three times.

Analysis of Issatchenkia orientalis and Pichia kudriavzevii resistance to ethanol

The yeast strains were tested for their capacity to grow under ethanol stress using a slightly modified method described by Carrasco et al. [48]. The strains were transferred from Sabouraud-chloramphenicol plates to a pre-culture medium of 10 ml YPD broth (10 g/L yeast extract, 10 g/L peptone, 10 g/L D-glucose) and cultured for 24 h at 30 °C. The pre-cultures were used to inoculate 50 mL of YPD broth supplemented with 0%, 5% percent, 10%, and 15% ethanol (v/v) at an initial cell concentration of 0.2 OD600nm. The cultures were incubated at 30 °C with rotation at 150 rpm for 24 h and tested for vitality at the end of the incubation period. The experiments were replicated three times.

Traditional sorghum beer production from Issatchenkia orientalis and Saccharomyces cerevisiae

Starter preparation

A dense suspension of each specie Issatchenkia orientalis and Saccharomyces cerevisiae from a Sabouraud-Chloramphenicol plate was prepared in 4 ml of sterile distilled water using a loop. The suspension was mixed with 40 mL of sterile sorghum wort taken from a traditional brewer in Williamsville Macaci (district of Abidjan). Each starter inoculum was incubated at 30 °C for 24 h. The S. cerevisiae F12-7 yeast strain was employed was from the culture collection of the Food Technology Department of University Nangui-Abrogoua, Abidjan, Côte d’Ivoire. It was isolated from sorghum beer from the Abidjan district (Southern Côte d’Ivoire). Polymerase Chain Reaction-Restriction Fragment Length polymorphism (PCR–RFLP) of the Internal Transcribed Spacer (ITS) region and sequencing of the 26S rRNA gene’s D1/D2 domains were used for its identification [14]. The yeast strain was stored at 20 °C in 20% glycerol.

Alcoholic fermentation

Fermentations were carried out in 500 mL Erlenmeyer flasks containing 400 mL sterile wort and closed with a cotton cap. The flasks were inoculated with each specie (Issatchenkia orientalis and Saccharomyces cerevisiae) at O.D. 0.5 and shaken at 120 rpm for 16 h at 30 °C. The fermentations were carried out three times.

Analytical Assays

Physicochemical analysis

Specific gravity, pH, titratable acidity (TA), total solids soluble (TSS), alcohol rate have been determined as previously described.

Organic acids analysis

Organic acids (tannic acid, citric acid, fumaric acid, acetic acid, tartric acid, and lactic acid) were separated and quantified using an HPLC system (Shimadzu Corporation, Japan) fitted with a pump (Shimadzu LC-6A Liquid Chromatograph), a detector (Shimadzu SPD-6A UV Spectrophotometric detector), and an Integrator (Shimadzu C-R 6A Chromatopac). An ion-exclusion ORH-801 column was used for chromatographic separation (300 mm × 6.5 mm, Interchrom, France). The eluant was 0.004 N H2SO4 at a flow rate of 0.8 mL/min, with a 210 nm detector. For HPLC samples, a 20 µL injection volume was used. The analysis was carried out twice and the mean values were used. The standards for organic acids were dissolved.

Statistical analysis

Statistical analysis was used to process the data obtained. Analysis of variance (ANOVA) was performed using XLStat software (version 2016). Mean values physicochemical parameters of fermenting wort and beers were analyzed with Duncan and Tukey’s tests. Differences were considered significant for values of P < 0.05. Principal component analysis (PCA) was performed using XLSTAT in order to visualize relationships among variables represented by beers compounds.

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