Selection of Potential Yeast Probiotics and a Cell Factory for Xylitol or Acid Production from Honeybee Samples

Abstract

Excessive use of antibiotics has detrimental consequences, including antibiotic resistance and gut microbiome destruction. Probiotic-rich diets help to restore good microbes, keeping the body healthy and preventing the onset of chronic diseases. Honey contains not only prebiotic oligosaccharides but, like yogurt and fermented foods, is an innovative natural source for probiotic discovery. Here, a collection of three honeybee samples was screened for yeast strains, aiming to characterize their potential in vitro probiotic properties and the ability to produce valuable metabolites. Ninety-four isolates out of one-hundred and four were able to grow at temperatures of 30 °C and 37 °C, while twelve isolates could grow at 42 °C. Fifty-eight and four isolates displayed the ability to grow under stimulated gastrointestinal condition, at pH 2.0-2.5, 0.3% (w/v) bile salt, and 37 °C. Twenty-four isolates showed high autoaggregation of 80-100% and could utilize various sugars, including galactose and xylose. The cell count of these isolates (7-9 log cfu/mL) was recorded and stable during 6 months of storage. Genomic characterization based on the internal transcribed spacer region (ITS) also identified four isolates of Saccharomyces cerevisiae displayed good ability to produce antimicrobial acids. These results provided the basis for selecting four natural yeast isolates as starter cultures for potential probiotic application in functional foods and animal feed. Additionally, these S. cerevisiae isolates also produced high levels of acids from fermented sugarcane molasses, an abundant agricultural waste product from the sugar industry. Furthermore, one of ten identified isolates of Meyerozyma guilliermondiii displayed an excellent ability to produce a pentose sugar xylitol at a yield of 0.490 g/g of consumed xylose. Potentially, yeast isolates of honeybee samples may offer various biotechnological advantages as probiotics or metabolite producers of multiproduct-based lignocellulosic biorefinery.

Keywords: acid production; functional food; honey; probiotics; xylitol; yeast.

Figure 1

Isolation and characterization of yeast isolates obtained from raw honeybee samples. (A) Honeybee products; (B) selected images of yeast cells by microscopy; (C) thermo-tolerance (30, 37, 42 °C) of KTF, RBF and RSO isolates grown on YPD plates containing 300 mg/L of Ampicillin; (D) autoaggregation assays of some selected isolates performed after 2, 4 and 24 h. incubation at 37 °C; (E) growth of KTF and RBF isolates on 2% galactose containing plates and (F) spot test of RSO isolates, BY4742 and SB strains on Yeast peptone (YP) plates, containing 20% glucose, 20% or 2% galactose. Results were obtained from at least two independent experiments performed in triplicates.

Figure 2

Phylogenetic tree displaying the relationships between some identified yeast isolates (KTF3, KTF5, KTF16, BRF37, BRF47 and BRF56) of Meyerozyma and (RSO1, RSO2, RSO3 and RSO4) of Saccharomyces obtained from honeybee samples (Table 1). For comparison, based on ITS region sequences from type strains of different species of Meyerozyma and Saccharomyces which are retrieved from the literature and NCBI database are shown. The phylogenic tree was constructed using the neighbor-joining method. Bootstrap values were calculated from 1000 replicates. ^T symbol indicates the “Type Strain” that is used as a reference.

Figure 3

Organic acids production of isolated S. cerevisiae RSO2 and RSO3 strains from fermentation of 5% sugarcane molasses. Concentrations of oxalic, citric, malic, succinic, formic and acetic acids (g/L) were determined by HPLC-UV or RI analysis and expressed as a percentage of the maximum concentration observed for each metabolite. Results were obtained from at least two independent experiments performed in triplicate. Significant differences of organic acid concentrations produced at different timepoints in comparison with the initial timepoint (at 0 h) were determined by one-way ANOVA with the Tukey HSD method (*, p < 0.05; **, p < 0.01; ****, p < 0.0001). Error bars indicate SD.

Figure 4

Xylitol production during fermentation of xylose by a selected M. guilliermondii isolated from the Apis dorsata Fabricius raw honeybee sample. (A) The growth curve indicated the optical density of cells at OD₆₀₀ and changes in xylose and xylitol concentration (g/L) of M. guilliermondii during 10 days of fermentation with 10% (w/v) xylose at 30 °C. (B) Survivability of M. guilliermondii and S. cerevisiae BY4742 on YP plates containing 2% (w/v) glucose, 2% or 10% (w/v) xylose as a carbon source with or without 12 mM lignocellulosic inhibitor furfural. Results were obtained from at least two independent experiments performed in triplicates.