Probiotic yeast characterization and fungal amplicon metagenomics analysis of fermented bamboo shoot products from Arunachal Pradesh, northeast India

Abstract

This study investigates the diverse fungal community and their probiotic functions present in ethnic fermented bamboo shoots of Arunachal Pradesh. Among 95 yeast isolates, 13 demonstrated notable probiotic attributes. These included growth at pH 3, bile tolerance, autoaggregation, co-aggregation, hydrophobicity, lysozyme tolerance and antimicrobial activity. Confirmation of some of the probiotic properties through specific primers enabled the detection of genes associated with acid and bile tolerance, antimicrobial activity, and adhesion. Probiotic yeasts were finally identified based on D1 and D2 sequences of large ribosomal subunit as Meyerozyma guilliermondii (BEP1, KGM1_3, NHR3), Meyerozyma caribbica (GEP7), Candida orthopsilopsis (ES1_2, EB1_2, EEGM2_4, GEP2, NEK9), Candida parasilopsis (HD1_1), Pichia kudriavzevii (NHR12), Pichia fermentans (BEP2), and Saccharomyces cerevisiae (NEP2). Fungal amplicon sequencing highlighted the predominance of Ascomycetes, particularly Pestalotiopsis and Penicillium genera. In this study we have perfomed a culture dependent isolation and probiotic study of yeasts and culture independent analysis of the fungal community present during the fermentation of bamboo shoots of Arunachal Pradesh which provides information about the beneficial properties of bamboo shoots as the reservoir of probiotic microorganisms.

Keywords: Fermented bamboo shoot; Fungal amplicon sequencing; Predictive functionality; Probiotic; Yeast.

Fig. 1

Flow chart representing the methodology involved in the culture dependent and culture independent study performed in the FBS of Arunchal Pradesh.

Fig. 2

Yeast isolated showing tolerance to acid at pH 2, and pH 3 and pH 6.8 (control). Data represent the mean ± SD, standard deviation of three independent replication as indicated by the vertical line. The vertical bars with different alphabets are statistically significant at p < 0.05.

Fig. 3

Yeast isolates exhibiting tolerance to cholic acid, oxbile and taurocholic acid at 0.3 %, 0.5 %, and 1 %. Data represent the mean ± SD, standard deviation of three independent replicates.

Fig. 4

Co-aggregation property of selected yeast isolates with B. cereus, E. coli, S. aureus, and S. enterica. Data represent the mean ± SD, Standard deviation of three independent replicates as indicated by the vertical line. The vertical bars with different alphabets are statistically significant at p < 0.05.

Fig. 5

Autoaggregation percentage of yeast isolates. Increase in autoaggregation percentage of yeast isolates are statistically significant at p < 0.05.

Fig. 6

The hydrophobicity percentage shown by the selected yeast isolates. Data represent the mean ± SD, standard deviation of three independent replicates as indicated by the vertical line. The vertical bars with different alphabets are statistically significant at p < 0.05.

Fig. 7

Bar plot shows the survival percentage of yeast isolates in lysozyme. Data represent the mean ± SD, Standard deviation of three independent replicates as indicated by the vertical line. The vertical bars with different alphabets are statistically significant at p < 0.05.

Fig. 8

The selected isolates showing sensitivity against selected antifungal agents.

Fig. 9

Phylogenetic tree of yeast isolates constructed using MEGA11 and iTOL software where names in blue colour font represent yeast isolates obtained from FBS of Arunachal Pradesh, whereas the names in the red colour font represent the Refseq extracted from NCBI. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

a. Probiotic genes identified using specific primers TPS1 for acid tolerance. b.Probiotic genes identified using specific primers FLOR5 for adhesion. L is the ladder well and rest of the wells were for the yeast isolates.

Fig. 11

a. Probiotic genes identified using specific primers Apid for antimicrobial activity. Probiotic genes identified using specific primers YOR1 for bile tolerance. L is the ladder well and rest of the wells were for the yeast isolates.

Fig. 12

a. PCA plot of Beta diversity obtained using Bray-Curtis algorithm. b. PCA plot of Beta diversity obtained using Jaccard algorithm. c. Rarefaction curves showing common species.

Fig. 13

Abundance profiling at (a) phylum and (b) genus level.

Fig. 14

Random forest plot showing Calcium and moisture as the key component; Fe, Cu, Zn, acidity follow; magnesium least.

Fig. 15

Correlation heat map of functional profiling and samples.

Plate assay for deconjugation of bile salt of 13 selected yeast isolates.

Antimicrobial activity of 13 selceted isolates against selected MTCC cultures.