# Detection and characterization of the Pichia manshurica biofilm on the traditionally produced homemade apple vinegar

## TL;DR

This study examines how Pichia manshurica biofilms affect homemade apple vinegar, showing they can spoil the product by forming harmful structures.

## Contribution

The study identifies and characterizes P. manshurica biofilms in homemade vinegar for the first time.

## Key findings

- P. manshurica was found to form biofilms on the cellulosic film in vinegar.
- Contaminated biofilms showed higher polysaccharide content and altered structure.
- The presence of P. manshurica negatively impacts vinegar quality during fermentation.

## Abstract

Pichia yeasts are capable of forming biofilms during vinegar production and causing spoilage in various beverages. In addition, there exists a significant likelihood of encountering yeast contamination which can prevent vinegar production. The present study investigates the detection and characterization of the Pichia manshurica (P. manshurica) biofilm on traditionally produced homemade apple vinegar. The unique characteristics of vinegar were analyzed with a focus on the constituent, known as the “mother of vinegar”, whose composition is comprised of cellulosic biofilm and acetic acid bacteria, including Gluconobacter oxydans (G. oxydans) Briefly, P. manshurica was isolated from apple vinegar and characterized in terms of the effect of biofilm formation on the surface of the cellulosic film on vinegar production. Microbial identification of vinegar with/without contamination by P. manshurica was analyzed through MALDI-TOF mass spectrometry (MS), and biofilm was characterized by Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), and crystal violet staining. Accordingly, MS spectrum of isolates was identified as G. oxydans and P. manshurica with a ratio of 2.01 and 1.94, respectively. The FTIR analysis indicated that the peaks within the range of 1150–900 cm−1 revealed a high content of polysaccharide in P. manchuria-contaminated biofilm, which is attributed to the stretching vibration of C-C and C-O bonds. The spectral region from 2921.51 to 2853.71 cm−1 exhibited the characteristic of lipids in bacterial cell walls and membranes. SEM images of bacterial biofilms revealed a three-dimensional network composed of ultrafine fibers with a ribbon-like shape; however, the condensed reticulated structure was observed in contaminated biofilms. The presence of two microbial populations was detected regarding the morphological analysis. Crystal violet staining of contaminated-cellulosic biofilms visualized bacterial and yeast colonization. Concisely, this study emphasizes that the proliferation of Pichia during apple fermentation has the potential to adversely affect the quality of the homemade vinegar, due to its distinct biofilm characteristics.