Abstract

   Microbial interactions are essential for maintaining the stability and
   functionality of microbiota in fermented foods. In this study,
   representative strains of predominant lactic acid bacteria and acetic
   acid bacteria in Sichuan bran vinegar were selected, and their
   interactions in a simulated solid-state fermentation system were
   investigated. The results reveal that the biomass of A. pasteurianus
   LA10 significantly increased in both the co-culture and the pure
   culture, whereas the biomass of L. amylovorus LL34 in the co-culture
   (6.44 ± 0.30 lg CFU/g) was significantly lower than that in the pure
   culture (7.28 ± 0.30 lg CFU/g) (p < 0.05), indicating a partially
   harmful symbiosis between these two strains. The metabolic analysis
   shows that total acid (21.82 mg/g) and acetic acid (9.53 mg/g) contents
   in the co-culture were lower than those in the pure culture of LA10,
   suggesting that LL34 inhibited the acid-producing activity of LA10 to
   some extent. The interaction between the two bacteria also influenced
   the production of volatile compounds and non-volatile compounds, as
   revealed by GC-MS and untargeted UHPLC-MS/MS, respectively. Significant
   enrichment of acid and amino acid metabolism pathways was observed in
   the co-culture, revealing the impact of bacterial interactions on
   flavor development. This study provides valuable insights into the
   advancement of vinegar brewing technology.

   Keywords: Sichuan bran vinegar, Acetobacter pasteurianus, Lactobacillus
   amylovorus, interaction, volatile compounds

1. Introduction

   Cereal vinegar is a vital acidic condiment in the daily Chinese diet,
   with a history spanning over 3000 years [[44]1,[45]2]. Various types of
   vinegar have been developed, particularly Sichuan Baoning vinegar,
   Zhenjiang aromatic vinegar, Shanxi aged vinegar, and Fujian Monascus
   vinegar [[46]3]. Traditional production involves solid-state
   fermentation; however, a more efficient approach involving liquid-state
   fermentation followed by solid-state fermentation is now preferred
   [[47]4]. Sichuan Baoning vinegar is a representative Sichuan bran
   vinegar that distinguishes itself from other vinegar due to its whole
   process of traditional solid-state fermentation using uncooked wheat
   bran as the primary raw material. This vinegar undergoes
   saccharification, alcohol fermentation, and acetic acid fermentation
   simultaneously in a single fermentation pond, with Daqu (also called
   great koji, a traditional fermentation starter made from a blend of
   grains that have been moistened and allowed to ferment for several
   days) incorporated with Chinese herbs serving as a saccharifying agent
   [[48]5].

   Lactic acid and acetic acid are the predominant organic acids in
   solid-state vinegar fermentation, comprising over 90% of the total
   organic acid content [[49]6,[50]7,[51]8]. Lactic acid is characterized
   by its mildness, while acetic acid imparts a more pungent flavor. The
   acid ratio plays a crucial role in determining the sensory
   characteristics of vinegar [[52]9]. Sichuan bran vinegar is notable for
   its higher lactic acid content than acetic acid [[53]10,[54]11]. As is
   known, the microbial composition of vinegar is complex, with microbial
   interactions essential for its stability and functionality. The
   dominant bacteria in the solid-state fermentation of vinegar differ at
   different periods, such as mold in the early stage of fermentation
   (starch saccharification fermentation), yeast in the middle stage
   (alcohol fermentation), and acetic acid bacteria in the late stage
   (acetic acid fermentation). At the same time, there are other dominant
   bacteria, such as Lactobacillus, Bacillus, Stenotrophomonas,
   Methyloversatilis, and Amycolatopsis, etc., among which lactic acid can
   produce lactic acid to neutralize the irritation of acetic acid
   [[55]12]. Bacillus can secrete a variety of hydrolases that play an
   important role in liquefaction and saccharification and can produce
   organic acids through the tricarboxylic acid cycle [[56]13]. However, a
   comprehensive understanding of these interactions in vinegar remains
   limited [[57]14]. Lactic acid and acetic acid are key metabolites
   produced by lactic acid bacteria and acetic acid bacteria,
   respectively. Metagenomic sequencing revealed Lactobacillus amylovorus
   (L. amylovorus) and Acetobacter pasteurianus (A. pasteurianus) as
   dominant strains in Cupei (the mixture of ingredients used for cereal
   vinegar fermentation) during Sichuan Baoning vinegar fermentation
   [[58]15].

   In this study, L. amylovorus LL34 and A. pasteurianus LA10 [[59]16],
   isolated from Sichuan Baoning vinegar for their high lactic acid and
   acetic acid yields and robust growth capacities, were employed as
   representative strains. This study aimed to investigate the interaction
   between these two strains during fermentation using a simulated
   fermentation system and its impact on strain growth, acid production,
   and volatile and non-volatile compounds. This study offers insights
   into microbial interactions during fermentation and provides
   theoretical guidance for enhancing the quality of Sichuan bran vinegar.

2. Materials and Methods

2.1. Strains and Growth Conditions

   L. amylovorus LL34 and A. pasteurianus LA10, isolated from Cupei of
   Sichuan Baoning vinegar, were maintained at the Laboratory of
   Microbiology, College of Food Science, Sichuan Agricultural University.
   The strains were individually cultured in 5 mL of MRS broth (10 g
   peptone, 8 g beef extract, 4 g yeast extract, 20 g glucose, 2 g
   K[2]HPO[4], 5 g CH[3]COONa, 2 g ammonium citrate, 0.2 g MgSO[4]·7H[2]O,
   0.05 g MnSO[4]·H[2]O, 1 mL Tween 80, pH 6.2–6.5, and 1 L H[2]O; L.
   amylovorus LL34) and 5 mL of GYE broth (1% glucose, 1% yeast extract,
   and 3% ethanol; A. pasteurianus LA10). L. amylovorus LL34 was
   statically incubated at 37 °C for 24 h, while A. pasteurianus LA10 was
   cultured in a shaking incubator at 30 °C for 72 h at 160 rpm.

2.2. Interaction Between Lactic Acid Bacteria and Acetic Acid Bacteria

2.2.1. Solid-State Fermentation and Sampling

   A Cupei stimulation medium comprising wheat bran (30%), wheat flour
   (20%), and MRS broth (50%) was developed for solid-state fermentation.
   The medium was sterilized at 121 °C and subsequently supplemented with
   2% ethanol, 1.3% lactic acid, and 0.3% acetic acid. The concentrations
   of ethanol and organic acids were based on our previous report
   [[60]17]. L. amylovorus LL34 (10^6 CFU/g, obtained by MRS broth
   culture) and A. pasteurianus LA10 (4 × 10^5 CFU/g, obtained by GYE
   broth culture) were co-inoculated into 200 g of the medium and
   statically incubated at 37 °C for 60 h. Samples were collected at 12 h
   intervals (0, 12, 24, 36, 48, and 60 h) after thoroughly mixing the
   medium. Each strain was individually inoculated as a control, and
   triplicate parallel experiments were conducted for each experimental
   group.

2.2.2. Enumeration of Strains

   The number of L. amylovorus LL34 and A. pasteurianus LA10 in each
   sample was determined by plating on an MRS agar plate [[61]18]. The two
   bacterial strains were distinguished based on colony morphology. The
   colony of L. amylovorus LL34 was larger and irregularly circular, while
   the colony of A. pasteurianus was smaller and exhibited a regular
   circular shape.

2.2.3. Determination of Total Acid, Lactic Acid, and Acetic Acid Content

   Five grams of Cupei was mixed with 45 mL of sterile water and kept at
   60 °C for 1 h. After cooling to 30 °C, the mixture was centrifuged at
   10,000 g for 10 min, and the supernatant was collected for analysis.
   The total acid content was tested by titration with 1.0 mol/L NaOH
   using phenolphthalein as an indicator and expressed as g/100 g lactic
   acid.

   Organic acid contents were analyzed with modifications from a previous
   report [[62]17]. Briefly, 4 mL of supernatant was mixed with 1 mL of
   potassium ferrocyanide (106 g/L) and 1 mL of zinc sulfate (300 g/L),
   followed by centrifugation at 10,000 g for 10 min. The supernatant was
   extracted using Sep-Pak C18 cartridges, filtered through a 0.22 μm
   membrane, and analyzed by HPLC. A Hichrom Alltech OA-1000 column (300 ×
   6.5 mm, 9 μm) was employed with 9 mmol/L H[2]SO[4] as the mobile phase
   at a flow rate of 0.6 mL/min. The injection volume was 10 μL, and the
   UV detector was set at 210 nm. A calibration curve was established
   using organic acid standard solutions, with concentration calculated
   versus peak area using the least-squares method.

2.2.4. Volatile Compounds

   Volatile compounds were analyzed using gas chromatography–mass
   spectrometry (GC–MS; 5975C/6890N; Agilent, CA, USA) with an HP-5MS
   column (60 m × 0.32 mm, 1 μm) according to a previous report [[63]17].
   Compound identification was performed by comparing obtained data with
   the National Institute of Standards and Technology (NIST) Library 2020
   (similarity index > 80%) and literature references. Methyl heptanoate