Short-Chain Carboxylates Facilitate the Counting of Yeasts in Sub-High Temperature Daqu
, , oraz | 28 kwi 2024
Article Category: Original Paper
Data publikacji: 28 kwi 2024
Zakres stron: 167 - 176
Otrzymano: 04 sty 2024
Przyjęty: 13 mar 2024
DOI: https://doi.org/10.33073/pjm-2024-015
Słowa kluczowe
© 2024 Zhiqiang Ren et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Sub-high-temperature Daqu (STD) is a saccharifying and fermenting agent derived from wheat, commonly used in producing Chinese strong-aroma Baijiu. It plays a pivotal role in the fermentation process (Yan et al. 2019a; Wang et al. 2020), facilitating the degradation of complex natural substrates such as starch and protein. Moreover, it catalyzes the transformation of these degraded products into a diverse array of compounds, including alcohols, acids, esters, aldehydes, and other flavor substances (De Vuyst and Leroy 2020), which significantly influences the quality and distinctive characteristics of Baijiu brewing (Yan et al. 2019a; Zhu et al. 2022).
STD is produced through natural inoculation, harboring a rich diversity of microorganisms, including numerous molds, yeasts, bacteria, and a smaller proportion of actinomycetes (Zou et al. 2018; Yan et al. 2019b). A recent study employing second-generation sequencing technology to investigate the fungal community structure within STD revealed that yeasts comprise approximately 60% of the total fungi population (Yang et al. 2018). A total of 420 fungal strains were isolated from 30 Daqu starter samples using the plate culture method and identified through ITS region sequencing. Among these strains, 386 (92%) were yeasts, while 34 (8%) were filamentous fungi. The dominant species identified were
Effectively isolating and counting yeasts in STD presents challenges due to interfering microorganisms, especially molds (Xu et al. 2020). Supplementing antibiotics in culture media can effectively inhibit bacterial growth and circumvent the interference of bacteria (Liu et al. 2014). Using the classical plate culture method, sixteen pure yeast cultures and various yeast strains with specific functions have been successfully isolated from STD samples (Hu et al. 2020; Ma et al. 2022; Li et al. 2023). However, the number of pure yeast species obtained through classical methods is notably lower than those identified through high-throughput sequencing, It is imperative to develop methods that inhibit the growth of molds while preserving yeast growth to isolate and analyze the entire yeast population.
Previous studies have demonstrated the inhibitory effects of organic acids on the growth of molds and yeasts. Valerate, propionate, and butyrate at concentrations ranging from 0.5 to 2.5 g/l have been shown to completely suppress mold growth, while higher concentrations of acetate and lactate are required for similar inhibitory effects (Moon 1983; Higgins and Brinkhaus 1999; Dijksterhuis et al. 2019). Various organic acids, including formic acid, acetic acid, propionate, lactate, and caproic acid, have been found to inhibit yeast growth in a concentration-dependent manner. For instance, yeasts can naturally grow when the lactate concentration is below 100 mM, but growth rates decrease by 50% when lactate concentration reaches 400 mM (Hundová and Fencl 1977).
While it is well-established that adding organic acids to the culture medium can regulate the growth of molds and yeasts, there is a lack of studies investigating whether organic acids exhibit differential effects on the growth of molds and yeasts at varying pH values and concentrations. This study aims to fill this gap by investigating the effects of short-chain carboxylates (C1-C6) on the growth of yeasts and molds in sub-high temperature Daqu. The goal is to identify a culture condition that favors yeast growth while inhibiting mold growth, thus enabling efficient isolation and counting of yeasts from mixed cultures. To our knowledge, this is the first study to examine the inhibitory effects of various short-chain carboxylates on molds and yeasts in sub-high temperature Daqu.
Sampling was conducted at a strong-flavor Baijiu production factory in Yibin, Sichuan Province, China. Daqu intended for production was gathered from ten fermentation workshops, with 0.5 kg samples collected from each workshop on November 3, 2022. These samples comprised a mixture of small particles and powder. After thoroughly mixing and milling, the samples were sifted through a 60-mesh screen and transferred to sterile bags. The bags were sealed, frozen at –20°C, and shipped to Sichuan University of Science and Engineering, Yibin, China, on dry ice for further analysis.
Five types of basal media were used in this study. Malt extract agar (MEA) (Heard and Fleet 1986) was prepared by dissolving 20 g malt extract, 10 g glucose, 5 g peptone, and 20 g agar in 1 l of distilled water. For potato dextrose agar (PDA) (Madbouly et al. 2020), 15 g of potato extract, 20 g of glucose, and 20 g of agar were dissolved in 1 l of distilled water. For rose Bengal agar (RBA) (Echevarría and Bello 2023), 5 g peptone, 10 g glucose, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate (MgSO4 · 7H2O), 100 ml of a 1/3,000 aqueous solution of rose Bengal, 20 g agar were dissolved in 1 l of distilled water. For Wallerstein laboratory nutrient agar (WL) (Li et al. 2010), 4 g yeast extract, 5 g tryptone, 50 g glucose, 0.425 g potassium chloride, 0.125 g calcium chloride, 0.125 g magnesium sulfate (MgSO4 · 7H2O), 0.55 g potassium dihydrogen phosphate, 0.0025 g ferric chloride, 0.0025 g manganese sulfate (MnSO4 · H2O), 0.022 g bromocresol green, 20 g agar were dissolved in 1 l of distilled water. For yeast-extract peptone dextrose agar (YPD) (Gerard et al. 2023),10 g yeast extract, 20 g peptone, 20 g glucose, and 20 g agar were dissolved in 1 l of distilled water. When the sterilized culture medium was cooled to approximately 50°C, antibiotics chloramphenicol was added to all culture medium to the final concentration of 50 μg/ml.
To select an appropriate culture medium for studying the effects of short-chain carboxylates on yeast and mold growth in Daqu, microbial suspensions of STD with three serial dilutions (10–2, 10–3, and 10–4) were plated onto five basal media: MEA, PDA, RBA, WL, and YPD. According to the screening results, MEA was chosen as a basal medium in this study. The short-chain carboxylate was supplemented to MEA with final concentrations of 0.05 M, 0.1 M, and 0.2 M. The pH value of the medium was adjusted within appropriate ranges: formate (4.2 to 5.2), acetate (4.8 to 5.8), propionate, butyrate, valerate, and caproate (5.4 to 6.4), pyruvate (2.6 to 4.0), and lactate (3.0 to 4.4), by adding 1 M HCl or 1 M NaOH. To compare the inhibitory effects of short-chain carboxylates on the growth of molds and yeasts in STD, the MEA medium was supplemented with formate, acetate, propionate, butyrate, valerate, caproate, lactate, and pyruvate at a final concentration of 0.05 M.
Additionally, MEA was supplemented with butyrate, valerate, and caproate at final concentrations of 0.01 M, 0.02 M, 0.03 M, and 0.04 M to investigate their effects on mold and yeast growth at low concentrations. Considering that the pH of the control group, consisting of MEA culture without short-chain carboxylates, was 5.7, the pH of the culture medium with different shortchain carboxylates was adjusted to the same pH value.
To validate their inhibitory effects in five different types of yeast medium, acetate, butyrate, and valerate were added at final concentrations of 0.05 M, 0.03 M, and 0.02 M. The pH value was adjusted to 5.0 for the media containing acetate and to 5.7 for the media containing butyrate or valerate. The pH value of control groups is the natural pH of these media without any adjustment.
Diluent saline peptone (SPO) was prepared by dissolving 8.5 g sodium chloride, 0.3 g disodium hydrogen phosphate (Na2HPO4 · 12H2O), and 1 g peptone in 1 l of distilled water. The pH of SPO was adjusted to 5.6 by adding 1 M HCl or 1 M NaOH. Samples (10 g) were mixed with 90 ml of SPO, soaked at 4°C for 30 minutes, and homogenized with a Vortex-Genie2 (Scientific Industries, Inc., USA) at “10” speed for 2 minutes, duplicate counting plates were prepared using 10–2, 10–3, and 10–4 serial dilutions. For spread plating, 0.1 ml of the dilution was spread on the surface of a dry plate. Agar plates were incubated at 30°C for 48 hours, 60 hours, or 72 hours until colonies appeared. Subsequently, the colonies were counted and calculated as colony-forming units (CFU) per gram of Daqu sample. In cases where plates were covered with molds that the naked eye could not directly count, the plates were inverted on a strong light source, a photograph was taken, and yeast counting was performed.
The yeast counting numbers from three independent experiments are presented as means ± standard error of the mean (SEM). Statistical significance was determined using a two-sided unpaired
Microbial suspensions of STD with three dilutions were plated onto five commonly used yeast culture media types, including MEA, PDA, RBA, WL, and YPD. When the dilution factor is 100, many yeast colonies grow on plates, making yeast counting difficult. Moreover, excessive mold growth interferes with yeast counting (Fig. 1A). When the dilution factor is increased to 1,000, a few hundred yeast colonies can be counted, and mold interference is attenuated (Fig. 1A). More importantly, the number of yeast colonies in this range meets the yeast isolation and counting criteria, which typically range from 30 to 300 CFU (Deak et al. 1998). Only a few yeast colonies can be counted when the microbial suspension is diluted 10,000 times, which falls far below the counting and culture criteria (Fig. 1A). Therefore, a dilution factor of 1,000 was used in subsequent experiments.
Fig. 1.
The microbial growth of STD in different yeast culture media.
A) representative agar plates;
B) yeast colony counting numbers at 1,000 times dilution of Daqu. MEA – malt extract agar; PDA – potato dextrose agar;
RBA – rose Bengal agar; WL – Wallerstein laboratory nutrient agar; YPD – yeast extract peptone dextrose agar.
10–2 – 100 times dilution;
10–3 – 1,000 times dilution; 10–4 – 10,000 times dilution.
ns – no statistically significant difference;
* – statistically significant difference,
** – statistically significant difference,
MEA showed the highest number of yeast colonies among the five tested yeast culture media, followed by PDA, RBA, WL, and YPD (Fig. 1B). There was no statistically significant difference in the number of yeast colonies between MEA and PDA. The primary nutrients in MEA are derived from malt juice, which aligns well with the raw materials used to prepare STDs. Therefore, MEA was chosen as the medium to explore the effects of short-chain carboxylates on mold and yeast growth.
In this study, the MEA medium was supplemented with short-chain carboxylates at concentrations of 0.05 M, 0.1 M, and 0.2 M, with the pH of the medium adjusted within appropriate ranges. The pH was adjusted within the range of 4.2 to 5.2 for the medium supplemented with formate. Both the pH value and formate concentration were observed to affect the growth of molds and yeasts. At pH below 4.8, minimal biomass was observed on agar plates (Fig. 2A). Molds and yeasts began to grow at pH 4.8, with increased biomass observed at pH 5.0 and 5.2. Agar plates with higher pH values exhibited a more significant number of yeasts. When the final concentration of formate added in the medium was 0.05 M, the number of yeast colonies at pH 4.8, 5.0, and 5.2 was (2.17 ± 0.51) × 105 CFU/g Daqu, (8.43 ± 0.21) × 105 CFU/g Daqu, and (12.17 ± 0.21) × 105 CFU/g Daqu, respectively (Fig. 2C). Furthermore, formate inhibited the growth of molds and yeasts in a concentration-dependent manner. For agar plates at pH 5.2 and formate concentrations of 0.05 M, 0.1 M, and 0.2 M, the number of yeast colonies was (12.17 ±0.21) × 105 CFU/g Daqu, (8.37 ± 0.87) × 105 CFU/g Daqu, and (2.30 ± 0.26) × 105 CFU/g Daqu, respectively (Fig. 2C).
Fig. 2.
The effect of formate or propionate on microbial growth of STD.
A) representative agar plates at different pHs and supplemented with different amounts of formate;
B) representative agar plates at different pHs and supplemented with different amounts of propionate;
C) yeast colony counting numbers from the agar plates at different pHs and supplemented with different amounts of formate;
D) yeast colony counting numbers from the agar plates at different pHs and supplemented with different amounts of propionate.
For the medium supplemented with propionate, the pH was adjusted within a range of 5.4 to 6.4. Molds and yeasts began to grow at pH 5.4, with increased biomass observed at pH 6.0, 6.2, and 6.4 (Fig. 2B). When the final concentration of propionate added in the medium was 0.05 M, the number of yeast colonies at pH 5.6, 5.8, and 6.0 was (8.53 ± 0.55) × 105 CFU/g Daqu, (12.23 ± 0.47) × 105 CFU/g Daqu, and (18.73 ± 0.75) × 105 CFU/g Daqu, respectively (Fig. 2D). Consistent with the observations from agar plates supplemented with formate, higher propionate concentrations resulted in higher cellular toxicity to the yeasts. For agar plates at pH 6.0 and propionate concentrations of 0.05 M, 0.1 M, and 0.2 M, the number of yeast colonies was (18.73 ± 0.75) × 105 CFU/g Daqu, (11.77 ± 0.76) × 105 CFU/g Daqu, and (6.17 ± 0.40) × 105 CFU/g Daqu, respectively (Fig. 2D).
It has been reported that acetate and butyrate affect the growth of yeast and mold (Perna et al. 2018; Dijksterhuis et al. 2019; Rodrigues and Pais 2000). Molds predominated in the microflora that grew on agar plates supplemented with acetate when the pH was higher than 5.4 (Fig. 3A). In contrast, lower pH values favored the growth of yeast colonies and strongly inhibited mold growth. Only yeast colonies were observed when the pH of agar plates reached 4.8 and 5.0 (Fig. 3A). A high concentration of acetate suppressed the growth of yeasts. When agar plates were at pH 5.0, and the concentrations of acetate in the medium were 0.05 M, 0.1 M, and 0.2 M, the number of yeast colonies was (15.70 ± 0.20) x 105 CFU/g Daqu (10.90 ± 0.85) x 105 CFU/g Daqu, and (7.50 ± 0.44) x 105 CFU/g Daqu, respectively (Fig. 3C).
Fig. 3.
The effect of acetate or butyrate on microbial growth of STD.
A) representative agar plates at different pHs and supplemented with different amounts of acetate;
B) representative agar plates at different pHs and supplemented with different amounts of butyrate;
C) yeast colony counting numbers from the agar plates at different pHs and supplemented with different amounts of acetate;
D) yeast colony counting numbers from the agar plates at different pHs and supplemented with different amounts of butyrate.
Butyrate showed stronger cellular toxicity compared to acetate. In a pH range of 5.4 to 6.4, no yeast colonies or mold were observed when the butyrate was added at a concentration of 0.2 M (Fig. 3B). Yeasts and molds began to grow when the pH value of the medium was 5.8, and the final concentration of butyrate in the medium was 0.05 M. For agar plates supplemented with butyrate, higher pH values favored microbial growth. When the final concentration of butyrate added to the medium was 0.05 M, the number of yeast colonies at pH 5.8, 6.0, and 6.2 was (12.83 ± 0.21) × 105 CFU/g Daqu, (15.53 ± 1.32) × 105 CFU/g Daqu, and (16.17 ± 0.61) × 105 CFU/g Daqu, respectively (Fig. 3D).
Acetate and butyrate exhibited different effects on microbial growth in STD. At pH 5.8, a large number of molds grew on agar plates supplemented with acetate. At the same time, only yeast colonies were observed on agar plates supplemented with butyrate, indicating that besides their distinct cellular toxicity, short-chain carboxylates may regulate the growth of yeasts and molds through altering metabolic pathways. The growth pattern of yeasts and molds could be modulated so that molds were suppressed, and yeasts were unaffected, facilitating the study of yeasts in a mixed culture.
Lactate and pyruvate are common organic acids produced during microbial fermentation. Previous studies showed that they affect the growth of molds and yeasts (Narendranath et al. 2001; Zhang et al. 2018). Molds appeared on all agar plates supplemented with lactate, indicating that lactate cannot effectively inhibit mold growth (Fig. 4A). At low pH values, a higher lactate concentration inhibited yeasts’ growth. When agar plates were at pH 3.0, and the concentrations of lactate in the medium were 0.05 M, 0.1 M, and 0.2 M, the number of yeast colonies was (2.67 ± 0.42) × 105 CFU/g Daqu, (0.60 ± 0.10) × 105 CFU/g Daqu, and (0.03 ± 0.06) × 105 CFU/g Daqu, respectively. However, a higher lactate concentration did not have stronger inhibitory effects on yeast growth when agar plates were at pH 3.4 (Fig. 4C).
Fig. 4.
The effect of lactate or pyruvate on microbial growth of STD.
A) representative agar plates at different pHs and supplemented with different amounts of lactate;
B) representative agar plates at different pHs and supplemented with different amounts of pyruvate.
C) yeast colony counting numbers from the agar plates at different pHs and supplemented with different amounts of lactate;
D) yeast colony counting numbers from the agar plates at different pHs and supplemented with different amounts of pyruvate.
Little biomass was found on agar plates supplemented with pyruvate when the pH of the medium was adjusted to 2.6 or 2.8. The number of yeast colonies started to increase at pH 3.0, but molds predominated in microbial growth at higher pH values (Fig. 4B). Consistent with what has been found in other shortchain carboxylates, pyruvate inhibited the growth of yeast in a concentration-dependent manner. As the concentration of pyruvate increased, the number of yeast colonies decreased. When agar plates were at pH 3.2 and the concentrations of pyruvate in the medium were 0.05 M, 0.1 M, and 0.2 M, the number of yeast colonies was (15.53 ± 0.81) × 105 CFU/g Daqu, (9.53 ± 0.38) × 105 CFU/g Daqu, and (7.73 ± 0.47) × 105 CFU/g Daqu, respectively (Fig. 4D). Interestingly, molds and yeasts grew at very low pH levels when agar plates were supplemented with lactate or pyruvate, in contrast to agar plates supplemented with other shortchain carboxylates where no molds or yeasts grew when the pH was below 4.2.
The MEA medium was supplemented with formate, acetate, propionate, butyrate, valerate, caproate, lactate, and pyruvate at a final concentration of 0.05 M to compare the inhibitory effects of short-chain carboxylates on the growth of yeasts and molds in STD. Given that the natural pH of MEA culture without short-chain carboxylates is 5.7, the pH of the culture medium with different short-chain carboxylates was adjusted to 5.7. Many molds grew on agar plates supplemented with formate, acetate, lactate, and pyruvate (Fig. 5A). Isolated yeast colonies were found on agar plates supplemented with propionate or butyrate. No yeast colonies or molds were found on agar plates supplemented with valerate or caproate, suggesting stronger inhibitory effects on microbial growth than other short-chain carboxylates (Fig. 5A). Notably, butyrate differentially suppressed the growth of molds and yeasts. Molds did not grow on agar plates supplemented with butyrate, while the number of yeast colonies reached (9.97 ± 0.45) × 105 CFU/g Daqu, much lower than that in the control (Fig. 5B).
Fig. 5.
Short-chain carboxylic acids have different inhibitory effects on microbial growth.
A) representative agar plates at pH 5.7 and supplemented with formate, acetate, propionate, butyrate, valerate, caproate, lactate, and pyruvate in a final concentration of 0.05 M and control without supplementing short chain carboxylic acid;
B) yeast colony counting numbers from the agar plates at pH 5.7 and supplemented with various short-chain carboxylic acids and control.
C) representative agar plates at pH 5.7 and supplemented with different amounts of butyrate, valerate, and caproate;
D) yeast colony counting numbers from the agar plates at pH 5.7 and supplemented with different amounts of butyrate, valerate, caproate.
Considering their concentration-dependent inhibitory effect on fungal growth, lower amounts of butyrate, valerate, and caproate were added to the MEA medium. For agar plates supplemented with butyrate at a final concentration of 0.03 M, the number of yeast colonies reached (20.07 ± 0.60) × 105 CFU/g Daqu, which is higher than that in the control (Fig. 5B, 5C, and 5D). For agar plates supplemented with valerate at a final concentration of 0.01 M, the number of yeast colonies was (20.67 ± 0.35) × 105 CFU/g Daqu (Fig. 5B, 5C, and 5D), and decreased at higher concentrations of valerate. Even though lower amounts of caproate (0.01 M) were added to the medium, no microbial growth was found on agar plates, indicating the potent toxicity of caproate to the microbes in STD (Fig. 5C and 5D).
Based on the above results, acetate, butyrate, and valerate were shown to suppress mold and yeast growth differentially, and isolated yeast colonies can be counted on agar plates supplemented with these short-chain carboxylates. To validate their inhibitory effects in five different types of yeast medium, acetate, butyrate, and valerate were added at concentrations of 0.05 M, 0.03 M, and 0.02 M. The results showed that all three shortchain carboxylates effectively inhibited mold growth on agar plates compared to the control group (Fig. 6A). The agar plates supplemented with 0.03 M butyrate showed a larger number of yeasts than the control group in all five yeast culture media (Fig. 6B), indicating that it is possible to find a condition that suppresses mold growth and favors yeast growth.
Fig. 6.
The inhibitory effects of acetate, butyrate, and valerate on microbial growth in different yeast culture media.
A) representative agar plates supplemented with acetate in a final concentration of 0.05 M at pH 5.0, with 0.03 M butyrate or 0.02 M valerate at pH 5.7.
B) yeast colony counting numbers from the agar plates supplemented with acetate in a final concentration of 0.05 M at pH 5.0, with 0.03 M butyrate or 0.02 M valerate at pH 5.7.
The numbers in the table are the yeast colony counting numbers divided by 105; “ / ” indicates no colony.
In this report, we investigated the effects of shortchain carboxylates on the growth of molds and yeasts in sub-high temperature Daqu. We observed that the inhibition of yeast and mold growth on agar plates by short-chain carboxylates depends on the culture medium’s pH value and the concentration of shortchain carboxylates. Notably, adding specific short-chain carboxylates to yeast culture media resulted in differential mold and yeast growth regulation. This finding suggests that manipulating short-chain carboxylate concentrations could be a simple and feasible strategy for improving yeast counting and isolation from mixed cultures. It is important to note that different Daqu starters contain diverse microorganism compositions. Therefore, an optimization process is required to determine the optimal conditions that effectively suppress mold growth while preserving yeast growth.