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. 2022 Feb 16;31(3):357–364. doi: 10.1007/s10068-022-01038-7

Growth characterization of Propionibacterium and propionic acid production capabilities at different temperatures and pH levels

Jung Kyu Chae 1,#, Sangha Han 1,#, Duk Hyun Kim 1, Si Hong Park 2, Sang-Do Ha 1,✉
PMCID: PMC8885949  PMID: 35273826

Abstract

Bacteria from the Propionibacterium genus were cocktailed to investigate growth and production of propionic acid at different temperatures and pH levels. A gas chromatograph with a flame ionization detector was also used for instrumental analysis. The Propionibacterium cocktails did not produce propionic acid at 10 and 20 °C for 10 days, but produced propionic acid at concentrations of 3265.32, 3670.76, and 1926.04 μg/mL at 25, 30, and 40 °C for 18 days, respectively. In pH tests, the cocktails did not produce propionic acid at pH 3 and 9 for 14 and 7 days, respectively. However, they produced propionic acid at concentrations of 2596.66, 2952.66, 3321.35, and 3586.95 μg/mL at pH 4, 5, 6, and 7 for 18 days, respectively. Growth characteristics of Propionibacterium cocktails by temperature and pH were set so that they reached the extinction stage after four days in the logarithmic phase.

Keywords: Propionic acid, Propionibacterium, Cocktail, Gas chromatograph with flame ionization detector (GC-FID), Logarithmic phase

Introduction

Consumers consider food additives to be the most harmful risk factor related to food safety, over pesticide residues, microbial contamination, and environmental pollutants; accordingly, they want stricter regulations on the use of food additives and more information about food additives (Jin et al., 2014). Food additives are classified according to their use as preservatives, sweeteners, antioxidants, etc., and are managed according to the designated criteria found in the Food Additives Codes. The purpose of these criteria is to ensure safety regarding the use of food additives and to contribute to public health by establishing a standard for manufacturing, processing, usage, and preservation methods for food additives (Food Additive Codes, MFDS 2019–31). Among food additives, preservatives are managed by setting the standard of use for each food, and if they are detected in foods that cannot involve their use, the business involved will be suspended under the Food Sanitation Act (Kim et al., 2016). It is difficult to determine whether to intentionally use preservatives for the purpose of food preservation, because they are not distinguished from naturally occurring preservatives found in raw food materials (Jang et al., 2010). Consequently, in the food industry, preservatives, which were naturally present in trace amounts in raw materials, increase in the process of manufacturing and processing, such as drying or concentration of food, and there are cases in which high concentrations are detected in processed foods. The Ministry of Food and Drug Safety (MFDS) recognized this change in the content of preservatives and has disclosed about 1500 cases of recognition of natural origin of food additives from 2010 to the present. Also, the MFDS allowed 0.1 g/kg of propionic acid to be present in food (Food Additive Codes, MFDS 2020-59). Nevertheless, studies on the amount of propionic acid change and the mechanism of production during food processing are insignificant, therefore in-depth studies are required.

Typical strains of propionic-acid-producing bacteria are Propionibacterium spp., which are gram-positive, non-motile, non-sporulating, rod-shaped, facultative anaerobes (Kumar and Babu, 2006). The Propionibacterium genus consists of two principal groups: cutaneous and classical. Cutaneous Propionibacterium are considered primary pathogens to humans, whereas classical Propionibacterium are widely used in the food and pharmaceutical industries (Piwowarek et al., 2018). All Propionibacterium genus also have fermentation capability and they have own valuable sources of metabolites, such as propionic acid, vitamin B12, bacteriocin, and trehalose. They can use different carbon sources, including glucose, fructose, maltose, sucrose, xylose, lactose, glycerol, molasses, and lactate, to produce propionic acid (Ahmadi et al., 2017). Propionic acids are known to exist as naturally occurring preservatives in many foods where fermentation or aging processes occur (Lee et al., 2003; Lim et al., 2013; Oh et al., 2000). Propionic acid is a preservative that controls the growth of molds in cheeses (vaccine components Swiss cheeses and Swiss-style Dutch cheeses), butter, bread, etc., and inhibits the growth of bacteria and yeast in syrup, apple sauce, and fresh fruits (Ray, 2004). Propionic acid has been reported to be naturally occurring in fermented foods such as vinegar, soybean products (Jang et al., 2010; Lee et al., 2003, 2010), noodles, and grains (Kim et al., 2016).

Several factors influence fermentation of propionic acid including microorganism species, temperature, pH, carbon sources, and conditions of fermentation. Among them, pH and temperature remarkably affect the propionic acid production, however a few studies have conducted and no systematic study on pH and temperature has been performed for Propionibacterium spp. Recently, it has been proven that naturally occurring propionic acid is produced in rice cakes made of starch (Park et al., 2018a).

However, most studies have focused on proving that naturally occurring propionic acids are produced in various foods, and there is a lack of analysis on the environmental characteristics for propionic-acid-producing bacteria. Specifically, this study aims to investigate the growth characteristics of Propionibacterium spp. and analyze their propionic-acid-producing capabilities at different temperatures and pH levels.

Materials and methods

Bacterial strains

Four bacterial strains identified from microbiome analysis of Joraengyi rice cakes by Chunlab Inc. (Seoul, Korea) were used in this experiment (Park et al., 2018b). The strains are P. thoenii KCTC 5343, P. cyclohexanicum KCTC 5755, P. jensenii KCTC 5340, and P. freudenreichii subsp. shermanii KCTC 5753, which produce propionic acids naturally in foods (Ahmadi et al., 2017; Gonzalez-Garcia et al., 2017; Macy et al., 1978; Ray and Bhunia, 2013). They were purchased from the Korean Collections for Type Cultures (KCTC).

Cultures and growth conditions

Propionibacterium cyclohexanicum KCTC 5755 (optimal growth at 37 °C), P. freudenreichii subsp. shermanii KCTC 5753, P. thoenii KCTC 5343, and P. jensenii KCTC 5340 were cultured for two days in an anaerobic jar at an optimal growth temperature of 30 °C. After two days of incubation, the four strains were diluted to a concentration of 5 Log CFU/mL using 0.1% Peptone Water (PW), and then cocktailed. In the pH tests, Tryptic Soy Broth (TSB, BD, USA) supplemented with glucose (10 g/L) was adjusted with 1 N HCl or 1 N NaOH to pH 3, 4, 5, 6, 7, and 9 using a pH meter (BP3001, Trans Instrument (s) Pte Ltd, Singapore). Propionibacterium cocktails were incubated at 30 °C for a maximum of 18 days in anaerobic conditions (Table 1). To eliminate the possibility of contamination during pH control, the samples were filtered using a 500-mL vacuum filter system (Corning, 0.22 μm pore) after completing the adjustment of the pH level. In the temperature tests, TSB supplemented with glucose (10 g/L) was used to culture the Propionibacterium cocktail at 10, 20, 25, 30 and 40 °C for a maximum 18 days in anaerobic conditions (Table 1).

Table 1.

Temperature and PH variables related to the growth of Propionibacterium cocktails and their propionic acid production capabilities in the experiment

Temperature (°C) Time (days)—microbial growth Time (days)—propionic acid production
10 0, 1, 2, 4, 7, 10 0, 1, 2, 4, 7, 10
20 0, 1, 2, 4, 7, 10 0, 1, 2, 4, 7, 10
25 0, 1, 2, 4, 7, 10, 14, 18 0, 1, 2, 3, 4, 5, 6, 7, 10, 14, 18
30 0, 1, 2, 4, 7, 10, 14, 18 0, 1, 2, 3, 4, 5, 6, 7, 10, 14, 18
40 0, 1, 2, 4, 7, 10, 14, 18 0, 1, 2, 3, 4, 5, 6, 7, 10, 14, 18
pH Time (days)—microbial growth Time (days)—propionic acid production
3 0, 1, 2, 4, 7, 10, 14 0, 1, 2, 4, 7, 10, 14
4 0, 1, 2, 4, 7, 10, 14, 18 0, 1, 2, 4, 7, 10, 14, 18
5 0, 1, 2, 4, 7, 10, 14, 18 0, 1, 2, 4, 7, 10, 14, 18
6 0, 1, 2, 4, 7, 10, 14, 18 0, 1, 2, 4, 7, 10, 14, 18
7 0, 1, 2, 4, 7, 10, 14, 18 0, 1, 2, 4, 7, 10, 14, 18
9 0, 1, 2, 4, 7 0, 1, 2, 4, 7
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Microbial growth

The 100 μL of the 5 Log CFU/mL Propionibacterium cocktail was inoculated into TSB supplemented with 10 g/L of glucose in both the temperature and pH tests. In the temperature test, it was incubated at 10, 20, 25, 30, and 40 °C, for a maximum 18 days in anaerobic conditions as shown in Table 1. In the pH test, it was incubated at 30 °C for a maximum 18 days in anaerobic conditions as shown in Table 2. Cocktails of Propionibacterium grown in each condition were diluted to 0.1% PW and applied to Reinforced Clostridial Medium (RCM, BD, USA) supplemented with agar powder (14.5 g/L) (Daejung Chemicals & Metals, Shiheung, Korea). After spreading, it was incubated at 30 °C for 48 h.

Table 2.

The analytical conditions of the GC-FID for propionic acid

Instruments GC (HP 7890B, USA) with FID
Column HP-FFAP
(30.0 m x 320 μm × 0.25 μm)
Injector temperature 180 °C
Detector temperature 230 °C
Oven temperature 80 °C → 10 °C↑ → 150  °C (5 min) → 20 °C↑ → 230 °C
Injection volume 1 μL
Carrier gas flow rate 1 mL/min (N2)
Split ratio 5:1
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Analysis of propionic acid production

Reagents and standards

Deionized water with a value of resistance of 18.2 Ω cm−1 and standard materials such as propionic acid (99.5%, Sigma-Aldrich, St. Louis, USA) and trans-crotonic acid (98%, Sigma-Aldrich) were used in the experiment. Acetone (for HPLC, GC and residue analysis, 99.9%), sodium chloride (99%, Sigma-Aldrich), phosphoric acid (85%, Wako, Japan), and ethyl ethers (Daejung Chemicals & Metals) were used to analyze the propionic acid using GC-FID (Son et al., 2011).

Preparation of standard solution

0.2 g of propionic acid is dissolved in 100 mL of acetone to make a standard solution and diluted with acetone to 5, 15, 30, 60, 120, 240, 480, and 960 ppm. Trans-crotonic acid was used as an internal standard material, and the final concentration in the standard stock solution was adjusted to 100 ppm (Kim et al., 2009).

Preparation of test solution and instrumental analysis

The method of preparing the test solution for the analysis of propionic acid was performed as previously described with some modifications (Son et al., 2011). The 5 mL sample (TSB supplemented with glucose) was accurately weighed in a conical tube and 10 mL 0.05 M phosphoric acid, 2 g NaCl, and 1 mL trans-crotonic acid (2000 mg/L) were then added. The cocktail was then sonicated for 30 min and vortexed for 10 min. Then, 20 mL Ethyl ether was added to the solution and vortexed for 2 min. After layer separation formed, the layer of Ethyl ether was filtered through a 0.2-μm PVDF filter (Agilent Technologies, Santa Clara, California, USA). After a test solution was completed, instrumental analysis was conducted by Agilent 6890 GC-FID. The analytical conditions using GC-FID are shown in Table 2 (Kim et al., 1999).

Recovery test

The recovery test was conducted (Kim et al., 2016) for TSB with 10 g/L glucose and the results were compared at three times for the instrument analysis. The TSB of 5 mL with 10 g/L glucose was added to the standard solution of 2000 mg/L, and the concentrations of propionic acid were adjusted to 30, 120, and 240 mg/L.

Results and discussion

Effect of temperature on growth and propionic acid production by Propionibacterium cocktail

Previous studies have shown that temperature is an important factor which influences bacterial growth and propionic acid formation (Walker and Phillips, 2007; Ahmadi et al., 2017; Koussémon et al., 2003). The results from this study showed that Propionibacterium cocktails did not produce propionic acid at 10 °C and 20 °C during incubation for 7 days and 14 days, respectively (Fig. 1A and B). However, the production of propionic acid at 25 °C started at 170.12 μg/mL on the 5th day and reached to 3265.32 μg/mL on the 18th day (Fig. 1C). The amount of propionic acid produced at 30 °C reached 3670.76 μg/mL on the 18th day (Fig. 1D), which is the maximum amount of propionic acid among the conditions; this is in agreement with previous studies. The optimum temperature for growth in the Propionibacterium cocktail was previously observed to be 30 °C (Farhadi et al., 2013). In addition, it has been found that most Propionibacterium spp. are mesophiles, and the optimum temperature for Propionibacteirum cocktails is 30 °C (Piwowarek et al., 2018). The production of propionic acid at 40 °C reached about 2000 ppm on the 18th day, but this was below the production at 30 °C (Fig. 1E). These results support the finding that the propionic acid production rate is better at 30 °C than any temperatures above (Coral et al., 2008). Farhadi et al. (2013) also suggested that propionic acid production decreases from 30 to 35 and 40 °C. Additionally, the production of propionic acid in each condition begins between the second and fifth day (Fig. 1C–E). Other studies have reported that propionic acid production began at different times such as 48, 96, 120, 144, and 168 h (Coral et al., 2008; Cho and Shuler, 1986; Quesada‐Chanto et al., 1998). However, no comprehensive study about the optimum time has been reported. The importance of this factor is directly related to the productivity of propionic acid (Ahmadi et al., 2017).

Fig. 1.

Fig. 1

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Bacterial growth of Propionibacterium cocktails and their propionic acid production capabilities at different temperatures. Propionibacterium cocktails at (A) 10 °C, (B) 20 °C, (C) 25 °C, (D) 30 °C, and (E) 40 °C were incubated in TSB supplemented with 10 g/L glucose. Bacterial populations (●) were monitored by measuring Log CFU/mL. Propionic acid concentrations (○) were monitored by measuring μg/mL using GC-FID. The data represent the mean ± SD of double experiments

Microbial growth characteristics of the Propionibacterium cocktails were reached the stationery and death stages after four days in the logarithmic stage (Fig. 1C–E). This result follows the four steps of a typical growth curve (lag, logarithmic, stationary, and death phases). It is important to note that by changing some environmental parameters (e.g., temperature), the growth rate of some microbial species can be slowed, but after a long time, the population can reach high numbers, which causes problems in foods (Ray, 2004). In relation to this, Silliker (1980) reported that microbial growth by temperature is accomplished through enzymatic reactions. Temperature probably affects the specificity of enzymatic reactions, cell membrane permeability, and metabolic regulatory mechanisms (Forage, 1985).

Effects of pH on growth and propionic acid production by Propionibacterium cocktail

pH is the most important environmental factor in propionic acid production. Coral et al. (2008) reported that the Propionibacterium species are strongly dependent on pH and usually require pH control. Previous research has reported that most productions of propionic acid were conducted at a neutral pH (Feng et al., 2010; Himmi et al., 2000; Roberto and Mayra, 2002).

The Propionibacterium cocktails did not produce propionic acid at pH 3 and 9 during incubation periods of 14 and 7 days, respectively (Fig. 2A and F). The production of propionic acid at pH 4 reached 2596.66 μg/mL on the 18th day, which is the minimum production amount of propionic acid among other pH conditions (Fig. 2B). The production of propionic acid at pH 5 reached 2952.66 μg/mL on the 18th day (Fig. 2C). Feng et al. (2010) reported that neither a lower pH, of 5.5, nor a higher pH, of 7.0, were beneficial for propionic acid production. On the other hand, the production of propionic acid at pH 6 and 7 reached 3321.35 μg/mL and 3586.95 μg/mL on the 18th day, respectively (Fig. 2C and D); these values show the maximum production of propionic acid among pH conditions. Considering the curves of pH 6 and 7, the cocktails produced much higher amounts of propionic acid overall than those at pH 4 and 5. Hsu and Yang (1991) also support the finding that the optimum pH range for propionic acid production is between pH 6 and 7.

Fig. 2.

Fig. 2

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Bacterial growth of Propionibacterium cocktails and its propionic acid production capabilities at different pH levels. Propionibacterium cocktails in TSB supplemented with 10 g/L glucose, adjusted to (A) pH 3, (B) pH 4, (C) pH 5, (D) pH 6, (E) pH 7, and (F) pH 9, were incubated at 30 °C. Bacterial populations (●) were monitored by measuring Log CFU/mL. Propionic acid concentrations (○) were monitored by measuring μg/mL using GC-FID. The data represent the mean ± SD of double experiments

Similar to the results of the temperature tests regarding growth characteristics, Propionibacterium also reached the stationery and death stages after four days in the logarithmic stage (Fig. 2C–E). Cell growth at pH 3 and 9 reached the stationery and death phases long before the substrate was depleted, perhaps due to the strong inhibition of acidic and alkaline pH (Fig. 2A and F). Ray (2004) reported that when the pH in a food is reduced below the lower limit for growth of a microbial species, the cells not only stop growing but also lose viability. The principal underpinning this is that it is more apparent with weak acids, especially those that have higher dissociation constants (pK) such as acetic acid and lactic acid. This is because, at the same pH, acetic acid has more undissociated molecules compared to lactic acid. The undissociated molecules, being lipophilic, enter the cell and dissociate to generate H + in the cytoplasm. This causes a reduction in internal pH, which ultimately destroys the proton gradient between the inside and the outside of the cells.

Additionally, it was found that the curves of propionic acid production and bacterial growth have a common feature in both the temperature and pH tests. When bacterial growth reached the stationary stage, propionic acid production increased rapidly (Figs. 1C–E, 2B–E). Propionic acid is produced by propionic bacteria in the dicarboxylic acid pathway, with acetate, succinate, and carbon dioxide as byproducts (Piwowarek et al., 2018). The accumulation of propionic acid as the predominant product in a mixed acid bioprocess confers, which is the advantage of a high theoretical conversion efficiency from substrate. Moreover, conversion of total carbon and dry matter are better in propionic acid dominant processes (Playne, 1978).

In conclusion, this study analyzed the growth characteristics of Propionibacterium and its capability to produce propionic acid at different temperatures and pH levels. The results of this study can be used as a guideline for food producers and distributors engaged in the food industry who oversee the production and storage environment conditions of foods. The results also can be used as safety management for the detection of propionic acid which is naturally occurring in foods.

Acknowledgements

This research was supported by the Chung-Ang University Research Scholarship Grants in 2021.

Author contributions

JKC Methodology, Data curation, Writing-Original draft preparation. SHH Investigation, Writing-Original draft preparation, Writing-Reviewing and Editing. DHK Methodology, Data curation. SHP Writing-Reviewing. SDH Conceptualization, Supervision, Funding acquisition.

Funding

Funding was provided by The Chung-Ang University Research scholarship Grants in 2021.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.s

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jung Kyu Chae and Sangha Han have contributed equally to this work.

Contributor Information

Jung Kyu Chae, Email: larblue102@naver.com.

Sangha Han, Email: lllls3640@gmail.com.

Duk Hyun Kim, Email: june7370@naver.com.

Si Hong Park, Email: sihong.park@oregonstate.edu.

Sang-Do Ha, Email: sangdoha@cau.ac.kr.

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