Influence of culture media, pH and temperature on growth and bacteriocin production of bacteriocinogenic lactic acid bacteria

Lactic acid bacteria (LAB) are a diverse group of gram positive, catalase negative, oxidase negative, and micro-aerophilic organisms (Carr et al. 2002). They are generally recognized as safe bacteria (GRAS status) and play an important role in food and feed fermentation and preservation, either as the natural microflora or as starter cultures under controlled conditions (Daeschel 1989). According to Magnusson (2003), the antimicrobial effects of LAB contribute to the yield of organic acids, competition for nutrients and production of antagonistic compounds. Bacteriocins are a group of antimicrobial compounds, which are ribosomally synthesized peptides produced by bacteria to inhibit the growth of similar or closely related bacterial strains either in the same species, or across genera (Bowdish et al. 2005; Cotter et al. 2005). LAB bacteriocins have potential biotechnological applications because they are easy to produce, non-toxic to humans, stable at low pH values, and sensitive to proteases (Todorov 2009). Many bacteriocins are heat-stable and retain their activity after several months of frozen or refrigerated storage and after drying. For instance, nisin is already used as food preservative, and pediocins produced by LAB have bactericidal activity against gram-positive bacteria and are also suitable candidates for use as food bio-preservatives (Bari et al. 2005). Usually bacteriocins are produced in complex culture media including MRS, TGE, APT, BHI, TSB and TSBYE (Guerra et al. 2001; Balciunas et al. 2016; Renye et al. 2016). However, bacteriocin production can be influenced by culture conditions, such as incubation atmosphere, pH, temperature and microbial growth phase (Gänzle et al. 1999; Todorov et al. 2012; Zhang et al. 2012; Zhou et al. 2015; Turgis et al. 2016). Several studies have shown that bacteriocin production is dependent on the biomass (De Vuyst et al. 1996; Mataragas et al. 2003, 2004; Todorov and Dicks 2009; Todorov et al. 2010). However, Mataragas et al. (2003) reported that the highest bacteriocin production is determined at temperatures and pH lower than that of the optimal cell growth. Because high bacteriocin production may or may not occur at optimal growth conditions and high cell mass (Todorov and Dicks 2009; Masuda et al. 2016), this necessitates study on the relationship between bacterial growth conditions and bacteriocin production (Leroy and De Vuyst 2002).

Microbial growth curves and mathematical models can provide useful information to understand microorganism’s growth behavior and select the optimal growth conditions. Turbidimetric method is a good alternative used to study bacterial growth since optical density (OD) measurement gives a real time values of bacterial population and have practical significance when dealing with bacteria samples in high cell densities (Carlos et al. 2009; Dalgaard and Koutsoumanis 2001). Estimation of microbial growth parameters based on absorbance measurements has the advantages of being rapid, non-destructive, inexpensive and relatively easy to automate as compared to the other techniques such as classical viable counts methods (Dalgaard and Koutsoumanis 2001). In present research, turbidimetric method with Bioscreen C was used to determine how different growth conditions affected LAB growth and bacteriocin production. Four bacteriocinogenic LAB strains, Arla-10 (GenBank accession number JX275803), JFR-1 (GenBank accession number JX275814), JFR-5 (GenBank accession number JX275817) and TSB-8 (GenBank accession number JX275827), isolated from cheeses and yogurt products with anti-listerial effect were selected (Yang et al. 2012). The influences of culture media, initial pH of culture media and temperature on LAB growth and bacteriocins production were investigated, and the relationship between growth kinetics and bacteriocins production were determined.

Lactic acid bacteria are generally recognized as safe bacteria in fermented foods like dairy products, processed vegetables and play an important role in preservation. We need to better understand the factors that affect LAB growth and bacteriocin activity (production) in order to apply bacteriocinogenic LAB and/or bacteriocins in the food products. We found that initial pH level of culture medium was one of the key factors influencing the growth of the four bacteriocinogenic LAB strains. The optimal pH for the four LAB growth were pH 7.4 and 8.5 (Table 1), while they were unable to grow at a low pH of 4.5, which was supported by other researches. LeBlanc et al. (2004) reported that the growth of La. fermentum CRL 722 was noticeably slower at pH 4.5 (μ_max = 0.78 h⁻¹) than at other pH values including pH 5.0, 5.5, and 6.0 (μ_max = 1.15 − 1.25 h⁻¹). Similar results were found by Mataragas et al. (2003) that at pH 4.5, the final biomass and bacteriocin activity of Luconostoc mesenteroides L124 and L442 were very low, and the optimum pH for these LAB to grow were between pH 6.0 and 6.5. It was suggested that the LAB growth was suppressed at pH < 5.0.

Temperature also played an important role in LAB growth, particularly influenced the latency time (p ≤ 0.001, Table 2). Carlos et al. (2009) reported that Enterococci sp. grew in both 2YT (2× yeast tryptone medium) and skim milk at 30 and 37 °C, however, the growth curve at 30 °C presented longer lag phase compared to 37 °C. Gardini et al. (2001) found that the most important factor influencing the lag phase of E. faecalis was temperature, although its influence on the final cell yield was low.

Bacteriocin production is strongly dependent on medium composition (Gänzle et al. 1999). MRS medium is a rich medium with a mixture of different carbon sources and complex nitrogen sources. BHI is a general purpose nutrient medium recommended for the cultivation of a variety of microorganisms including bacteria, yeasts and mold. Growth kinetics results showed that BHI broth with initial pH 7.4 was an ideal growth condition for LAB (Table 1). However, the bacteriocin activity was not found when LAB strains were cultured in BHI broth with initial pH 7.4 at 37 °C for 16 h. This finding was consistent with the report by Khalil et al. (2009) that the highest bacteriocin inhibitory effect was obtained in MRS broth incubated at 30 °C for 12–18 h while BHI medium was not suitable for bacteriocin production. Moreover, the lower antimicrobial activity (200 AU ml⁻¹) of bacteriocin ST151BR was recorded in M17 broth, BHI broth, soymilk and molasses in comparison to in MRS broth (6400 AU ml⁻¹) despite relatively good growth (Todorov and Dicks 2004). Similar results were reported by De Kwaadsteniet et al. (2005) that MRS broth was the optimal medium for bacteriocin production of E. mundtii ST15 compared to BHI broth, M17 broth, soymilk and molasses. In our previous studies, the bacteriocin activity of LAB was not detected following incubation for 16 h in M17 medium. Therefore, some culture media used for the cultivation of microorganisms may not be suitable medium for bacteriocins production.

Some research found that the produced antimicrobial metabolites were pH-dependent, but the antifungal activity could not be attributed to a low pH effects (De Muynck et al. 2004; Elsanhoty 2008). In addition, the dependence of bacteriocin production on pH suggested that the expression of the biosynthetic genes may be regulated by pH, as has been reported previously for several classes of genes (Olson 1993). Guerra and Pastrana (2003) reported that higher pH drops, defined as the difference between initial and final pH, enhanced both nisin and pediocin production until a final pH was inappropriate for survivability and cell growth of LAB. Similar result was reported by Cabo et al. (2001), they concluded that pH drop gradient (VpH) enhanced nisin production (approximately fourfold), and increased the efficiency of nutrient consumption. This may be related to the need for a low final pH for an efficient post-translational processing of L. lactis and P. acidilactici to produce active bacteriocins (Yang and Ray 1994). It is also interesting to note that the buffer capacity of the different media because these bacteriocinogenic LAB strains likely acidify the medium. In addition, pH is known to have an influence on cationic peptides to associate with the cell membrane of the producer strains.

Bacteriocin production by LAB has been reported as a temperature-sensitive process (Leroy and De Vuyst 1999). In this research, bactriocins produced were strongly influenced by temperatures, especially in MRS broth, pH 6.2. Although, cultured at 44 °C resulted in a fast cell growth, which did not significantly affect final OD values and the highest bacteriocin production. It has been suggested that bacteriocin production by LAB was enhanced by sub-optimal fermentation condition (De Vuyst et al. 1996; Delgado et al. 2005). It is interesting to note that MRS broth, pH 6.2, at 37 °C provided the optimal condition for the four LAB to produce highest bacteriocin production, while the optimal pH for growth (highest OD values) was in MRS broth with pH 7.4 (Table 1). Moreover, LAB grew better in BHI broth, pH 6.2 than in BHI, pH 5.5, higher bacteriocin activity (BLS) was recorded in BHI broth, pH 5.5 (Figs. 2d, 3d, 4d, 5d). Messens et al. (2003) found that the optimum pH 6.0 for cell growth differed significantly from the optimum pH 5.1 for maximum curvacin A activity produced by L. curvatus. At a constant temperature of 35 °C, maximum cell yields were achieved at pH of 7.5–8.0, but this did not coincide with high enterocin production, and the highest enterocin activity of the supernatant was found at pH 5.5–6.5 (Leroy and De Vuyst 2002). Mataragas et al. (2003) also reported that the optimum pH and temperature for Leuconostoc mesenteroides L124 and L. curvatu L442 growth were 6.0–6.5 at 30 °C and for bacteriocin production was 5.5 at 25 °C.

Moreover, the bacteriocins activity of the four LAB strains was increased continuously during the exponential growth phase and the highest AU was reached by the end of this phase (Figs. 2d, 3d, 4d, 5d). The maximum bacteriocins produced in the middle or at the end of the exponential growth phase or at the beginning of the stationary phase were reported by other researchers (Guerra and Pastrana 2003; Todorov and Dicks 2004). Only a few bacteriocins were produced during the stationary phase (Lisboa et al. 2006; Khalil et al. 2009). Therefore, it is necessary to determine the correlations of pH, OD, bacteriocin activity and viability of LAB strains.

The BLS from the four bacteriocinogenic LAB were treated with 1 mg ml⁻¹ of proteolytic enzymes, including proteinase K (33 U mg⁻¹), α-chymotripsin (66 U mg⁻¹), and trypsin (105 U mg⁻¹) at 37 °C for 2 h (Yang et al. 2012). We found that the BLS lost their antilisterial activity following treatment with these enzymes indicating the proteinaceous nature of the BLS. We also investigated the effect of pH on the adsorption/desorption of BLS to LAB cells. Bacteriocins produced by JFR-1, JFR-5, Arla-10 and TSB-8 were maximally adsorbed to their cells at pH 6, 5, 5 and 4, respectively. Tricine–SDS-PAGE is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. Using this system, we found that the molecular weights of the bacteriocins produced by these four LAB strains ranged from 4.5–6 kDa. However, it remains to be determined whether these bacteriocins will be functional in foods or feeds. Bacteriocins may be used as biopreservatives either as powdered food ingredients, purified-or partially purified-peptides or through bacteriocinogenic LAB cultures. The application of combinations of different LAB-bacteriocins may contribute to reducing possible development of resistant bacterial populations and improving the safety/quality and shelf-life of food products. Further research is required to gain insights into the molecular mechanisms involved in bacteriocin production, immunity and mode of action.