In recent years, the isolation of lactic acid bacteria from traditional fermented foods has increased promoting the production of the regional healthy foods and recovering the traditions which allows to improve the nutrition of the population[1].The microorganisms involved in these artisanal fermentation processes, in their great majority correspond to the group of lactic acid bacteria, many of these are generally recognized as safe by the food industry (GRAS), and some also have the name of probiotic microorganisms that provide, in addition to the technical benefits, specific health benefits to the hosts [2][3].

Among the traditional products of Colombia, the “Suero Costeño” (SC) is an artisanal soured cream produced in the Northern-Caribbean regional coast of Colombia. It is widely produced by the rural population in the regions of Córdoba, Sucre and Bolívar, manufactured mainly in households on a small scale, and represents a gastronomic heritage of Colombian cuisine [4] (Simanca et al., 2010). Microorganisms that are not commonly used in dairy products have been isolated from this product [5]. After distinguishing the existent microorganisms in the local products and identifying the shared probiotic technical characteristics and their molecular identification [6], it is necessary to think about the application of biotechnological scaling techniques in order to bioaugment the specific ferments and consequently make them available to improve the quality during the production of the specific products, which often is the financial support of many families depending on this production.

Scaling techniques of native potential probiotic/starter dairy strains involves the study of basic growth dynamic microorganisms, such as kinetic parameters, which is not very documented with lactic acid bacterial in Colombia; probably due to biotechnological dairy starter production of these microorganisms, has been restricted to few industrialized companies that offer starter cultures for massive use to develop food products.

This shows how the kinetic parameters of each microorganism in ideal conditions provide information that can be used to establish suitable conditions in the inoculum, among other purposes for: suggest alternative industrial culture methods, production of specific metabolites, [7], establish conditions in the concentration of cells in order to standardize probiotic and technological tests and / or cell conservation.

The purpose of this research is to establish the kinetic parameters of lactic acid bacteria isolates obtained from the fermented milk “Suero Costeño” of artisan manufacture.

The results of the kinetic growth for the three species of microorganisms are presented in Figs. 1A, 1B and 1C.

Figs. 1A, 1B and 1C present the biomass growth and substrate consumption during the fermentation and substrate consumption in commercial media for each of the isolations. The initial glucose concentration initial concentration in the BHI+glucose broth was 14,5g/L, while the initial concentration of glucose for the MRS broth was 16g/L. During the first 12 hours of fermentation they were consumed 59,6%, 65,9% and 68,9% leaving a remnant at the end of the process of 29%, 31,5% and 7% substrate for strains L. lactis 25-2, S. infantarius 46-3 and L. plantarum 60-1, respectively.

Although the initial concentration of the inoculum in the three strains did not present statistically significant differences, at the end of fermentation L. plantarum 60-1 was the isolation with the highest growth (6,46 +/- 0,71) (Fig. 1).

Figure 1 Source: Author.

The independent terms and the slope obtained for the curves of OD vs Log UFC/mL and OD Vs Dry weight g/mL. In addition, the correlated coefficients and the R² for each curve are presented on Table 2.

Table 2

Source: Author.

In general, for all microorganisms, a greater adjustment was observed between the curves that correlate the OD vs LogUFC/mL (0,94 - 0,97) that the curves that correlate OD Vs dry weight g/mL (0,85 - 0,88). For the isolates, highest correlation obtained was L. lactis 25-1 (0,97).

L. lactis 25-2 and S. infantarius 46-3 have a similar behaviour and decreasing of pH was faster than L. plantarum 60-1. Therefore, L. lactis 25-2 and S. infantarius 46-3 strains isolated from the fermented milk “Suero Costeño” can be considered starter culture, because in five hours the pH decreased two pH-metric units, after this time pH was maintained stable.

The behaviour of the L. plantarum 60-1 did not show a fast decrease in pH, but after 10h decrease more than 4 pH-metric units, reaching a better ΔpH with a value of 3.17 units.

Fig. 3 presents the results of the pH values at time 0, 6, 12 and 24 hours.

Figure 3 Source: Author.

Fig. 2 presents the results of the three LAB strains pH change studied during the time.

Figure 2 Source: Author.

During the first six hours, pH changes more faster for the L. lactis 26-2 and S. Infantarius 46-3, (1,87 y 2,13 respectively) isolations while the L. plantarum 60-1 was of 0,36 units; after 12 hours of fermentation the changes were L. lactis 26-2 (0,19), S. Infantarius 46-3, (0,04) and L. plantarum 60-1(2,02), after 24 hours of process, the change was observed only in the L. plantarum 60-1 (0,17) isolation.

Table 3 presents the results of the acidity calculated (mg/L) from equation 3.

Table 3

Source: Author.

The highest acidifying activity was for L. plantarum with a 24-hour value of 58.43 (mg /L), the values for L. lactis and S. infantarius were 0.89 and 0.76 respectively.

Table 4 presents the results of biomass performance based from the substrate, specific growth rate and doubling time for the studied isolates.

Table 4

Source: Author.

The highest yield biomass was for L. plantarum 60-1 with a value of 0.3 (gX/gS), followed by L. lactis 26-2 and S. infantarius 46-3 with a value of 0,24 (gX/gS). The specific growth velocity (μ) in the exponential phase was greater for S. infantarius 46-3. The doubling time of cells was similar for L. lactis 26-2 and L. plantarum 60-1 and almost 1.5h less for S. infantarius 46-3 which obtained the shortest doubling time.

The selected commercial culture media are suitable for the lactic acid bacteria growth of fermented milk “Suero Costeño” (Fig. 1), since adaptation phase is not evident from the moment of inoculation. Logarithmic phase is concluded between 10 and 12h for the microorganisms in discontinuous growth, which does not coincide with others authors: Escobar et al. carried out the fermentations with enriched buttermilk for lactobacillus species for almost 170 hours [18]; Peñuela et al. performed fermentations in yogurt for the L. plantarum and L. lactis growth with almost a duration of 72 hours; Agudelo et al. develop fermentations with time duration of 48 hours [19]. However, it was observed that after hour 13, the pH was stabilized as well as the biomass production, and at 24 hours, no change in the biomass. As a result, the early stationary phase is adequate, for the performance of many tests or procedures which is achieved in a time shorter than 13h for the established growth conditions.

The results of the pH curves indicates that the strains studied are good candidates to be used in industrial processes as starter microorganisms, some as primary cultures and others as adjuvants [10]. In the elaboration of dairy products a rapid decrease in pH causes the destabilization of casein micelles (milk protein) due to the solubilization of their calcium phosphate, the pH values reached are close to the isoelectric point of the milk, with which coagulation takes place. L. plantarum 60-1 having a decrease in the higher pH but need more time compared to the other two strains, could be used as an adjunct culture [20]

L. plantarum 60-1 had the highest yield biomass probably to resistance pH stress. According to Chungking et al (2017), L. plantarum could maintain constant pH gradient between the inside of the cell (cytoplasm) and culture media (extracellular) in the presence of high lactate concentrations, therefore it is capable of supporting acid culture media in comparison with the other microorganisms and finish completely the fermentation [21].

In the growth and substrate consumption pH curves and due to the lactic acid nature of the isolates such as the fermentation metabolites, it is possible for either L. lactis 25-2 or S. infantarius 46-3 in addition to the carbón source, the occurrence of an inhibition per product, that even though with a decrease evidence in the substrate concentration, did not allow the microorganisms the glucose consumption, therefore; there was a decrease in the expected growth, this is due to the fact that most of the bacteria have an optimal pH value and when the decreasing occurred in the first 8 hours this could inhibit cell growth.

L plantarum 60-1 not show an inhibition by the acidity, probably, this microorganism use a mechanism known as F1-F0 ATPase as protection under acidic conditions. The F1-F0 ATPase is a multiple enzymatic subunit, which contains a catalytic portion (F1), incorporating subunits for the hydrolysis of ATP; and an integral membrane portion (F0) that includes subunits that function as membrane channels for proton translocation [22]. To obtain a higher yield biomass and improve the performance of the strains L. lactis 25-2 and S. infantarius 46-3, it is necessary to carry out a pH control in the optimal growth values, since the cellular metabolism tends to turn slower when it is not operating in optimal conditions [23].

The kinetic parameters values obtained in this work differ from those reported in the literature; Georgieva et al. present a Yx/s of 0,143 for L. plantarum in MRS culture médium with glucose [24] obtaining 48% better results. The speed depends on several factors among which are the nutrients concentration, products, pH and temperature. The results of the three strains studied show a specific growth rate lower than that reported by other authors. Agudelo et al. 2010, reported a 0,53 (h^-1) for L. plantarum [19]. When the specific growth rate depends only on a limiting substrate, when the substrate is added in high concentrations, the specific velocity reaches maximum values, up to the point at which substrate inhibition occurs. The results obtained seem to indicate that, although we worked with a simple substrate (glucose) we would expect a higher specific growth rate, therefore, it is assumed that the kinetics growth was of order 1 where the low substrate concentration makes that the values of u are below the maximum growth rate.

The doubling time values indicate the time it takes for LABs to double their population. For L. plantarum 60-1 doubling times between 1.1 and 1.98 hours have been reported, therefore, the effect of some variables such as the change in pH over the doubling time could be evidenced.

The culture media used was suitable for the growth of the microorganism, since the adaptation phase was not observed in the studied isolates, despite being bacterias that can be used as starter culture. For its production and bioaugmentation it is necessary to carry out a pH control during the fermentation since it is not possible to consume all the substrate and an inhibition by product is observed without reaching the best performance in biomass.

Kinetic parameters were obtained from lactic acid bacterial isolates from the fermented milk “Suero Costeño”, which can be used in different fermentation processes in the food industry, taking into consideration the acidifying capacity and kinetic growth.