3.1. Kinetic Analysis of L. plantarum Culture on Plasma-Based Media at Laboratory and Bench Scale
Figure 1a,b show the experimental data of
L. plantarum ATCC 8014 cell growth on two media based on bovine blood plasma with hydrolyzed proteins at the laboratory and bench-scale levels, respectively. It is observed that cellular biomass increases significantly in the first 36 h of culture for the laboratory scale, while at bench scale the biomass steadily increases in the first 26 h reaching higher values. Regarding the concentration of total carbohydrates in both fermentations, it is observed that their concentration at the laboratory-scale fermentation takes up to 10 h to begin the reduction and to subsequently exhibit a traditional decreasing substrate behavior. For bench-scale fermentation, the carbohydrate content increases from the beginning until 12 h and then decreases until 36 h. From this moment, the concentration of carbohydrates is stabilized, which coincides with the beginning of the stationary phase of lactobacilli growth. It is important to note that the method used to determine the concentration of the carbon source corresponded to the quantification of total carbohydrates [
29]. This method not only quantifies the sucrose content, but also includes the content of different types of carbohydrates including monosaccharides, oligosaccharides, and polysaccharides. The above explains the atypical behavior of carbon source kinetics at bench scale since different researchers have demonstrated the formation of exopolysaccharides during lactic fermentation [
37,
38,
39], which represent a mechanism of defense of bacteria against desiccation, phagocytosis, phage attack, antibiotics, toxic compounds, protozoan predation, and osmotic pressure, among other factors, in addition to playing a role in cell recognition. This ability of lactobacilli to synthesize exopolysaccharides may explain the increase in carbohydrate concentration at the beginning of fermentation, especially if considering that the culture conditions in a bioreactor are more severe and controlled than in a laboratory flask.
With the experimental data from
Figure 1, the non-linear regression algorithm was applied to find the four parameters of the mathematical model proposed (see
Table 3). The specific cell growth rate obtained for the culture model at bench scale (0.0545 h
−1) was higher than the average of the
µmax values for the laboratory scale (0.0111 h
−1). The maximum biomass
Xmax also showed higher values at bench scale (9.4136 CFU/mL) than at the laboratory scale (7.44 log CFU/mL). The above is explained by the particularities of the technological system of fermentation at the laboratory and bench-scale levels, since in the latter the conditions of the environment are more homogeneous, which translates into better availability of nutrients, substrates, and dispersion of products. On the other hand, the
qp coefficient showed substantially higher absolute values at the lab-scale fermentation, compared to the bench-scale fermentation. This suggests that fermentation at a laboratory scale produces a more tangible decrease in the concentration of total carbohydrates in the medium, considering that it does not seem to indicate a greater production of exopolysaccharides than the consumption of carbohydrates, a phenomenon that can be seen in the fermentation at bench scale. On the other hand, the values of the
n factor indicate that the microorganism is relatively resistant to self-inhibition in both cultivation scales.
When solving the system of two ordinary differential Equations (1) and (2) that make up the mathematical model with these parameters values, the kinetic curves were generated as depicted in
Figure 1 through continuous lines. The proposed deterministic, non-segregated, and dynamic model adequately described the growth kinetics of
L. plantarum ATCC 8014 on a discontinuous culture at bench scale and laboratory level, as well as the dynamics of carbohydrates for these two cases. Considering the above, the proposed kinetic model captures well the complexity of these phenomena. In particular, it is suggested that the sucrose present in the medium is hydrolyzed into its two constituent monosaccharides (glucose and fructose). These monosaccharides are absorbed by bacterial cells for their energy metabolism and, subsequently, for the synthesis of exopolysaccharides, which leads to a complex balance of total carbohydrates present in the culture broth. Equation (2), proposed in a previous work [
31], applied a kinetic relationship that describes the formation and consumption of reducing sugars during the degradation of lignocellulosic materials by white rot fungi. In this work, the aforementioned equation was applied to model this carbohydrate balance based on the growth rate of cells and, to a lesser extent, on their concentration. Regarding the biomass concentration, the obtained adjustment allows us to conclude that, at both, laboratory and bench-scale levels, the logistic equation appropriately captures growth behavior at its exponential and stationary phase for the fermentation systems studied.
A submerged fermentation process was implemented at bench scale using the proposed medium for
L. plantarum growth based on bovine blood plasma with enzymatically hydrolyzed proteins (see
Table 1). The studied medium was then compared to a commercial medium (MRS) for
L. plantarum propagation as can be observed in
Figure 2. It is evident that the cell biomass curve during fermentation at bench scale has a behavior similar to that obtained at the laboratory level (150-mL working volume). However, the comparison with the biomass formation curve in the MRS medium indicates that there is an appreciable latent phase in the plasma-based medium. This latency is due to the period of adaptation required by lactobacilli to assimilate the carbon and nitrogen sources available in the plasma-based medium, which are different from the MRS commercial medium: glucose as a source of carbon and peptone, yeast and meat extracts as nitrogen sources. This means that time required for reaching the maximum values of cell biomass, increases from 24 h in the commercial MRS medium to 52 h in the plasma-based medium. Although it is evident that the growth rate in the medium based on the hydrolyzate of BBP proteins at bench scale has a lower rate than that of the MRS medium, it achieves higher concentrations of viable biomass of
L. plantarum (9.58 log CFU/mL or 3.80 × 10
9 CFU/mL versus 9.53 log CFU/mL or 3.39 × 10
9 CFU/mL of the MRS medium). This result is relatively similar to that reported by Hyun and Shin [
19] for a medium based on an enzymatic hydrolyzate of bovine blood plasma mixed with MRS broth, with a protein content close to that of this study of 30.2 g/L, in which a maximum concentration of 9.71 log CFU/mL (5.13 × 10
9 CFU/mL) was obtained at 24 h of fermentation.
It should be noted that the MRS medium is already optimized for the proliferation of lactobacilli in general at the laboratory, but its use at bench, pilot, or industrial levels is limited by its high costs. It is precisely for this reason that a plasma-based medium from a residual effluent, such as bovine blood from slaughterhouses, is an attractive alternative to use a waste from the meat industry, especially when the final biomass concentrations reached are comparable to those of a medium used for laboratory level research.
3.2. Validation of the Use of the Biomass Obtained for the Elaboration of a Meat Product
The cell biomass of
L. plantarum obtained during bench-scale fermentations was recovered from the culture broth and washed in order to be used for preparing pepperoni, a maturated meat product. For comparison, pepperoni was prepared by using a commercial starter culture widely employed in meat industry. The results of the physical-chemical and microbiological requirements of both the meat raw material and finished products are consolidated in
Table 4 and
Table 5.
The data presented in
Table 4 and
Table 5 were within the indices allowed by the Colombian standard NTC 1325, which indicates adequate management of hygienic conditions in pre-processing and subsequent stages of pepperoni preparation, including cutting and packing. In this way, raw materials have the appropriate sanitary conditions to be subjected to the maturation process, and they can also go through the stages of conditioning, drying, and ripening adequately to obtain a product that meets the sanitary requirements for its consumption. The values of pH, acidity, moisture, and count of altering microorganisms correspond to a level of good quality and absence of pathogens and provide a suitable medium for the development of starter cultures (LAB), allowing these bacteria to more easily colonize the pepperoni mixture and develop its characteristic fermentative metabolism without competing with other microbial flora. The comparative results of the physical-chemical and microbiological analyses of pepperoni made with the studied strain and the control pepperoni made with the commercial starter culture are shown in
Table 5. It is observed that the control product and the pepperoni made with
L. plantarum have a good quality level according to NTC 1325. In addition, moisture and pH meet the composition and formulation requirements for matured or fermented products.
The results obtained for the triangle sensory analysis of pepperoni using
L. plantarum ATCC 1084 and the control with commercial culture are presented in
Table 6. These outcomes show that 70% of the testers identified pepperoni made with
L. plantarum as a different sample. However, 50% of the testers rated the samples as slightly different and 20% of them thought the differences were moderate.
Table 7 shows the results of the Kruskal Wallis test for multidimensional profile analysis obtained from the sensory evaluation of the nine descriptors applied to pepperoni and performed by ten semi-trained testers. It can be observed that there are no significant differences in the perception of the intensity of each descriptor for pepperoni made with
L. plantarum and with the commercial culture, given that there are no differences between the means.
Figure 3 shows the multidimensional sensory profile obtained for the product inoculated with
L. plantarum and for the product inoculated with the commercial culture. The result shows that the sensory profile of pepperoni in the evaluation overlaps for all descriptors, except for the color and characteristic odor or aroma, which are still close.
The appearance of products formulated with the commercial culture and with
L. plantarum is shown in
Figure 4. It is evident that the color and general appearance of the control product and that made with
L. plantarum do not show an appreciable visual difference.
Regarding the overall quality, the results of the semi-trained panel were as follows: 70% of the testers considered pepperoni made with L. plantarum with a high-quality level and the remaining 30% evaluated it with medium quality. For the control product, 80% of the testers evaluated it with a high-quality level and 20% with medium quality.