Low heat skim milk powder (LHSMP) was obtained from Valio Ltd. (Helsinki, Finland). LHSMP was suspended in distilled water to yield a final concentration of 10% (w v⁻¹) milk solids, with addition of CaCl₂ in final concentration of 10 mM. The samples with LHSMP were mixed thoroughly for 1 h at room temperature, heated at 90°C for 30 min and cooled to 30°C immediately before the experiments and used as liquid RSM samples.

The strain of Streptococcus thermophilus ST12 (further St. thermophilus ST12) was kindly provided by Chr. Hansen (Hørsholm, Denmark). Deep-frozen cultures of St. thermophilus ST12 were thawed and pre-grown on M17 agar (LAB M, Lancashire, UK) for 24 h at 40°C. One colony from a M17 agar plate was inoculated into 10 mL of RSM and left at 40°C until coagulation (16 h). One per cent of the culture was used for inoculation of the 10 mL of RSM samples, left until coagulation (12 h) and further used for inoculation of the microcalorimetry samples. The number of viable cells in the inocula was determined by plating on M17 agar followed by incubation for 72 h at 40°C to be (1.15 ± 0.19) × 10⁹ CFU mL⁻¹.

RSM as growth media were prepared in 50 mL Erlenmeyer flasks. Milk samples were inoculated with 1% (v v⁻¹) of inoculum and stirred thoroughly. The concentrations of inocula in milk samples for microcalorimetry were varied from 10¹ to 10⁶ CFU mL⁻¹ with the 10-fold increment. Milk gels were prepared by addition of 100 μl of a 10 g L⁻¹ chymosin (CHY-MAX Powder Extra, Hørsholm, Denmark) aqueous solution to 10 mL of RSM. The enzyme (chymosin) was added immediately after the inoculation. Milk samples for the microcalorimetric experiments, 2 mL samples of RSM and r-RSM were transferred into the autoclaved microcalorimetric ampoules, inserted into microcalorimeter and maintained at 40°C. Monitoring of the growth of microorganisms was started in microcalorimeter about 1 h after the inoculation, after the temperature of the samples was in perfect equilibrium with the set temperature of the thermostat (40°C). Taking into account that the coagulation of the liquid milk samples was over in 40 min after the inoculation, the growth of the bacteria in case of the r-RSM samples was studied in practice in solid gel matrices from the beginning of the microcalorimetric monitoring.

The remaining after the preparing of the microcalorimetric samples inoculated and renneted milks were divided into 1 mL aliquots and incubated at 40°C in parallel with the samples in microcalorimeter and used for the determination of pH and the concentrations of carbohydrates, lactic acid and amino acids at appropriate time points, determined based on the peculiarities of power-time curves.

A 24 channel isothermal batch microcalorimeter TAM III Thermal Activity Monitor (TA Instruments, New Castle, DE, USA) was used for the monitoring of the growth of thermophilic starter St. thermophilus ST12. Data acquisition and analysis was carried out using TAM Assistant Program (v 0.9.1012.40, SciTech Software AB, Thermometric AB).

Incubated in parallel to the microcalorimetric samples milk samples were mixed at chosen times 1:1 with isopropanol for sedimentation of proteins. The precipitate was removed by centrifugation at 14,000 × g for 10 min. The supernatant was filtered through a 13 mm diameter and 0.2-μm pore-size regenerated cellulose (RC) membrane filter (Whatman, Maidstone, UK) and diluted with water before analysis. High-performance liquid chromatography (HPLC) system (Alliance 2695 system, Waters Corp., Milford, MA) with a Refractive Index Detector 2414 and column BioRad HPX-87H 300 × 7.8 mm (Hercules, CA) was used for measuring lactose, glucose, galactose and lactate concentrations.

Analysis of FAA was performed on an ultra-performance liquid chromatography (UPLC) system (Acquity UPLC; Waters Corp.) including a binary solvent manager, a sample manager and photodiode array (PDA) detector, connected to Waters Empower™ 2.0 software. Separations were performed on a 2.1 × 100 mm Waters Acquity UPLC AccQ•Tag Ultra Column operated at 55°C, the running time being 12 min. Sample derivatization procedure was as follows: 20 μl AccQ•Fluor reagent was added to the mixed solution of 70 μl AccQ•Fluor borate buffer and 10 μl sample or standard. The mixed solution was vortexed immediately for 10 s, transferred to an autosampler vial and allowed to stand at room temperature for 1 min. Then the vials were placed in a heating block at 55°C for 10 min, after which they were analyzed by the UPLC system. Empower software (Waters Corp.) was used for the data processing.

The pH of milk samples was measured with pH meter S20 Seven Easy equipped with InLab 413 electrode (Mettler-Toledo GmbH, Greifensee, Switzerland).

The growth characteristics of the bacteria (yield coefficient values, growth rates etc.) during the growth in milk were calculated on the basis of the heat evolution converted to biomass concentrations or cell numbers and concentrations of organic acids etc. in the culture media as follows:

where μ is the specific growth rate (h⁻¹), t is time (h), N is the number of cells calculated assuming that heat produced per cell formation is constant (Y_Q = 4.45 × 10⁻⁹ J per cell), X is the dry biomass (gdw L⁻¹), assuming that the mass of one cell is 0.2 × 10⁻¹² g (unpublished data), I_Lactose is the lactose consumption per biomass produced (mmol gdw⁻¹), O_Lactate is the lactate production per biomass produced (mmol gdw⁻¹); C designates the concentration of corresponding compound, for example C_Lactate (mM), Y_{Lactate/Lactose} shows the lactate yield per lactose consumed, Y_XS is the biomass yield per hexose consumed and I_AA is the consumption of corresponding amino acid per biomass produced (mmol gdw⁻¹).

All the microcalorimetric experiments were repeated twice and measurements were carried out with two or three parallel samples. The power-time curves presented on the Figures and used in the calculations are combined curves of two parallel experiments.

The other analyses (HPLC, UPLC, pH) were carried out in triplicate. All values of the parallel experimental points were averaged and reported along with the value of standard deviation (SD). The experimental data were submitted to single-factor analysis of variance (ANOVA), and the differences of the means were evaluated by Fisher's least significant difference (LSD) test. The difference of the mean values was accepted at the significance level p < 0.05. The Student's t-test was performed to evaluate statistically significant differences between the mean calorimetric values obtained in RSM and r-RSM.

Representative calorimetric power-time curves of the growth of ST 12 in RSM and r-RSM, at inoculation rate 10⁵ CFU mL⁻¹, are presented in Figure 2.

As was previously described (Stulova et al., 2013), a typical microcalorimetric power-time curve of inoculated RSM contained two peaks corresponding to two distinctively separated growth phases (first and second exponential growth phases). As seen in Figure 2, the general pattern with two major exponential growth phases was observed also in r-RSM. The growth curves of St. thermophilus ST12 in r-RSM and RSM were practically coinciding in the beginning, but starting from a certain number of the bacteria in the sample (N^* = 0.63 × 10⁷ CFU mL⁻¹ at N₀ = 10⁵ CFU mL⁻¹), at the “deviation moment” marked by the arrow in Figure 2 the maximal calorimetric growth rate (μ_max1) in r-RSM was decreased, branching from the curve of RSM, and the further growth in the coagulated matrix was clearly different from that in RSM. This led to the appearance of the second slower phase in the first exponential phase in comparison with the growth curve in milk (RSM). The splitting of the second exponential growth phase also into two was observed as well—see Figure 2.

As seen in Figure 2, acidification developed more slowly after 3.5 h of incubation in renneted milk compared to the liquid milk samples which was in a good correlation with the slower growth rate of the bacteria in r-RSM. Higher pH values in r-RSM samples accompanying the main part of the growth of the bacteria were observed in case of all inoculation rates studied (Figure 3B), in difference from a previous report (Favrot and Maubois, 1996) showing slower acidification in rennet curd only at lower level of inoculation (10² CFU mL⁻¹).

The studies of consumption of lactose and formation of organic acids were carried out in the case of inoculation rate of 10⁵ CFU mL⁻¹.

The changes of the concentration of carbohydrates (lactose, glucose, galactose) and lactic acid during fermentation of RSM and r-RSM with St. thermophilus ST12 at inoculation rate 10⁵ CFU mL⁻¹ are presented in Figure 4. As seen from this figure, the patterns of hydrolysis of lactose, release of glucose and galactose and formation of lactic acid were practically identical in both RSM and r-RSM (Figure 4). Approximately 130 mmol L⁻¹ of lactose was hydrolysed at the end of the second exponential growth phase (during active growth), 127 mmol L⁻¹ of glucose and 58 mmol L⁻¹ of galactose was utilized by the bacteria, and about 20 mmol L⁻¹ of lactic acid was produced.

Lactose consumption and lactate production per biomass formed (mmol gdw⁻¹) were calculated (see Section Calculation of Growth Parameters Characterizing Metabolism of the Growing Cells) and are presented in Figures 5A,B. During the growth of St. thermophilus ST12, lactose was consumed very fast—up to 2200 and 1800 mmol gdw⁻¹ in liquid and renneted RSM, respectively. Lactose consumption led to the accumulation of galactose, as well as glucose (Figure 5), due to low glycolytic activity of the bacteria, reflected by the low lactate yield per lactose below 0.2 mol mol⁻¹ in RSM as well as in r-RSM (theoretical maximum 4 mol mol⁻¹). It can be assumed that galactose was not metabolized by this strain; however, the accumulation of galactose was lower than predicted compared to the consumption of lactose. This indicated that either galactose was consumed through glycolysis, or it was used for polysaccharide production. Usually polysaccharides are produced in growth-restricted conditions to synthesize carbon storage substances for the future.

The changes of the concentrations of the individual FAA during fermentation of liquid and renneted RSM with St. thermophilus ST12 at inoculation 10⁵ CFU mL⁻¹ were evaluated and presented in Figure 6. As seen from Figure 6, glutamic acid was initially the most abundant free amino acid in both, RSM and r-RSM, accounting for 46% of the total.

There was a dramatic reduction of asparagine, glycine and valine at the end of exponential phase in both RSM and r-RSM. Leucine showed a clear decline in RSM during fermentation, whereas in the case of r-RSM a larger increase of this amino acid as well as phenylalanine was observed. It can be seen from the data presented in Figure 7 that the intensive liberation of the majority of amino acids in RSM and r-RSM was observed during the period from the end of exponential phase till the end of fermentation (from 5 h till 22 h).

The concentrations of the total free amino acids (TFAA) were 15% higher in r-RSM than those determined in RSM at the end of cultivations, 2024 and 1713 μmol L⁻¹, respectively. Glutamic acid, proline, lysine and aspartic acid were the most predominant FAA in RSM at the end of fermentation (at 22 h), accounting for 22, 12, 9, and 8% of the total, respectively. In contrast, leucine, glutamic acid, phenylalanine, arginine and proline, accounting for 18, 17, 12, 11, and 8% of the total were the dominant amino acids in r-RSM—see Figure 8.

Proteolysis in rennet curd is catalyzed by enzymes from coagulant and enzymes from the inoculated St. thermophilus The initial hydrolysis of caseins is caused by the chymosin which results in the formation of large (water-insoluble) and intermediate-sized (water-soluble) peptides which are degraded subsequently by the coagulant and enzymes from the starters (Sousa et al., 2001). The final products of proteolysis are FAA and their concentration in curd is the net result of the liberation of amino acids from casein, their degradation to catabolic products and also utilization and synthesis by the starter bacteria. Free amino acid concentrations changes were evaluated in liquid and renneted milk samples during the growth of St. thermophilus ST12—see Figures 6–8. Amino acid consumption during the 1st and 2nd exponential growth phases is presented in Table 5 Although St. thermophilus has not been shown to be auxotrophic for any single amino acid, growth is abolished when both Glu/Gln and Cys/Met are removed (Letort et al., 2002). In addition, the exhaustion of Leu and Val reduces the growth rate more than three times and only six amino acids (Asn, Ala, Ile, Gly, Ser, and Thr) can be individually omitted without any effect on the growth rate.

Most of the trends in consumption of FAA during the exponential growth of St. thermophilus ST12 in RSM and r-RSM were similar—see Tables 5, 6. The only amino acids consumed in amounts corresponding to the calculated requirements for biomass synthesis in the 1st exponential phase were Gly, Glx (Gln+Glu) and Asx. These amino acids were also the only amino acids whose concentrations in milk exceeded the amount required for biomass synthesis (Table 6). The amount of free His was also sufficient to satisfy biomass requirements, but it should be noted that the amount of this amino acid did not decrease during the exponential growth of the bacteria. Most of the concentrations of other FAA could not support the growth of cells more than 30%. Free Arg and Pro in milk could support amino acid requirements for over 40% but instead of decrease the concentrations of these amino acids increased during the 1st exponential growth phase. This pattern was quite general. Accumulation of amino acids in the media, especially in renneted milk samples due to their release from proteins, and most noticeably during the second exponential growth phase, made it impossible to analyse consumptions of amino acids in this growth phase.

Overconsumption of Glu 5–7 times exceeding the need for biomass synthesis indicated the potential of the synthesis of other amino acids from it. In total, 4.6 mmol g⁻¹ of amino acids were incorporated into the biomass. Of the total, 30% was covered by the consumption of FAA (excluding Glu). If we take into account Glu overconsumption and amino group transfer to other amino acids, Glu can provide ammonia for 25% of amino acids of the biomass. The remaining 45% of amino acids must be have been derived from peptides.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.