In recent years, there has been an increasing interest in using probiotic cultures from lactic acid bacteria (LAB) as additives in animal feed to prevent or treat diseases (Alvarez-Olmos and Oberhelman 2001) and enhance the production results, e.g. weight gain and feed conversion efficiency (Guerra et al. 2007a).
For a successful application in animal feed, the probiotic cultures should contain a high concentration of viable cells, bacteriocins and fermentation metabolites to control the growth of pathogenic bacteria in both the animal feed and gut of the animals (Guerra et al. 2007a; Costas et al. 2018). Three alternatives for this purpose are: i) the use of cheaper fermentation and feeding media (like wastes from the food industry), ii) the selection of an appropriate strain, and iii) the design of an adequate fermentation procedure.
Whey (a waste from the cheese-making process) and mussel processing wastes (MPW) contain lactose in the relatively high concentration (~ 50 g/l in case of whey), glycogen (~ 10 g/l in case of MPW), proteins (~ 5.0 g/l in case of whey and 3.5 g/l in case of MPW), as well as micronutrients, including amino acids, vitamins and minerals (Murado et al. 1994; Costas et al. 2018). For these reasons, both substrates have been used for productions of probiotic biomass (Costas et al. 2018) and bacteriocins (Garsa et al. 2014; Costas et al. 2018) by different LAB.
The comparison of the antibacterial activity of 38 bacteriocin-producing LAB (including
Considering that fed-batch fermentation allows obtaining cultures with a high concentration of viable cells (Cho et al. 2010; Costas et al. 2018), the use of this fermentation technique for production of highly concentrated probiotic cultures on whey and MPW could be very advantageous. With this approach, the high mean chemical oxygen demand of these wastes, about 70 g/l in case of whey (Slavov 2017) and 25 g/l in case of MPW (Murado et al. 1994), and consequently their contamination effects could be considerably reduced.
Previous studies showed that the growth and nisin synthesis by LAB in realkalized fed-batch cultures depend on the stepwise pH profiles generated in the cultures, due to i) the effect of pH on nutrient uptake, ii) the inhibitory effect of low pH values on biomass and product formation, and iii) the specific effect of pH on bacteriocin synthesis (Cabo et al. 2001; Costas et al. 2018). Then, the use of an adequate mathematical model describing the joint effect of the culture pH and nutrient (glucose) addition on the productions of biomass, nisin and fermentation products could provide a better understanding for controlling and optimizing the fermentation process.
However, to our knowledge, no information is available on the quantification of the joint effect of glucose addition with the feeding media and the pH gradients (
For these reasons, in the present study, a first glucose-limited realkalized fed-batch culture in DW medium was designed by using a medium prepared with mussel processing wastes (CMPW) and a 400 g/l concentrated lactose (CL) as feeding media. From the results obtained in this culture, the effects of glucose addition and
The culture media used in this study (Table I) were prepared with diluted whey (DW) and a concentrated mussel processing waste (CMPW) obtained from local dairy and mussel processing plants, respectively. The pretreatment of both substrates was described by Costas et al. (2018).
Mean composition (g/l) of the substrates used as culture media.
Nutrient | DW medium | CMPW medium | CMPWGP medium |
---|---|---|---|
Lactose | 22.62 ± 0.05 | − | − |
Glucose | − | 101.72 ± 0.17 | 400.00 ± 0.01 |
Total nitrogen | 0.433 ± 0.02 | 0.540 ± 0.01 | 0.431 ± 0.02 |
Total phosphorus | 0.227 ± 0.02 | 0.060 ± 0.00 | 3.210 ± 0.06 |
Proteins | 2.07 ± 0.01 | 3.47 ± 0.03 | 2.75 ± 0.02 |
The two fixed-volume realkalized fed-batch fermentations (cultures I and II) were carried out in duplicate in a 6-l bench-top fermenter (New Brunswick Scientific, Edison, NJ, USA) containing 4 l of sterilized DW medium (pH 7.0), at 30°C, 200 rpm and with an aeration level of 0.5 l/h. The first culture was fed a mixture of CMPW medium and 400 g/l concentrated lactose (CL), and the second fermentation was fed a CMPW medium supplemented with glucose and KH2PO4 up to concentrations of 400 g glucose/l and 3.21 g total phosphorus/l (CMPWGP medium) and sterile distilled water (if needed). In the two realkalized fed-batch cultures the feeding media were used to bring the cultures up to the initial total sugars (TS) concentration (22.62 g/l) in the fermentation medium in every realkalization and feeding cycle.
However, the CMPW medium has a mean total sugars concentration (101.72 g/l) considerably lower than that (400 g/l) of the CL substrate and a total phosphorus (TP) concentration (0.060 g/l) lower than that (0.227 g/l) of the fermentation medium (Table I). Therefore, the use of the unsupplemented CMPW medium as the unique substrate to feed the growing culture has two drawbacks. First, high volumes of the feeding substrate could be necessary to replenish the initial TS levels in the culture medium in every realkalization and feeding cycle, increasing considerably the volume of fermentation medium and provoking the dilution of the culture. Second, the use of CMPW medium jointly with the sample extraction every 12 h, could lead to the exhaustion of the phosphorus source in the fermentation medium, thus limiting the growth of
For these reasons, the CMPW medium was firstly supplemented with KH2PO4 up to 3.21 g TP/l to obtain the same C/TP relationship (31.7) as in the MRS medium, because this salt was found to be the best TP source for nisin synthesis (De Vuyst and Vandamme 1993). Then, the medium was supplemented with glucose up to a concentration of 400 g/l.
Samples were taken from the culture medium in the corresponding fermenter every 12 h and divided into three aliquots to measure the viable cell counts (first aliquot), the culture pH and concentrations of biomass, nutrients and fermentation products (second aliquot), and also the nisin activity (third aliquot).
The volume of fermentation medium (
Where
From equations [1.1] and [1.2] it follows that:
The reduction in the mass (in grams) of TS in the medium due to the joint effect of the extraction of samples and TS consumption by the growing strain (
Where [
Therefore, the mass of TS (in grams) that must be added to the fermenter to restore the initial TS concentration in the DW medium was calculated by the following expressions:
Where [
Substituting Eq. [2.1] into Eq. [4.1] gives:
Thus, the
The
The
The
These sampling, feeding, and realkalization procedures were repeated every 12 h in all the cultures.
The concentration of total sugars was measured using the phenol/sulfuric acid method (Dubois et al. 1956) according to Strickland and Parsons (1968a), with glucose (at concentrations between 12.5–125.0 µg/ml) as standard. Total nitrogen was quantified by the micro-Kjeldahl method, replacing distillation by the spectrophotometric method of Havilah et al. (1977), with ammonium sulfate (at concentrations between 12.5–500.0 mg/l) as standard. Total phosphorus was determined by the molybdate reaction (Murphy and Riley 1962) according to Strickland and Parsons (1968b), with KH2PO4 (at concentrations between 0.2–2.0 mg/l) as standard. Protein was measured by the method of Lowry et al. (1951), with bovine serum albumin (at concentrations between 0.05–0.50 g/l) as standard.
Concentrations of glucose (G), lactose (L), lactic acid (LA), acetic acid (AA) and butane-2,3-diol (B) were quantified by a high-performance liquid chromatography (HPLC) system equipped with an ION-300 Organic Acids column (length 300 mm, internal diameter 7.8 mm) with a precolumn IONGUARD™ (polymeric guard column), both obtained from Tecknokroma S. Coop. C. Ltda, Barcelona, Spain. Sugars and fermentation products were separated at 60–65°C using a 0.012 N sulfuric acid aqueous mobile phase flowing at 0.4 ml/min and detected using a refractometer with a refractive index detector. Solutions of glucose, lactose, lactic acid, acetic acid and butane-2,3-diol at a concentration between 0.5 and 10.0 g/l were used as standards. Prior to HPLC analysis, all samples and standards were filtered using syringe filters (0.22-μm pore size, 25-mm diameter disk filters, Membrane Solutions, Dallas, TX, USA) (Costas et al. 2018). All the analytical determinations were performed in triplicate.
Where
The corresponding values for the constants and their standard errors were obtained by using the nonlinear curve-fitting software of the SigmaPlot program, version 12.0 (Systat Software Inc., 2012). The coefficients of the models were considered statistically significant if their
The goodness-of-fit of model [7] for each product was evaluated by analyzing the determination coefficient (
Where
The experimental concentrations of X, VCC, Nis, LA, AA or B synthesized (
Where
Subsequently, the calculated X, VCC, Nis, LA, AA or B concentrations at the end of every realkalization and feeding cycle (c
Where
The differences between the predictions of model [7] and experimental data were minimized according to the sum of squares of errors (
Where
Time course of the culture pH (◊), pH gradient (VpH, ●), and remaining (
Time course of the culture pH (◊), pH gradient (VpH, ●), and remaining (○), consumed (£), added (∆) and extracted (▲) concentrations of glucose (G), lactose (L), proteins (Pr), total nitrogen (TN), and phosphorus (TP) in the realkalized fed-batch culture II. The data reported are means ± standard deviations of two repeated experiments and three replicate measurements.
Time course of the biomass (X), viable cell counts (VCC), nisin (Nis), lactic acid (LA), acetic acid (AA), and butane-2,3-diol (B) in the realkalized fed-batch cultures I (left side), and II (right side). The data reported are means ± standard deviations of two repeated experiments and three replicate measurements. The solid lines drawn through the experimental data for each variable were obtained according to the empirical model [7].
The nisin-producing strain was grown in two growth cycles (Fig. 3) composed of two exponential growth phases, separated by an intermediate lag phase, as observed before in previously realkalized fed-batch cultures in DW medium (Guerra et al. 2007b; Costas et al. 2016, 2018).
Since
The TN and TP concentrations decreased progressively in the first culture as a consequence of their consumption by the nisin-producing strain and the sample collection every 12 h. However, in the second fermentation, the growth slowed down at 144 h of incubation, even though the culture was fed with a substrate (CMPWGP) containing a relatively high TP concentration (3.21 g/l) that produced an increase in the concentration of this nutrient in the fermentation medium (Fig. 2).
Thus, culture II was stopped at 168 h because the cells entered in the second stationary phase of growth after 144 h of incubation and, taking into account the profiles described by the variables X and Nis in fermentation I (left side of Fig. 3), it is logical to consider that the extension of the fermentation would not produce significant increases in the concentration of both variables.
In addition, the TN source could be considered as a limiting substrate for the growth of
Nisin production paralleled both the biomass production and pH gradients generated in the two cultures (Figs. 1, 2 and 3), because this metabolite was produced as a pH-dependent primary product in this kind of realkalized fed-batch fermentations (Cabo et al. 2001; Guerra et al. 2007b; Costas et al. 2016; 2018). Lactic acid was also synthesized in parallel with biomass synthesis in both cultures, but the productions of acetic acid and butane-2,3-diol triggered after 84 h of fermentation (left and right sides of Fig. 3).
Although the incubation times in fermentation I (264 h) and fermentation II (168 h) were different, the final levels of X, VCC, Nis and AA synthesized in the first culture (3.07 g/l, 1.75 × 1010 CFU/ml, 105.61 BU/ml, and 1.78 g/l, respectively) were almost similar (
This observation indicates that the CMPWGP medium can be used as an appropriate feeding substrate for the production of probiotic biomass and nisin. However, further studies based on optimizing its TP concentration are required to avoid the accumulation of this nutrient in the fermentation medium.
Significant values (
Parameter | Biomass | Viable cell counts | Nisin | Lactic acid | Acetic acid | Butane-2,3-diol |
---|---|---|---|---|---|---|
4.12 ± 0.631 | 1.86 ± 0.084 | 1.13 ± 0.113 | 5.71 ± 0.235 | 1.16 ± 0.003 | 2.75 ± 0.277 | |
−3.60 ± 0.545 | −1.56 ± 0.072 | 8.67 ± 0.4244 | −4.04 ± 0.225 | −0.70 ± 0.012 | −2.00 ± 0.265 | |
0.83 ± 0.116 | 0.41 ± 0.015 | −2.07 ± 0.206 | 0.88 ± 0.054 | 0.12 ± 0.005 | 0.37 ± 0.064 | |
−1.09 ± 0.383 | −0.15 ± 0.051 | −9.25 ± 1.547 | −0.92 ± 0.040 | −0.46 ± 0.021 | −0.19 ± 0.021 | |
0.14 ± 0.062 | 0.03 ± 0.008 | 2.93 ± 0.314 | 0.26 ± 0.019 | 0.15 ± 0.011 | 0.03 ± 0.003 | |
0.35 ± 0.168 | NS | 0.74 ± 0.203 | NS | NS | NS | |
0.9950 | 0.9948 | 0.9993 | 0.9986 | 0.9949 | 0.9954 | |
3.335 | 0.228 | 1.121 | 3.760 | 1.701 | 5.809 | |
1.81 | 1.92 | 2.32 | 2.30 | 2.83 | 2.40 | |
5.19 | 5.08 | 4.68 | 4.70 | 4.17 | 4.60 | |
1.64 | 2.72 | 1.29 | 1.76 | 1.41 | 2.80 |
From a mathematical point of view, these results indicate that X and VCC increased for
The decrease in the bacterial growth at pH values lower than 5.19 or 5.08 could be related to a reduction in the metabolic activity of
Using the same argument as that used for the
With regard to the addition of glucose, it can be noted that the
Lactic acid production in the realkalized fed-batch culture I was minimal at final pH values between 4.62 and 4.83 (Fig. 2), which includes the maximum pH value (4.70) calculated from model [7]. Similarly, when the culture reached final pH values between 4.22 and 4.97 (12–84 h of incubation), the lowest AA and B productions were obtained, because, at these incubation times, the two fermentation metabolites had not been detected in the culture medium (Fig. 2). In fact, AA and B productions started when the culture reached a final pH value of 4.99 after 84 h of incubation (left side of Fig. 3).
In the same way, the minimum concentrations of glucose in the processes for high LA, AA and B production were 1.76, 1.41 and 2.80 g/l, respectively (Table II).
Significant values (
Parameter | Biomass | Viable cell counts | Nisin | Lactic acid | Acetic acid | Butane-2,3-diol |
---|---|---|---|---|---|---|
0.46 ± 0.043 | 0.58 ± 0.053 | 0.42 ± 0.040 | 10.19 ± 0.405 | 0.41 ± 0.032 | 0.23 ± 0.062 | |
−3.32 ± 0.351 | −1.75 ± 0.062 | −27.69 ± 1.011 | −8.10 ± 0.621 | −2.19 ± 0.027 | −3.15 ± 0.333 | |
0.58 ± 0.059 | 0.45 ± 0.011 | 6.53 ± 0.462 | 1.79 ± 0.109 | 0.44 ± 0.024 | 0.63 ± 0.097 | |
0.53 ± 0.038 | 0.26 ± 0.065 | 4.73 ± 0.516 | −0.35 ± 0.017 | 0.42 ± 0.023 | 0.72 ± 0.065 | |
−0.04 ± 0.005 | −0.01 ± 0.003 | −0.10 ± 0.013 | 0.03 ± 0.006 | −0.02 ± 0.001 | −0.04 ± 0.004 | |
0.11 ± 0.006 | NS | NS | NS | NS | NS | |
0.9973 | 0.9906 | 0.9950 | 0.9959 | 0.9794 | 0.9856 | |
0.987 | 0.326 | 3.775 | 2.609 | 3.988 | 3.602 | |
1.82 | 1.96 | 2.12 | 2.26 | 2.47 | 2.49 | |
5.18 | 5.04 | 4.88 | 4.74 | 4.53 | 4.51 | |
10.55 | 11.10 | 22.83 | 5.30 | 11.17 | 10.27 |
However, in the case of nisin synthesis, the optimum pH value calculated (4.88) could be considered as the maximum final pH value for high bacteriocin production in every realkalization and feeding cycle, since the value of the coefficient for
As shown in Table IV, the optimum final pH values for nisin production by other
The optimal final culture pH values for different bacteriocins produced by lactic acid bacteria.
Bacteriocin | Producing strain | Optimum final pH | Culture medium | Reference |
---|---|---|---|---|
Mesenterocin 5 | 4.24 to 4.34 | Whey | Daba et al. 1993 | |
Pediocin AcH | 3.70 | TGE broth | Yang and Ray 1994 | |
Nisin | 5.80 | TGE broth | Yang and Ray 1994 | |
Leuconocin Lcm1 | 5.00 | TGE broth | Yang and Ray 1994 | |
Sakacin A | 4.50 | TGE broth | Yang and Ray 1994 | |
Nisin | 4.90 | Whey permeate | Flôres and Alegre 2001 | |
Carnocin KZ213 | 4.80 to 5.08 | MRS broth | Khouiti and Simon 2004 | |
Nisin | 4.60 | MRS + milk | Penna et al. 2005 | |
4.80 | M17 + milk | |||
Nisin | 4.65 to 4.96 | Whey + YE (5 g/l) | Jozala et al. 2011 |
YE: yeast extract
This different effect of the final pH values on the bacteriocin synthesis has been related with the need of an appropriate final pH range for the post-translational conversion of prebacteriocin to active bacteriocin (Yang and Ray 1994).
With regard to the glucose addition, it could be noted that with the exception of LA, the signs for the coefficients of
These observations are consistent with the results obtained for the fed-batch production of nisin by
The results obtained with the use of model [7] are in perfect agreement with the affirmation that nisin is produced as a pH-dependent primary metabolite since its production depends on both the biomass synthesis and the final culture pH in the medium (Yang and Ray 1994; Guerra et al. 2007b). Thus, biomass production by
From a practical point of view, the modeling procedure used in this work could allow: i) determining the optimum pH and glucose ranges to obtain high levels of biomass, nisin and fermentation metabolites in realkalized fed-batch cultures, ii) providing an accurate interpretation of the fermentation kinetics taking into account the effects of the amount of glucose added and the pH gradient generated in every realkalization and feeding cycle on the growth and product synthesis by