Process Biochemistry xxx (2004) xxx–xxx

4 Modelling the fed-batch production of pediocin using

5 mussel processing wastes
6 Nelson P. Guerra, Ana Torrado Agrasar, Cristina López Macı́as, Lorenzo Pastrana∗
7 Departamento de Bioquı́mica, Xenética e Inmunoloxı́a, Facultade de Ciencias de Ourense, Universidade de Vigo,
8 As Lagoas, 32004 Ourense, Spain
9 Received 9 December 2003; accepted 23 March 2004

10 Abstract

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11 Cell growth and pediocin production by Pediococcus acidilactici NRRL B-5627 were compared using batch (on MRS broth and a culture
12 medium from mussel processing wastes (MPW)) and two fed-batch fermentations on MPW with re-alkalization cycles. This last fermentation
13 technique yielded the highest biomass and pediocin productions as compared to batch fermentations. Mathematical models were set up to

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14 describe fed-batch production of biomass and pediocin by P. acidilactici. While cell growth was dependent on pH change, nitrogen and
15 phosphorous availability and product inhibition (lactic acid, ethanol and butane-2,3-diol), pediocin production was dependent on both growth
16 and the final pH reached in each re-alkalization period. The models developed offer a better fit and a more realistic description of the
17 experimental biomass and pediocin production data than the logistic and Luedeking and Piret model. Therefore, they could be used to design
18 feeding strategies for enhancing and controlling fed-batch pediocin production.
19 © 2004 Published by Elsevier Ltd.
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20 Keywords: Pediocin; Fed-batch fermentation; Mussel processing wastes; Modelling; Homolactic fermentation; Mixed acid fermentation.

21 1. Introduction Since bacteriocin synthesis is considered to be growth 39

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associated, factors affecting biomass production (such as 40

22 In recent years, there has been an increased interest in the temperature, pH, media composition, availability of certain 41
23 use of bacteriocins as natural food preservatives and antimi- essential compounds, presence of inhibitory compounds) 42
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24 crobial agents [1]. Bacteriocins are biologically active pro- also affect bacteriocin production [10,11]. However, in some 43
25 teins that have an antibacterial activity against Gram-positive cases the pH can also produce a specific effect on bacteriocin 44
26 bacterial species related to the producer strain. Some of these synthesis without affecting biomass production [10,12–14]. 45
27 have a very narrow spectrum of activity but others have a In batch fermentations, when the optimal initial culture con- 46
ditions for cell growth are fixed (pH, temperature and me-
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28 relatively broad spectrum of antibacterial activity [2–4]. 47

29 Pediocins (that are produced by Pediococcus strains, or- dia composition), bacteriocin production was found to be 48
30 ganisms generally recognized as safe (GRAS)) have a wide strongly dependent on the initial pH [15–18], the pH-time 49
31 inhibitory spectrum of activity which includes both spoilage course [13,19], the pH drop [12,19] and the final pH reached 50
32 and pathogenic organisms, such as Listeria monocytogenes, in the cultures [19–22]. However, this effect of pH on bac- 51
Enterococcus faecalis, Staphylococcus aureus and Clostrid- teriocin production depends on the culture medium [17,18]
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33 52
34 ium perfringens [5]. The use of these bacteriocins in combi- and the bacteriocin-producing strains or species [10,23]. 53
35 nation with other stress inducing processes (such as heating, Studies on factors affecting bacteriocin production by lac- 54
36 freezing, acid treatment, chelating agents, high hydrostatic tic acid bacteria (LAB) are usually carried out in rich, un- 55
37 pressure and electroporation) can also be effective against defined media. However, such media are too expensive and 56
38 Gram-negative or resistant Gram-positive bacteria [6–9]. have high peptone contents, which make difficult the sub- 57
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sequent purification of bacteriocins [10]. For these reasons, 58

residual effluents from food industry have been used as in- 59
∗ Corresponding author. Tel.: +34 988 387062; fax: +34 988 387001. expensive substrates for bacteriocin production [13,14]. In 60
E-mail address: pastrana@uvigo.es (L. Pastrana). a previous work [14], we demonstrated the suitability of 61
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1 0032-9592/$ – see front matter © 2004 Published by Elsevier Ltd.

2 doi:10.1016/j.procbio.2004.03.014

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62 glycogen-rich mussel processing wastes [(MPW); average Batch cultures of P. acidilactici on MRS and MPW 102
63 COD: 25 g O2 /l; glycogen as main component: 5–10 g/l] to medium were performed in 250 ml Erlenmeyer flasks con- 103
64 be used as culture medium for batch production of nisin and taining 50 ml of the fermentation medium, on a rotary 104
65 pediocin. Since the production of some bacteriocins can be shaker (200 rpm) at 30 ◦ C for 18 h. The inoculum consisted 105
66 increased by employing fed-batch rather than batch fermen- on 2% (v/v) of an exponentially growing culture on MRS 106
67 tation methods [25–27], we used a fed-batch fermentation broth or MPW medium. Samples were withdrawn (each 2 107
68 technique based on successive re-alkalizations of the culture or 4 h) during incubation periods to perform the analytical 108
69 medium [12] to improve nisin production on mussel pro- determinations. 109
70 cessing wastes [24]. Fed-batch and re-alkalized cultures on MPW medium 110
71 Some mathematical models have been proposed to de- were carried out at a controlled temperature of 30 ◦ C in a 6 l 111
72 scribe bacteriocin production in batch cultivations [16,19]. bench top fermentor (New Brunswick Scientific, New Jer- 112
73 These models were set up based on the Luedeking and sey) with an agitation of 200 rpm and continuous-record of 113
74 Piret-like equation [28], with a term for growth associated pH. The fermentor was filled with 4 l working volume of 114
75 bacteriocin production and a term for bacteriocin degrada- medium. The aeration level (0.5 l/h) was obtained by con- 115
76 tion or adsorption. However, models describing fed-batch trolling the air supply by a flowmeter. Stepwise-pH profiles 116
77 bacteriocin production are lacking and those used in batch were obtained by re-alkalizing the cultures repeatedly up to 117
78 cultivations were not suitable for the simulation of fed-batch initial pH (6.3) with 4N NaOH each 8 h. The feeding sub- 118
79 bacteriocin production [12,26]. strate was added at the same time as re-alkalizations. The 119
80 In this paper, we report on the use of the fed-batch culture inoculum consisted of 2% (v/v) of an exponentially grow- 120
81 technique with re-alkalization cycles to increase pediocin ing culture on MPW medium. Samples (100 ml) were with- 121

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82 production by Pediococcus acidilactici NRRL B-5627 on drawn at 8 h intervals to perform analytical determinations. 122
83 mussel-processing wastes. Furthermore, mathematical mod- The total sugar determination was used to calculate the fresh 123

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84 els were set up to describe the kinetics of P. acidilactici feeding substrate volumes needed to restore the initial total 124
85 NRRL B-5627 cell growth and bacteriocin synthesis during sugars concentration (5.33 g/l) in the fermentation medium. 125
86 fed-batch fermentation. The culture volume was kept constant by feeding the fer- 126
mentor with 100 ml fresh substrates containing the amounts 127
of sugars consumed in each cycle and distilled water when 128

87 2. Materials and methods this was necessary. 129

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Two fresh substrates were used to feed the fermentor: a 130

88 2.1. Microorganisms 240 g/l concentrated glucose (fermentation I), and a concen- 131
trated MPW (CMPW, Table 1) medium (fermentation II) 132

89 Pediococcus acidilactici NRRL B-5627, the pediocin- which was prepared in the same conditions as the fermenta- 133

90 producing strain, and Carnobacterium piscicola CECT tion medium (MPW). The feeding media were added to the 134

91 4020, the target organism, were obtained from the Northern fermentor using a peristaltic pump (LKB, Pharmacia). 135
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92 Regional Research Laboratory (NRRL, Peoria, IL, USA)

93 and the Spanish Type Culture Collection (CECT), respec- 2.3. Analytical determinations 136

94 tively. These bacteria were maintained at 4 ◦ C on agar slants

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95 (MRS). Methods for determination of cell growth, total phospho- 137

rus, nitrogen, protein and sugars were described or referred 138

96 2.2. Culture medium, inoculum preparation and to in a previous paper [24]. 139

97 fermentation conditions The concentrations of lactic and acetic acids, butane-2,3- 140
EC

diol and ethanol were measured by HPLC using an ION-300 141

98 The composition of the mussel processing wastes used as Organic Acids column (length 300 mm, internal diam- 142

fermentation medium is shown in Table 1. The preparation eter 7.8 mm) with a pre-column IONGUARDTM (poly- 143
99
100 of this waste to be used as culture medium was described meric guard column), both obtained from Tecknokroma 144

previously [14]. S. Coop. C. Ltda., Barcelona, Spain. The mobile phase 145
101
consisted of 0.006N H2 SO4 at a flow rate of 0.4 ml/min
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146
at (60–65) ◦ C and the refractive index of the peaks was 147
Table 1 measured by a refractometer with a refractive-index detec- 148
Mean composition (g/l) of the culture media obtained from MPW
tor [24]. All analytical determinations were performed in 149
MPW CMPW triplicate. 150
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Total sugars 5.33 101.33

Reducing sugars 5.33 101.33 2.4. Antibacterial activity assay 151
Proteins 1.82 3.47
Total nitrogen 0.65 0.54
Aliquots from P. acidilactici NRRL B-5627 cultures were 152
Total phosphorous 0.14 0.06
adjusted to pH 3.5 with 5N HCl to avoid the adsorption 153
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154 of molecules of bacteriocin onto the producer cell surfaces broth and on two supplemented MPW media (Fig. 1). In 172

155 [22]. Subsequently, they were heated at 100 ◦ C for 3 min to the first medium the concentrations of biomass and pe- 173

156 kill the cells and centrifuged at 27 200×g for 15 min at 4 ◦ C. diocin were higher than those reached in the unsupple- 174

157 The culture supernatants (cell-free broth) containing overall mented MPW medium after 18 h of incubation (Table 2). 175

158 antibacterial activity were frozen until further use. Since bacteriocin production is strongly affected by the type 176

159 Quantitative antibacterial activities of cell-free broths and level of carbon, nitrogen and phosphate sources, cations 177

160 were estimated using a photometric assay on culture tubes and inhibitors [10], the different pediocin productions ob- 178

161 [24,29] using C. piscicola CECT 4020 as target organ- served can be attributed to the different composition of the 179

162 ism. Antibacterial activity was expressed as Activity Units media. 180

163 (AU/ml, 1 AU/ml = amount of antibacterial compound However, when the MPW medium was supplemented 181

164 causing 50% growth inhibition (inhibitory dose 50: ID50 with 2% complex nitrogen sources like Casitone or yeast ex- 182

165 obtained from triplicate samples)), compared with a control tract (Fig. 1), the pediocin levels were two-fold higher than 183
166 without bacteriocins. those obtained in MRS broth. Thus, the pediocin-promoting 184
properties of both nitrogen sources could be related with 185
the additional supplement of vitamins, minerals and amino 186
167 3. Results and discussion acids [30]. 187
Nevertheless, both yeast extract and Casitone are too ex- 188

168 3.1. Batch cultures of Pediococcus acidilactici NRRL pensive nitrogen sources for the large-scale production of pe- 189
169 B-5627 on MRS broth and MPW medium diocin. For this reason, it was necessary to find other fermen- 190
tation techniques more productive than the batch mode. Tak- 191

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170 The ability of P. acidilactici NRRL B-5627 to produce ing into account that the fed-batch fermentation enhanced 192

171 bacteriocins was firstly tested in batch cultures on MRS nisin production on MPW [24] and on TGE broth [12], two

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Fig. 1. Kinetics of growth of Pediococcus acidilactici NRRL B-5627 on MRS broth (䊉), non-supplemented MPW (䊊) and MPW supplemented with
a 2% yeast extract (䉱) and 2% Casitone (). CDW: cell dry weight; Ped: pediocin titres; LA: lactic acid; Pr: proteins; RS: reducing sugars; TN: total
nitrogen; TP: total phosphorous. Means of three analytical replications.
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Table 2
Growth and fermentation parameters of non-realkalized (batch) and realkalized (fed-batch) cultures of Pediococcus acidilactici NRRL B-5627 for pediocin
production
Parameter Batch cultures Fed-batch cultures

MRS MPW MPW + 2% YE MPW + 2% CT MPW (I) MPW (II)

CDW∗ (g/l) 1.76 0.79 1.39 1.41 2.28 2.82

Ped∗ (AU/ml) 493.21 368.40 939.27 912.11 1034.01 1395.14
YPed/TSc (AU/mg) 75.07 151.41 210.15 216.33 27.13 77.98
YPed/TNc (AU/mg) 604.3 3185.1 806.19 695.44 2839.2 2790.9
ETS 0.339 0.537 0.507 0.258 0.951 0.670
ETN 0.268 0.207 0.360 0.335 0.552 0.655
CDW∗ and Ped∗ are the maximum biomass and pediocin levels produced in the cultures. YPed/TSc and YPed/TNc are the pediocin yield coefficients
expressed as the amount of pediocin produced (AU) per mg of total sugars (TSc) or nitrogen (TNc) consumed. ETS and ETN are the efficiencies expressed
as g of substrate consumed per g of substrate supplied.

193 fed-batch and re-alkalized fermentations for pediocin pro- strain continued growing. Due to this specific effect of pH on 232
194 duction on MPW were carried out. pediocin synthesis, a deviation in linearity between biomass 233
and pediocin production was produced (Fig. 2). 234
195 3.2. Re-alkalized culture of Pediococcus acidilactici NRRL The same specific effect of final pH on pediocin produc- 235
196 B-5627 on MPW with feeding with glucose (fermentation I) tion has been observed before [13,14,20,21,24]. These re- 236

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searchers obtained the highest levels of pediocin at final pH 237
197 The growth kinetics of P. acidilactici NRRL B-5627 in values below 5.0 as compared to the negligible amounts of 238

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198 the fed-batch culture on MPW fed with a concentrated glu- bacteriocin obtained at final pH values above 5.0, even in the 239
199 cose solution (240 g/l) and re-alkalized up to initial pH (6.3) presence of a substantial increase in cell density. This was 240
200 is shown in Fig. 2. From a kinetic point of view, it can be related to the need of low pH for posttranslational process- 241
201 noted that biomass and pediocin production and nutrients ing of prepediocin to produce the active pediocin [20,21]. 242
202 consumption described wavy profiles. Moreover, the final As it can be observed in Fig. 2, a mixed acid fermenta- 243
203 pHs at the end of each re-alkalization cycle increased almost tion phase began after 56 h of cultivation, with the accumu- 244
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204 linearly throughout the fermentation and the pH recovery lation of butane-2,3-diol in the medium. In addition, ethanol 245
205 capacity collapsed after 96 h of incubation when the growth became detectable in the medium after 88 h of incubation. 246
206 stopped. However, the final productions of biomass and pe- As a consequence of the production of these mixed acid 247
207 diocin (2.28 g/l and 1034.01 AU/ml) were higher than those metabolites, the glucose consumption rate throughout this 248
208 obtained in batch culture on MRS broth and on the two sup- phase (0.48 g l−1 h−1 ) was higher than that observed in the 249
209 plemented MPW media. previous phase. 250
The nitrogen (35 mg l−1 h−1 ) and phosphorus
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210 During the first 56 h of cultivation, P. acidilactici NRRL 251

211 B-5627 developed a homolactic fermentation (only lactic (383.4 mg l−1 h−1 ) consumption rates, which were constant 252
212 acid was produced). In this period, lactic acid and glucose from 56 to 96 h, decreased to 0.76 and 38.85 mg l−1 h−1 253
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213 were produced and consumed at constant rates of 0.18 until the 112 h of cultivation. 254
214 and 0.20 g l−1 h−1 , respectively. The biomass concentra- The profiles described by both nitrogen and phosphorus 255
215 tion profile displayed two exponential growth phases (0–24 consumption paralleled the evolution of biomass and pe- 256
216 and 40–56 h) with similar biomass production rates (0.028 diocin production throughout the mixed acid fermentation 257
and 0.026 g l−1 h−1 ), separated by an intermediate station-
EC

217 phase. Biomass and pediocin production rates were constant 258
218 ary phase (24–40 h of incubation). The final biomass level (0.021 g l−1 h−1 and 8.99 AU ml−1 h−1 ) from 56 to 96 h, but 259
219 reached in this homolactic phase was 1.35 g/l. In addition, they decreased to 0.006 g l−1 h−1 and 4.09 AU ml−1 h−1 af- 260
220 the profiles of nutrient (protein (Pr), total phosphorous terwards. A similar trend was observed for the final pH 261
221 (TP) and nitrogen (TN)) consumptions paralleled with that reached at the end of each re-alkalization cycle. 262
described by biomass production. The decrease in biomass production observed from 96 h
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222 263
223 In contrast, pediocin production increased to a level of could not be associated with the complete exhaustion of the 264
224 608.9 AU/ml, describing a logistic-type profile. A steep ini- nitrogen source, since at the end of the cultivation, the re- 265
225 tial slope in the first 16 h of fermentation, when the final maining concentration of nitrogen was 0.17 g/l. Neither bac- 266
226 pH in each re-alkalization cycle reached a value of 4.0, teriocin autoinhibition nor the accumulations of by-products 267
227 was followed by a second slow but steadily near-linear bac- with antibacterial activity seem to be causes for the cessa- 268
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228 teriocin synthesis, owing to increasing levels in the final tion of growth. This is because the producing strains possess 269
229 pH levels (from 16 to 56 h). Therefore, the pediocin pro- specific immunity to their own bacteriocin [31,32] and the 270
230 duction rates decreased from 28.02 UA ml−1 h−1 (0–16 h) amounts of inhibitory by-products synthesized (lactic acid, 271
231 to 4.01 UA ml−1 h−1 (16–56 h), even though the producing butane-2,3-diol or ethanol) produced by P. acidilactici were 272
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Fig. 2. Time course of re-alkalized cultures of Pediococcus acidilactici NRRL B-5627 on MPW with feeding with a 240 g/l concentrated glucose. B:
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butane-2,3-diol; Et: ethanol; Pr, TS, TN, TP: proteins, total sugars, nitrogen and phosphorous supplemented () remaining (䊐) and consumed (䊊). I:
phase of homolactic fermentation; II: phase of mixed acid fermentation. Other notations as in Fig. 1.
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273 lower than those considered damaging for lactic acid bacte- phate concentrations lower than 1%. In contrast, increasing 291
274 ria [17]. the initial phosphate concentrations to values between 1 and 292
275 In this case, a possible explanation could be the low avail- 5% led to an increase in both the growth and nisin activity 293
ability or the exhaustion of one or several amino acids, pep- levels. This fact was related to a possible stimulatory effect
EC

276 294
277 tides, vitamins or cations essential for the growth of P. acidi- of high phosphate concentrations on the formation of ATP, 295
278 lactici [30]. In this way, Kim et al. [32] also observed that the which leads to a high energy charge of the cells [33]. 296
279 growth of Lactococcus lactis and nisin production decreased The growth of P. acidilactici NRRL B-5627 on MPW 297
280 with decreasing organic nitrogen concentrations. This was seems to be regulated and controlled by the nutrient (ni- 298
attributed to low nutrient availability, which occurs when trogen and phosphorus sources) availability or limitation,
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281 299
282 a strain grows well in a medium and quickly exhausts the whereas pediocin production, which depends on biomass 300
283 available nutrients leading to a cessation in both the growth production, is also regulated by the final pH reached in each 301
284 and bacteriocin production. re-alkalization cycle. 302
285 Other possible explanation for growth cessation could be Taking into account that both nitrogen and phosphorus 303
286 the lower phosphorus concentration (0.016 g/l) reached at were almost exhausted due to both the consumption and 304
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287 the end of the fermentation. This observation agrees with the the successive extraction of samples, another fed-batch and 305
288 results obtained by De Vuyst and Vandamme [33] for L. lac- re-alkalized culture of P. acidilactici NRRL B-5627 was 306
289 tis subsp. lactis. These researchers observed a drastic limi- carried out, using a concentrated MPW medium as a feeding 307
290 tation in the cell yield and growth rate of this strain at phos- substrate. Thus, this feeding medium was not only used to 308
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309 replenish the glucose consumed in each re-alkalization cycle, the highest pH decreases. However, after this time, the final 363
310 but also as an additional source of nitrogen and phosphorus. pHs in each cycle were always higher than 4.0 and bacteri- 364
ocin production slowed down. 365
311 3.3. Fed-batch culture for pediocin production by As shown in Table 2, the results of this study showed that 366
312 Pediococcus acidilactici on MPW medium with feeding the fed-batch and re-alkalized cultures (fermentations I and 367
313 with CMPW medium (fermentation II) II) enhanced pediocin production on MPW as compared to 368
batch fermentations on MRS and MPW (non-supplemented 369
314 The results of this second fed-batch fermentation are and supplemented with a 2% of yeast extract or Casitone). 370
315 shown in Fig. 3. As a consequence of feeding the fermenta- The values of YPed/TSc were lower in the two cultivations 371
316 tion medium with a concentrated MPW, biomass (2.82 g/l) with steps of pH with respect to the four batch cultures. 372
317 and pediocin (1395.1 AU/ml) levels were, respectively, 1.26 This could probably be due to an overconsumption of sug- 373
318 and 1.37 times higher than those obtained in the previous ars to support higher growth, metabolite production (lactic 374
319 culture with the same pH decrease values. Moreover, from acid, ethanol and butane-2,3-diol) as well as to compensate 375
320 96 h of incubation, the final pH before each re-alkalization the stress produced by the re-alkalizations each 8 h [24]. 376
321 cycle was stabilised in a value slightly higher than 4.5. Contrarily, the values of YPed/TN in the fed-batch cultures 377
322 In addition, probably as a consequence of the joint sup- were higher than those values obtained in batch cultivations. 378
323 plement of glucose, nitrogen and phosphorous throughout This fact suggests a more economic utilization of the ni- 379
324 the feeding with CMPW medium, the pediocin producing trogen sources for pediocin production in the fed-batch and 380
325 strain developed a metabolism typically homofermentative re-alkalized cultures. 381
326 in this second fermentation. With regard to nutrient consumptions, it can be appreci- 382

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327 In Fig. 3, it can be noted that at 64 h of incubation, the ated that the efficiencies (ETSc and ETNc ) in the batch cul- 383
328 remaining concentration of nitrogen in the medium reached tures on MRS and MPW were lower than those obtained in 384

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329 the same value (0.21 g/l) as that observed at the end of the the two fed-batch cultures (Table 2). Thus, this last fermen- 385
330 former fed-batch fermentation. However, P. acidilactici con- tation technique contributes to obtaining lower amounts of 386
331 tinued growing and consuming nitrogen and phosphorous nutrients (total sugars and nitrogen) in the medium at the 387
332 until the end of the fermentation. Thus, nitrogen and phos- end of the fermentation. As reported by Murado et al. [38] 388
333 phorus consumption were so highly stimulated, that the fi- the mussel processing wastes represent a serious environ- 389
334 nal concentrations of both nutrients after 120 h of incuba- mental problem in the Rias Baixas (Galicia, NW of Spain). 390
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335 tion were 0.08 and 0.014 g/l, respectively. In contrast, in the Therefore, the use of this effluent as culture media for bac- 391
336 first culture, the growth stopped (from the 96 h of incuba- teriocin production could be a suitable treatment to reduce 392
337 tion), when the concentrations of nitrogen and phosphorous its contamination effect. 393
338 in the medium were 0.17 and 0.017 g/l. This suggests that The beneficial effect that this fed-batch fermentation tech- 394
339 the CMPW medium provided growth-stimulating molecules nique with re-alkalization cycles produced on pediocin pro- 395
340 (essential amino acids, peptides, vitamins or cations (Mg2+ , duction seems to be a common phenomenon among the lac- 396
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341 Ca2+ , Mn2+ )) which probably were exhausted in the first tic bacteria, since the same positive effects were observed 397
342 cultivation. before for nisin production on TGE [12] and MPW [24]. 398
343 In this way, it has been observed that for growth of lac-
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344 tococci on 10 g lactose/l, the essential amino acids were 3.4. Stoichiometry 399
345 required at different concentrations. While glutamic acid
346 was needed in the largest amounts (77 mg/l), the other It has been reported that the carbohydrates and the 400
347 amino acids were required in concentrations between 20 aminoacids: arginine (which is converted to ornithine with 401
EC

348 and 40 mg/l [34]. However, other researchers [35] observed the production of 1 mol of ATP per mol of arginine utilised) 402
349 that for optimal growth of L. lactis and L. cremoris at a and serine are the only energy sources for lactic acid bac- 403
350 lactose concentration of 12 g/l, the minimal concentration teria [39,40]. Taking into account the low carbohydrate 404
351 of essential amino acids was in the range of 10–80 mg/l. content (5 g/l) in the MPW medium (fermentations I and 405
352 In addition, when the MPW was supplemented with a II), it is reasonable to suppose that P. acidilactici obtained 406
2% of glutamic acid [36], biomass production by L. lac-
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353 additional ATP from the catabolism of both amino acids, 407
354 tis subsp. lactis CECT 539 was four-fold higher than that which are present in this medium [41], for both growth and 408
355 obtained in the non-supplemented MPW medium. From maintenance. 409
356 these observations, it could be suggested that the relative In an attempt to prove the above observations, the theo- 410
357 amounts of the essential amino acids seem to be probably retical glucose utilization by P. acidilactici in MPW cultures 411
358 more important than their actual concentrations [37]. was determined by using the corresponding metabolic reac- 412
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359 The stimulatory effect of pH decrease on pediocin synthe- tions, which can be described by the following stoichiomet- 413
360 sis was also observed in this fermentation. Thus, pediocin ric equations [42]: 414
361 production increased quickly after 16 h of incubation in con-
362 comitance with a rapid increase in biomass production and Glucose + 2ADP + 2Pi → 2lactate + 2ATP + 2H2 O 415
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Fig. 3. Time course of re-alkalized cultures of Pediococcus acidilactici NRRL B-5627 on MPW with feeding with CMPW (101.33 g of glucose/l).
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Notations as in Figs. 1 and 2.

416 Glucose + 2ADP + 2Pi and YCO2 were calculated from the above stoichiometric re- 435

417 → butane-2, 3-diol + 2CO2 + 2ATP + 2H2 O lationships. The molar coefficient for biomass production 436

418 (YX ) was obtained from bibliography data and it was as- 437
sumed to be 0.5. In the same way, rX was calculated assum- 438
Glucose + 2ADP + 2Pi
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419 ing a molecular weight for biomass of 25 g/mol [43]. 439

420 → 2ethanol + 2CO2 + 2ATP + 2H2 O Since the experimental design used did not allow the cal- 440
culation of the glucose consumed for the maintenance and 441
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421 From these equations, it possible to obtain expressions the production of CO2 , both parameters were calculated 442
422 for determining the glucose utilization rate (rG ) for biomass jointly from the Eqs. (1) and (2) as follows: 443
423 (X), by-product formation (lactic acid (LA), butane-2,3-diol
for fermentation I: 444
445
424 (B) and ethanol (Et)), CO2 and maintenance energy:


EC

for fermentation I: 1
425
426
mX + rCO2
YCO2 /G 446
1 1 1 
−rG = rLA + rEt + rB + mX 1 1 1
427 YLA/G YEt/G YB/G = −rG − rLA + rEt + rB
1 1 YLA/G YEt/G YB/G 447
+ rCO2 + rX (1) 
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428 YCO2 /G YX/G + rX

1
(3)
YX/G 448
429
430 for fermentation II:
for fermentation II: 449
450
1 1 1
−rG = rLA + mX + rCO2 + rX  
431 YLA/G YCO2 /G YX/G 1
mX + rCO2
CO

432 (2) YCO /G 451

 2 
1 1
= −rG − rLA + rX (4)
433 The term mX accounts for the amount of glucose used YLA/G YX/G 452
434 for maintenance energy. The molar coefficients YLA , YB , YEt
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acidilactici NRRL B-5627, this variable was considered in 471

the overall model developed for cell growth. 472

3.5. Development of the kinetic models 473

In this work, the volume of medium in the two fed-batch 474

fermentations was maintained constant ((dV/dt) = 0) by 475
matching the feeding volume with the sampling volume. 476
Biomass and pediocin production in the fermentor are 477
given by the following expressions: 478
   
VS
X(tn ) = X(tn−1 ) 1 − + rX dt (5)
VF 479

   
VS
Ped(tn ) = Ped(tn−1 ) 1 − + rPed dt (6)
VF 480

where X(tn ) and Ped(tn ) are the biomass (in g/l) and pediocin 481
(in AU/ml) produced at the end of each re-alkalization pe- 482
riod. X(tn−1 ) and Ped(tn−1 ) are the biomass (in g/l) and pe- 483
diocin (in AU/ml) at the beginning of each re-alkalization 484

F
period. VF is the volume (in l) of medium in the fermen- 485
tor and VS is the volume (in l) of sampling. rX and rPed 486

OO
are the biomass and pediocin production rates and dt is the 487
re-alkalization period (in h). 488
Usually, logistic-type models and Luedeking and Piret 489
model [28] have been, respectively, used to model bacte- 490
rial growth and bacteriocin production in batch fermenta- 491
tion [44,45]. However, these models have been found to be 492
PR
unsatisfactory to describe biomass and bacteriocin produc- 493
tion in fed-batch cultures [26] and in fed-batch cultures with 494
re-alkalization cycles [12]. This indicates that these mod- 495
els did not include all the variables affecting both biomass 496

Fig. 4. Glucose utilization rates (γ G ) for both the maintenance and CO2 and bacteriocin production. For these reasons, some modi- 497
production, and nitrogen consumption rates (NCR) determined from the fications for both the logistic-type and Luedeking and Piret 498
D

re-alkalized and fed-batch cultures of Pediococcus acidilactici NRRL models have been suggested recently [12,26]. 499
B-5627 on MPW. (A) Fermentation I and (B) fermentation II. In the As was discussed above, the growth rate of P. acidilactici 500
dotted lines γ G = 0.
growing in MPW medium seems to be a function of pH 501
TE

change, nutrient limitation (nitrogen and phosphorous) and 502

453 Then, the parameter (mX + rCO2 (1/YCO2 /G )) was named product inhibition (lactic acid, ethanol and butane-2,3-diol), 503
454 as γ G and plotted versus the time of fermentation (Fig. 4). variables that change in concentration in the course of the 504
455 The value of γ G was always negative for the two fed-batch fermentation. In addition, pediocin production seemed to 505
EC

456 fermentations (upper parts of Fig. 4A and B), indicating that be affected by the final pH reached in each re-alkalization 506
457 the glucose levels supplied were not enough to support both cycle. Therefore, in a first step, an overall equation was set 507
458 growth and metabolite production by P. acidilactici through- up to describe the effects of the main factors affecting the 508
459 out the fermentations I and II. In addition, it can be noted growth of P. acidilactici. A modification of the Luedeking 509
460 that the nitrogen consumption rate (NCR) increased (lower and Piret model including a term for the specific effect of 510
parts of Fig. 4A and B) as the parameter γ G decreased, sug- pH on pediocin production was then developed.
RR

461 511
462 gesting that P. acidilactici NRRL B-5627 utilized the argi- Although these models have little biological meaning be- 512
463 nine and serine to obtain additional energy for growth and cause their empirical nature, they can be used to develop 513
464 maintenance. an appropriate control strategy for optimizing the fed-batch 514
465 From the above discussion, it can be pointed out that the production of pediocin. 515
466 nitrogen consumption rate is an important variable that may
CO

467 be taken into account in the development of a model for de- 3.5.1. Effect of pH on the growth of 516
468 scribing the kinetics of biomass production in the fed-batch Pediococcus acidilactici 517
469 fermentations on MPW. In addition, since the phosphate Although the optimal pH for growth and product forma- 518
470 source seems to play an important role on the growth of P. tion by lactic acid bacteria has found to be around 6.0, a rapid 519
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 N.P. Guerra et al. / Process Biochemistry xxx (2004) xxx–xxx 9

520 decline in the growth is produced as pH decrease [15,46–49]. With regard to lactic acid, there are discrepancies related 563
521 Thus, an inadequate pH can cause the precipitation of nec- to its main mechanism of inhibition on cell growth. Thus, 564
522 essary nutrients, ions, etc. the denaturation of enzymes and some researchers consider that the inhibitory action lies in 565
523 structural proteins or cause membrane disruption or inacti- the presence of both the undissociated and dissociated forms 566
524 vation of transport or other protein functions [46,50–52]. of the molecule [48,49,57], but others believe that the inhi- 567
525 Since the growth rate is a function of pH [53], the model bition is produced only by the undissociated form [58,59], 568
526 proposed by Presser et al. [48], was set up to describe the or by the total concentration of the acid [52]. 569
527
528 relationship between both variables, as follows: Taking into account these discrepancies, the inhibitory 570
  effect of the undissociated and dissociated forms of lactic 571
10pHmin
RX = b0 1 − ; being : b0 = rX × 10−pHmin acid on cell growth as proposed by Presser et al. [48] were 572
529 10pHt considered in the following form: 573
574
530 (7)

[LA]
531 where RX is the growth rate (g l−1 h−1 ) affected by the pH, γ[ULA] = d 1 −   ;
532 pHmin is the minimum pH value beyond which no growth is [ULAmin ] 1 + 10pHt −pKa 575

533 possible, pHt is the pH over time. rX is the absolute growth being : d = c0 ULAmin (12) 576
534 rate (in g l−1 h−1 ) in each re-alkalization cycle. 577
535 The numeric integration of Eq. (7) with respect to time  
[LA]
536 provided the estimated values of biomass concentration over γ[DLA] = p 1 − ;
537 time: [DLAmin ](1 + 10pKa −pHt ) 578

t=t t=t   being : p = c1 DLAmin (13) 579

 

F
10pHmin
XR = RX = b0 1 − (8) where γ[ULA] and γ[DLA] are the inhibitory functions for
538 10pHt 580
t=0 t=0

OO
the inhibition of the undissociated and dissociated forms of 581
539 where XR is the biomass concentration (g/l) and t is the time the lactic acid, c0 and c1 are constants of proportionality to be 582
540 (h). Other terms are as previously defined. experimentally determined. [LA] is the total concentration 583
of lactic acid, [ULAmin ], [DLAmin ] are, respectively, the 584
541 3.5.2. Effect of nitrogen and phosphorus limitation minimum concentrations of undissociated and dissociated 585
542 Biomass production was strongly influenced by the time forms of the acid which completely prevents the growth, pKa 586
PR
543 course of both the nitrogen and phosphorus consumption is the pH at which the concentrations of the two forms are 587
544 profiles (Figs. 2 and 3). From the observation that biomass equal and pHt is the final pH in each re-alkalization cycle. 588
545 production and both nitrogen and phosphorous consumption Including the effects of the undissociated and dissociated 589
546 are directly correlated (Figs. 2 and 3), the simplest forms to species of lactic acid in Eq. (11) gives: 590
591
547 describe these relationships are:
t=t
 t=t
  
t=t
 10pHmin
XR = RX = b0 1 − (1 + b1 rNc )
D

RX = rX (1 + b1 rNc ) (9) 10pHt 592

t=0 t=0
548 t=0
× (1 + b2 rPc )γ[ULA]γ[DLA] (14) 593
TE

t=t

RX = rX (1 + b2 rPc ) (10) In the same way, the inhibitory effect of the total con- 594
549 t=0 centration of lactic acid was modelled using the following 595
expression [53,59]: 596
550 where rNc and rPc are the nitrogen and phosphorous con-  
sumption rates, b1 and b2 are constants of proportionality. [LA]
EC

551
γ[LA] = 1 − (15)
552 Combining the effects of pH and the rates of nitrogen and [LAmax ] 597
553
554 phosphorous consumption, the overall form of the model is:
where γ[LA] is an inhibition function that accounts for 598
t=t
 t=t
  
10pHmin the inhibitory effect of the total lactic acid concentration, 599
XR = RX = b0 1 − (1 + b1 rNc ) [LAmax ] is the maximum total lactic acid concentration from 600
10pHt
RR

555 t=0 t=0 which the growth stopped and [LA] is as previously defined. 601
556 × (1 + b2 rPc ) (11) Therefore, including Eq. (15) into model (11): 602
603

t=t
 t=t
  
10pHmin
557 3.5.3. Effect of product formation XR = RX = b0 1 − (1 + b1 rNc )
558 Taking into account that product accumulation (or- 10pHt 604
t=0 t=0
CO

559 ganic acids (mainly lactic and acetic acids), ethanol and × (1 + b2 rPc )γ[LA] (16) 605
560 butane-2,3-diol) produces inhibitory effects on bacterial
561 growth [54–56], in this work, the effects of these variables The ability of ethanol to produce perturbation in cell mem- 606
562 were also considered in the growth model. branes or the alteration in fatty acid composition contributes 607
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 10 N.P. Guerra et al. / Process Biochemistry xxx (2004) xxx–xxx

608 to an increase in the uptake of the undissociated form of lac- 3.5.4. Specific effect of the optimum final pH value on 649
609 tic acid [55,56]. On the other hand, it has been reported that pediocin production 650
610 butane-2,3-diol is able to deactivate enzymes from several As was discussed above, in the two fed-batch cultures of 651
611 microorganisms [60]. P. acidilactici, pediocin synthesis was strongly dependent on 652
612 The negative influence of the concentrations of ethanol or both biomass production and the final pH value reached at 653
613 butane-2,3-diol produced in the cultures may be described the end of each re-alkalization period. It was hypothesised 654
614 in the same form to that given for the effect of the total lactic that the increase in pediocin concentration is proportional to 655
615 acid concentration, so that: the increase in the pH drop rate (∂pH) which is less than the 656
  maximum ∂pH from which bacteriocin synthesis stops. To 657
[Et]
γ[Et] = 1 − (17) describe the effects of ∂pH on rPed , the equation proposed 658
616 [Etmax ]
by Guerra and Pastrana [19] was used: 659
 
[B] −b3 (∂pHmax −∂pH)
γ[B] = 1 − (18) rPed (∂pH) = rPed e (25) 660
617 [Bmax ]
where rPed (∂pH) is the pediocin production rate affected by 661
618 where γ[Et] and γ[B] are the inhibition functions for ethanol the pH drop rate in each re-alkalization cycle, b3 a constant 662
619 and butane-2,3-diol inhibition. [Et] and [B] are the total con- of proportionality, ∂pHmax is the maximum decrease in pH 663
620 centrations (mM) of ethanol and butane-2,3-diol. [Etmax ] and per unit of time, from which bacteriocin production stopped. 664
621 [Bmax ] are, respectively, the maximum concentrations (mM) The combination of Eqs. (24) and (25) yields Eq. (26): 665
666
622 of ethanol and butane-2,3-diol, which completely prevent
623 the growth. Including the effects of both variables in mod- t=t
 t=t

els (14) and (16), biomass formation could be expressed by Ped = rPed (αRX + βXR )(1 + e−b3 (∂pHmax −∂pH) )

F
624
t=0 t=0 667
625
626 the following equations:
  (26) 668

OO
t=t
 t=t
 10pHmin
XR = RX = b0 1− (1 + b1 rNc ) Then, the effects of all factors affecting biomass produc- 669
627 10pHt
t=0 t=0 tion by P. acidilactici were tested by fitting models (21) and 670
628 × (1 + b2 rPc )γ[ULA]γ[DLA]γ[B]γ[Et] (22) to the experimental cell growth data. Using the biomass 671

629 (19) formation estimates obtained from these two models, pe- 672
630 diocin production was then modelled using the models (24) 673
PR
t=t t=t   and (26). 674
  10pHmin For optimal estimation of the model parameters, a 675
XR = RX = b0 1− (1 + b1 rNc )
631 10pHt non-linear regression technique (method of Newton by 676
t=0 t=0
using a minimal non-linear squares method), was used to 677
632 × (1 + b2 rPc )γ[LA]γ[B]γ[Et] (20)
minimise the deviations between the model predictions and 678
633 Then, if µ was constant, Eqs. (19) and (20) could be experimental fed-batch data. 679
D

634
635 expressed in the integral form:
K
XR =
TE

(21)
1 + e(c−µ((1−(10 min /10 t ))(1+b1 rNc )(1+b2 rPc )γ[ULA]γ[DLA]γ[B]γ[Et])t)
636 pH pH

K
XR = (22)
1 + e(c−µ((1−(10 min /10 t ))(1+b1 rNc )(1+b2 rPc )γ[LA]γ[B]γ[Et])t)
637 pH pH
EC

638 The introduction of the values of RX and XR into the

As it can be seen in Figs. 5 and 6 and Table 3, mod- 680
639 Luedeking and Piret expression [28] results in:
els (21) and (22) described the trend well. From Table 3, 681
640 rPed = αRX + βXR (23) it can be noted that the inhibitory action of the dissociated 682
form of the lactic acid was lower than the inhibitory effect 683
641 where rPed is the pediocin (Ped) production rate, α a produced by the undissociated specie, since a higher con-
RR

684
642 growth-associated constant (BU/mg) that corresponds to centration of the dissociated form was needed to inhibit the 685
643 the yield product on biomass formed (YB/X ) for primary growth of P. acidilactici. Nevertheless, the inhibitory con- 686
644 metabolites, and β is the non-growth-associated constant centrations of both the undissociated and dissociated forms 687
645 (BU mg−1 h−1 ). of the acid predicted by model (21) were always higher than 688
646 Therefore, numeric integration of Eq. (23) results in those amounts produced in the two fed-batch fermentations 689
CO

647 Eq. (24), which provides the pediocin estimates (Ped): (Tables 3 and 4). 690
t=t
 t=t
 When the inhibitory action is considered to be due to the 691
Ped = rPed = (αRX + βXR ) (24) total concentration of lactic acid, the maximum inhibitory 692
648 t=0 t=0 concentration obtained from model (22) was also higher 693
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 N.P. Guerra et al. / Process Biochemistry xxx (2004) xxx–xxx 11

Table 3
Parameters in the models for fed-batch production of biomass (models 21 and 22) and pediocin (model 26) by Pediococcus acidilactici NRRL B-5627
in fermentations I and II
Parameter Fermentation I Fermentation II

Eqs. (21) and (26) Eqs. (22) and (26) Eqs. (21) and (26) Eqs. (22) and (26)

K (g/l) 1.89 1.94 2.89 2.94

µ (h−1 ) 0.055 0.058 0.047 0.031
pHmin 3.86 3.88 3.96 3.88
b1 0.31 0.36 0.31 2.67
b2 3.15 3.70 1.73 0.45
ULAmin (mM) 68.90 – 68.92 –
pKa 3.86 – 3.86 –
DLAmin (mM) 298.35 – 359.30 –
LAmax (mM) – 277.93 – 519.85
Etmax (mM) 150.45 150.45 150.45 150.45
Bmax (mM) 438.97 341.26 438.97 341.26
α (BU/mg) 2255.78 2253.55 2725.80 2620.54
β (BU/mg−1 h−1 ) 0 0 0 0
b3 10.89 11.54 4.22 3.33
∂pHmax 0.273 0.273 0.274 0.274
rX
2 0.9858 0.9851 0.9880 0.9861
rPed
2 0.9847 0.9836 0.9710 0.9747

F
rX
2 and r 2
Ped are the correlation coefficients between expected and experimental biomass and pediocin values.

OO
694 than those amounts produced by P. acidilactici in the two lated, the minimum pH (pHmin ) from which bacteriocin pro- 704
695 fed-batch fermentations (Tables 3 and 4). duction stopped was determined as 4.03 in fermentations I 705
696 The inhibitory effects of both butane-2,3-diol and ethanol and II. This pH value is in accord with the optimal final pH 706
697 in the first fed-batch culture seem to be very low, since value (below 5.0) reported by other researchers for pediocin 707
698 the inhibitory levels obtained from models (21) and (22) production [20,21]. In addition, these observations support 708
PR
699 were higher than those levels reached at the end of both the proposal of the existence of an optimum final pH for 709
700 cultivations (Table 3 and Fig. 2). bacteriocin production [13,14,20,21]. 710
701 On the other hand, pediocin production was best mathe- On the other hand, taking into account that in both 711
702 matically described using Eq. (26) as compared to Eq. (24) fed-batch cultures β = 0, and pediocin production was de- 712
703 in both fermentations. From the values of ∂pHmax calcu- pendent on the final pH reached in each re-alkalization cy- 713
D
TE
EC
RR

Fig. 5. Experimental data (symbols) of biomass (left part) and pediocin Fig. 6. Experimental data (symbols) of biomass (left part) and pediocin
CO

(right part) production by Pediococcus acidilactici NRRL B-5627 on
MPW (fed-batch fermentation I). The curves drawn through the biomass
data were obtained according to the models 21 (A) and 22 (B). The
curves drawn through the pediocin data were obtained according to the
models 24 (dashed lines) and 26 (solid line). Notations as in Fig. 1.
UN
(right part) production by Pediococcus acidilactici NRRL B-5627 on
MPW (fed-batch fermentation II). The curves drawn through the biomass
data were obtained according to the models 21 (A) and 22 (B). The
curves drawn through the pediocin data were obtained according to the
models 24 (dashed lines) and 26 (solid line). Notations as in Fig. 1.

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Table 4 the bacteriocins pediocin AcH and nisin. Lett Appl Microbiol 749
Concentrations of undissociated (U) and dissociated (D) forms of lac- 1992;15:239–43. 750
tic (LA) acid produced by Pediococcus acidilactici NRRL B-5627 [7] Kalchayanand N, Sikes T, Dunne CP, Ray B. Hydrostatic pressure and 751
at the end of both fed-batch fermentations calculated by using the electroporation have increased bactericidal efficiency in combination 752
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[8] Delves-Broughton J, Blackburn P, Evans RJ, Hugenholtz J. Applica- 754
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ULA (mM) 1.53 17.49 [9] Soomoro AH, Masud T, Anwaar K. Role of lactic acid bacteria 756
DLA (mM) 211.50 241.40 (LAB) in food preservation and human health. A review. Pakistan J 757
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Et (mM) 44.66 – [10] Parente E, Ricciardi A. Production, recovery and purification of 759
B (mM) 157.76 – bacteriocins from lactic acid bacteria. Appl Microbiol Biotechnol 760
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717 The kinetic data from fed-batch and re-alkalized cultiva- [14] Guerra NP, Pastrana L. Production of bacteriocins from Lacto- 772

F
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OO
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PR
725 continuous culture. J Ind Microbiol Biotechnol 1997;18:62–7. 782
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D

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TE

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