Effect of aeration and dilution rate on nisin Z production during continuous fermentation with free and immobilized Lactococcus lactis UL719 in supplemented whey permeate

International Dairy Journal 11 (2001) 943–951

Effect of aeration and dilution rate on nisin Z production during continuous fermentation with free and immobilized Lactococcus lactis UL719 in supplemented whey permeate P. Desjardins, J. Meghrous, C. Lacroix* ! Dairy Research Centre STELA, Pavillon Paul-Comtois, Universite! Laval, Quebec, Que., Canada G1K 7P4 Received 5 February 2001; accepted 4 August 2001

Abstract The influence of dilution rate (D) and aeration on soluble and cell-bound nisin Z production was investigated during continuous free (FC) and immobilized cell (IC) cultures with Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in supplemented whey permeate. Maximum total bacteriocin titres during non-aerated continuous FC and IC cultures were obtained for low D; with 1490 and 1090 IU mL 1 for 0.15 h 1 or 0.25 and 0.5 h 1, respectively. For both systems, aeration increased nisin total production with maximum titres of 2560 and 2430 IU mL 1 for low D; respectively, as well as specific production. Volumetric productivity was the highest for an intermediate D of 0.4 h 1 during FC cultures (460 IU mL 1 h 1 for both aerated and non-aerated cultures), while it increased continuously with D during IC cultures, reaching high values of 1090 and 1760 IU mL 1 h 1 at 2.0 h 1 without and with aeration, respectively. In comparison with previous data for FC batch cultures, data from this study may indicate that during continuous fermentations at steady state, some steps in nisin biosynthesis are limiting. In these conditions, nisin production by immobilized cells is reduced. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Nisin Z; L. lactis subsp. lactis biovar. diacetylactis; Continuous culture; Cell immobilization; Whey permeate

1. Introduction Bacteriocins are proteins or protein complexes with bactericidal activity directed against species that are usually closely related (Klaenhammer, 1988; Tagg, Dajani, & Wannamaker, 1976). Nisin is a type I lantibiotic composed of 34 amino acids and produced by Lactococcus lactis strains (Stringer, Dodd, Morgan, & Waites, 1995; De Vuyst & Vandamme, 1994). Nisin A and Z are two natural variants that differ by a single amino acid residue at position 27 (Mulders, Boerrigter, Rollema, Siezen, & De Vos, 1991). Nisin Z exhibits increased solubility at pH above 6 which is an important characteristic for use in foods (de Vos, Mulders, Siezen, Hugenholtz, & Kuipers, 1993). Nisin is a broad spectrum bacteriocin, exhibiting antimicrobial activity against a wide range of Gram-positive vegetative cells and spores (Hurst, 1981; Meghrous, Lacroix, & Simard, 1999). Nisin is used currently in 50 countries for specific *Corresponding author. Tel.: +1-418-656-7445; fax: +1-418-6563353. E-mail address: [email protected] (C. Lacroix).

food applications (De Vuyst & Vandamme, 1994; Turtell & Delves-Broughton, 1998). Recently, there has been renewed interest in the use of nisin as a preservative as well as antilisterial and anticlostridial agent in foods, particularly in cheese (Delves-Broughton, 1998). A major limitation for application of bacteriocins is their low rate of production in fermentation broth. For several bacteriocins, such as nisin, production is associated with cell growth (De Vuyst & Vandamme, 1994; Amiali, Lacroix, & Simard, 1998). This has been found to be influenced by numerous factors, such as the microbial strain, composition of the fermentation medium, pH, temperature and aeration (De Vuyst & Vandamme, 1994). Maximum reported nisin productions, in the range from 2500 to 4000 IU mL 1 (corresponding to approximately 60–100 mg mL 1 of pure nisin) are obtained during pH-controlled batch cultures in rich media (De Vuyst & Vandamme, 1992; Matsusaki, Endo, Sonomoto, & Ishizaki, 1996; Van’t Hul & Gibbons, 1997; Chinachoti, Zaima, Matsusaki, Sonomoto, & Ishisaki, 1997). We have recently reported very high nisin Z production (10 000–20 000 IU mL 1)

0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 1 2 8 - 5

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during pH-controlled batch cultures of citrate positive Lactococcus lactis subsp. lactis (L. diacetylactis) UL719 in a supplemented whey permeate medium without (Goulhen, Meghrous, & Lacroix, 1999) and with (Amiali et al., 1998) aeration. Biomass growth and product formation during batch cultures with lactic acid bacteria are limited by the accumulation of metabolic inhibitory products, such as lactic acid. Continuous culture may overcome this limitation by providing a continuous replacement of growth medium. Cell immobilization also allows a large increase of cell density in the continuous reactor; continuous cultures can be carried out at dilution rates higher than the maximum specific growth rate of the strain, without cell washout (Lamboley, Lacroix, Champagne, & Vuillemard, 1997). Immobilized cells (IC) have many advantages over free-cell (FC) cultures which include higher productivity as a result of high cell densities, long-term operational stability, improved process control, protection against contamination, and improved plasmid stability (Huang, Lacroix, Daba, & Simard, 1996; Lamboley et al., 1997; Sodini, Boquien, Corrieu, & Lacroix, 1997). Limited work has been reported on bacteriocin production using IC in batch and continuous cultures. In general, maximum bacteriocin concentration was similar or lower to that obtained during FC-batch or continuous cultures. In some studies, very low dilution rates, close to 1 day 1, were used (Cho, Yousef, & Yang, 1996; Sonomoto, Chinachoti, Endo, & Ishizaki, 2000); in others, very low cell concentrations were measured in the immobilized cell systems (Zezza et al., 1993; Chinachoti et al., 1997) which may explain the low bacteriocin production. On the other hand, Huang et al. (1996) showed that cell immobilization increased cell concentration, pediocin volumetric productivity and plasmid stability during continuous cultures with Pediococcus acidilactici, compared with FC cultures. However, maximum pediocin production was lower during continuous IC compared to FC cultures. In this work, nisin Z production by L. diacetylactis UL719, an efficient nisin Z producer strain, was studied during continuous FC and IC cultures in a supplemented whey permeate (SWP) medium. Our objectives were to determine the effects of dilution rate (D), aeration and immobilization on biomass, soluble and cell-bound nisin production, and on sugar metabolism.

Lacroix, Bouksa.ım, Lapointe, & Simard, 1997). The indicator strain used for bacteriocin activity determination was Pediococcus acidilactici UL5 from our culture collection. Stock cultures were maintained at 801C in De Man, Rogosa and Sharpe (MRS) medium (De Man, Rogosa, & Sharpe, 1960) obtained from Rosell-Lallemand inc. (Montre! al, QC, Canada) with 20% glycerol added. 2.2. Fermentation medium Whey permeate powder (Edible Dry Whey Permeate, Foremost Ingredient Group, Baraboo, WI, USA) was rehydrated with distilled water to a final concentration of 6% (w/w) and supplemented with 0.2 m KCl for mechanical stability of the gel biocatalysts during continuous fermentations. Preliminary tests with free cells showed no influence of KCl on biomass and nisin Z production by L. diacetylactis UL719. The rehydrated whey permeate was allowed to settle for 18 h at 41C and the supernatant was filtered (filter no 2, Flojet, Buon Vino, Cambridge, ON, Canada). The whey permeate was heat treated at 1351C for 8 s (Spiratherm, MFG, Chicago, IL, USA) and stored at 41C. Yeast extract (Rosell-Lallemand inc.) and Tween 80 were prepared and autoclaved separately at 1211C for 15 min. Yeast extract (410 g) was rehydrated in 1 L of distilled water, supplemented with 41 g of Tween 80, and autoclaved separately at 1211C for 15 min. The sterile solution was added to 40 L whey permeate for a final concentration of 1% (w/v) yeast extract and 0.1% (w/v) Tween 80 (Amiali et al., 1998). 2.3. Cell enumeration Viable cell counts (cfu mL 1) were estimated by dilution plating on MRS medium supplemented with 0.5% Bacto-agar (Difco Laboratories, Detroit, MI, USA). Culture samples were treated with an UltraTurrax mixer (Janke & Kunkel, Ika-Labortechnik, Staufen, Germany) at 13 500 rpm for 1 min and serial dilutions were prepared in peptone water. For immobilized cell enumerations, approximately 0.7 g of gel beads accurately weighed was soaked in 5.3 g of peptone water and dissolved by treatement with Ultra-Turrax mixer at 13 500 rpm for 1 min. Cell counts were determined from the average number of colonies on two plates after 48 h incubation at 301C.

2. Materials and methods 2.4. Cell immobilization procedure 2.1. Bacterial strains Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 (L. diacetylactis UL719) isolated from raw goat milk cheese was used as nisinogenic strain (Meghrous,

A dispersion process in a two-phase system was used following the procedure reported by Arnaud and Lacroix (1991). A culture of L. diacetylactis UL719 was grown overnight in 10 mL MRS broth at 301C. The

P. Desjardins et al. / International Dairy Journal 11 (2001) 943–951

cells were washed three times with 10 mL of 0.1% (w/v) peptone and recovered by centrifugation at 10 000g for 10 min at 41C. The pellets were resuspended in saline solution (0.75% NaCl). A sterile polymer solution of 2.75% (w/v) k-carrageenan (Genugel X-0909, Copenhagen Pectin, Lille Skensved, Denmark) and 0.25% (w/v) locust bean gum (Satiagel MR150, Sanofi Bio-Industries, Waukesha, WI, USA) autoclaved at 1211C for 15 min, was inoculated at 451C with 2.5% (v/v) washed cell suspension. After hardening in a sterile solution of 0.3 m KCl and 0.03 m CaCl2 for 1 h, gel beads with diameters between 1.0 and 2.0 mm were selected by wet sieving. Batch preculturing was carried out following bead preparation in order to increase the cell population in gel beads. A volume of 250 mL of beads, measured by liquid displacement, was transferred into a 1.25 L vertical bioreactor (Bioflo III, New Brunswick, Edison, NJ, USA) containing MRS medium supplemented with 0.2 m KCl, for a total culture volume of 1 L. Temperature was maintained at 301C and pH at 6.0 by addition of 6 n sodium hydroxide. After 16 h, the fermented broth was replaced by fresh medium, incubated for 8 h, and the fermentor re-filled with fresh medium and incubation proceeded for additional 4 h. The beads were then recovered and stored in sterile hardening solution. 2.5. Culture conditions Continuous FC and IC fermentations were carried out at 301C and pH 6.0 (controlled by addition of 6 n NaOH) in the same 1.25 L bioreactor with a total culture volume of 1 L. Mixing was performed by a four inclined flat blade impeller at 100 rpm. Fresh medium was added using a peristaltic pump (503S, WatsonMarlow, Cornwall, ON, Canada), and fermented broth was harvested through a 0.5 mm grid in order to retain the beads in the reactor, using a peristaltic pump (Masterflex, Cole-Parmer Instrument Company, Chicago, IL, USA). For IC cultures, a periodic injection of nitrogen through the outlet was used to dislodge beads on the outlet grid. Continuous FC cultures were inoculated at 1% (v/v) with an overnight culture of L. diacetylactis UL719 in MRS broth at 301C. The IC reactor was inoculated at 25% (v/v) with pre-colonized beads. For aerated cultures, filter-sterilized air was supplied to the fermenter at a constant air flow rate of 3.5 v.v.m (volume of air/volume of fermenter min 1), which corresponded to the optimal aeration condition for nisin production during FC batch cultures (Amiali et al., 1998). For each dilution rate tested, two samples were collected at 1 h and 30 min intervals during steady or pseudo-steady state of continuous FC and IC cultures, respectively. For FC cultures, steady state was determined by constant absorbance measurements (600 nm,

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LKB diode array spectrophotometer, LKB, Cambridge, UK) at 1 h intervals, after a minimum of seven reactor volume changes. For IC cultures, pseudo steady state was attained 24 h after changing the D set point. Various dilution rates were studied during the same continuous culture, in randomized order, in the range from 0.15 to 0.8 h 1 and 0.25 to 2.0 h 1 for aerated FC and aerated and non-aerated IC cultures, respectively. Continuous FC and IC experiments were duplicated. For nonaerated FC cultures, D in the range from 0.15 to 1.0 h 1 were tested in duplicate in randomized order during four culture experiments, except for D of 0.15 or 0.60 h 1 and 0.35 and 0.40 h 1 which were replicated 3 and 4 times to test for strain stability.

2.6. Bacteriocin activity assay Broth samples were centrifuged at 12 000g for 15 min at 41C. The supernatant was filtered (0.2 mm disposable sterile nylon filter, Cameo 25 N, MSI, Westboro, MA, USA), stored at 201C for a maximum period of 5 days, and tested for soluble nisin activity. For cell-bound nisin activity measurements, the pellets were resuspended in 0.02 m HCl (pH 2.0) and boiled in water for 10 min (White & Hurst, 1968). The sample was then cooled on ice for 15 min, filtered (0.2 mm) and stored at 201C, until testing for bacteriocin activity within a period of 5 days. Total nisin activity was obtained by adding soluble and cell-bound activities. For total nisin activity determination in gel beads, 0.7 g beads withdrawn from the fermenter was added to 5 mL peptone water and 1.25 mL 0.02 m HCl (pH 2.0), and dissolved by Ultra-Turrax mixer at 13 500 rpm for 1 min. The mixture was boiled in water for 10 min, cooled on ice, and centrifuged at 12 000g for 15 min at 41C. The supernatant was filtered (0.2 mm filter), and stored at 201C until testing for bacteriocin activity within a period of 5 days. Nisin Z activity was determined in duplicate by a critical-dilution micromethod (Meghrous et al., 1997). Serial 2-fold dilutions of the sample were carried out in 125 mL volume of MRS in a 96-well Falcon microtitre plate (Becton Dickinson Labware, Lincoln Park, NJ, USA). Each well was then inoculated with 25 mL of a 100-fold diluted-overnight culture of the test organism, P. acidilactici UL5. Assay microplates were incubated at 301C for 18 h. Bacteriocin activity (AU mL 1) was determined using the following formula: (1000/ 125)  (1/d), where d is the highest dilution that prevented growth of the test organism after 18 h incubation. The correspondence between arbitrary units (AU) and international units (IU) was determined as above, using HPLC-purified nisin Z and Nisaplin (2.5% nisin A, Aplin and Barrett Ltd., Beaminster, UK); 1 AU corresponded to 1 IU (40 IU=1 mg of pure nisin).

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2.7. Determination of lactose and lactic acid concentrations Lactose and lactic acid concentrations were measured by HPLC (Waters, Milford, MA, USA) equipped with an Interaction ION 300 column (Phenomenex, Torrance, CA, USA), using H2SO4 (0.0064 n) as mobile phase at a flow rate of 0.4 mL min 1. Lactose was detected by refractive index and lactic acid by UV at 210 nm. 2.8. Statistical analysis

0.4 h 1. Cellular productivity was maximum at 3.9  109 cfu mL 1 h 1 for D of 0.4 h 1, and decreased for lower or higher D to 1.9  109 cfu mL 1 h 1 for 0.15 h 1 and 2.1  109 cfu mL 1 h 1 for 1.0 h 1. Aeration resulted in a 6–9-fold decrease of viable cell counts for DX0:4 h 1 and cellular productivity was much lower compared to non-aerated cultures (Table 1). Lactose consumption and lactic acid production decreased continuously with D (pa p0:01) during nonaerated cultures, from 32.9 and 22.3 g L 1 for 0.15 h 1 to 13.0 and 1.3 g L 1 for 1.0 h 1, respectively (Fig. 1). Aeration decreased both lactose consumption and lactic

Analysis of variance was carried out with Data Analysis Tools of Microsoft Excel. Significance of polynomial effects (linear, quadratic, cubic and quartic) for dilution rate were tested with the t-test. A logarithmic transformation was used for cell counts to ensure variance homogeneity of data. Bacteriocin activity data obtained with the serial 2-fold dilution micromethod are discontinuous and were not analyzed statistically. However, bacteriocin titres were highly reproducible for the repeated cultures and differed by no more than one well, corresponding to a 2-fold dilution.

3. Results 3.1. Cell production and lactose utilization during continuous FC and IC cultures A high and stable cell production was obtained during continuous FC cultures at pH 6.0 without aeration, in the range from 1.0 to 1.3  1010 cfu mL 1 for low dilution rates from 0.15 to 0.4 h 1 (Table 1). Cell concentration decreased significantly for D higher than

Fig. 1. Effect of dilution rate and aeration on lactose consumption (’, &) and lactic acid production (m, n) during continuous free cell cultures. Filled and open symbols correspond to non-aerated and aerated cultures, respectively. Vertical bars represent standard deviation.

Table 1 Effect of dilution rate and aeration on biomass production and lactose utilization during continuous free cell culturesa Dilution rate (h 1) No aeration Cell concentration (log10 cfu mL 1) Cellular productivity (109 cfu mL 1 h 1) Product yieldc (g g 1) Aeration Cell concentration (log10 cfu mL 1) Cellular productivity (109 cfu mL 1 h 1) Product yieldc (g g 1) a

0.40

0.60

0.80

1.00

SLb

0.15

0.20

0.25

0.30

0.35

10.1170.07

10.0970.11

10.0870.04

10.0770.16

10.0270.13

9.9870.05

9.7470.07

9.5770.15

9.3270.01

L***

1.9170.30

2.5070.62

3.0370.30

3.6071.26

3.7871.26

3.8870.44

3.3070.54

3.0471.04

2.1070.10

0.68370.073

0.68770.045

0.66770.136

0.60270.048

0.58970.072

0.65770.066

0.54970.174

0.27170.002

0.09770.001

L**, Q**, C* L***

10.0570.20

10.0170.03

9.4470.16

8.6070.33

L***

1.7770.78

2.5870.18

1.1470.40

0.3770.30

L**

0.74370.131

0.56870.044

0.58870.011

0.06170.055 1

Q* 1

Reported data are means and standard deviations of duplicated cultures, except for D of 0.35 or 0.40 h and 0.15 or 0.60 h which were tested with 3 and 4 replications, respectively, for non-aerated cultures. b Significance levels: *, pa p0:05; **, pa p0:01; ***, pa p0:001; n.s., not significant (pa > 0:05). L, linear effect; Q, quadratic effect; C, cubic effect. c Conversion yield of lactose to lactic acid.

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P. Desjardins et al. / International Dairy Journal 11 (2001) 943–951 Table 2 Effect of dilution rate and aeration on biomass production and lactose utilization during continuous immobilized cell culturesa Dilution rate (h 1)

0.25

0.50

1.25

2.00

SLb

No aeration Released cell concentration (log10 cfu mL 1) Immobilized cell concentration (log10 cfu g 1 gel) Cellular productivity (109 cfu mL 1 h 1) Product yieldc (g g 1)

10.1070.20 10.8970.15 2.4070.25 0.90170.086

9.9070.06 11.0970.13 4.0070.55 0.88270.102

9.7770.06 11.1470.06 7.3871.00 0.79770.236

9.6070.02 11.1970.02 8.0070.40 0.94670.030

L*** n.s. L* n.s.

Aeration Released cell concentration (log10 cfu mL 1) Immobilized cell concentration (log10 cfu g 1 gel) Cellular productivity (109 cfu mL 1 h 1) Product yieldc (g g 1)

9.8170.13 10.4870.07 1.6570.48 0.88570.022

9.7470.02 10.8170.01 2.7570.10 0.84970.152

9.4270.02 11.0270.11 3.2570.13 0.89570.037

9.2670.03 11.0370.12 3.6070.20 0.89270.112

L*** L* L**, Q* n.s.

a

Reported data are means and standard deviations of duplicated cultures. Significance levels: *, pa p0:05; **, pa p0:01; ***, pa p0:001; n.s., not significant (pa > 0:05). L, linear effect; Q, quadratic effect. c Conversion yield of lactose to lactic acid. b

acid production, and the effect was much more pronounced at high D of 0.4 and 0.8 h 1. For the IC cultures, a high concentration of released cells was measured in the fermented broth, decreasing from 9.6 to 4.0  109 cfu mL 1 and from 6.6 to 1.8  109 cfu mL 1 when D increased from 0.25 to 2.0 h 1 during non-aerated and aerated cultures, respectively (Table 2). Mean cell concentration in gel beads did not change significantly with D; and averaged 1.2  1011 cfu g 1 during non-aerated cultures (Table 2). On the other hand, immobilized cell counts increased from 3.1  1010 to 1.1  1011 cfu g 1 when D was increased from 0.25 to 2.0 h 1 for aerated IC cultures, and were 25–50% lower than for non-aerated cultures for the same D: Cellular productivity continuously increased with D in the tested range, from 2.4 to 8.0  109 cfu mL 1 h 1 without aeration, and from 1.6 to 3.6  109 cfu mL 1 h 1 with aeration (Table 2). Both lactose consumption and lactic acid production decreased with D (pa p0:01) during aerated and nonaerated continuous IC cultures, and aerobic conditions had a negative effect on both parameters (Fig. 2). 3.2. Nisin Z production during continuous FC and IC cultures Both soluble and cell-bound nisin activities decreased during continuous FC cultures without aeration, from 1325 to 256 IU mL 1 and from 128 to 18 IU mL 1 when D increased in the range of 0.15–0.8 h 1, respectively (Table 3). The same change of nisin production with D was observed for aerated cultures. However, soluble activity increased and decreased with aeration at low and high D; respectively. Furthermore, aeration contributed to an important 4-fold increase of cell-bound activity at low D of 0.15 and 0.25 h 1, but to a 4-fold decrease at D of 0.8 h 1, compared with non-aerated cultures. During non-aerated FC cultures, nisin produc-

Fig. 2. Effect of dilution rate and aeration on lactose consumption (’, &) and lactic acid production (m, n) during continuous immobilized cell cultures. Filled and open symbols correspond to non-aerated and aerated cultures, respectively. Vertical bars represent standard deviation.

tivity increased from 224 IU mL 1 h 1 at D of 0.15 h 1 to a maximum of 453 IU mL 1 h 1 at 0.4 h 1, and then decreased to 274 IU mL 1 h 1 at 1.0 h 1 (Fig. 3). Aeration had a positive effect on nisin productivity at low D; but this decreased at 0.8 h 1, compared to non-aerated cultures (Fig. 3). During continuous IC cultures without aeration, soluble nisin production slightly decreased with D from 1024 IU mL 1 at 0.25 and 0.5 h 1 to 512 IU mL 1 at 2.0 h 1, respectively (Table 4). Cell-bound activity was low and stable (64–96 IU mL 1) for D from 0.25 to 1.25 h 1, and decreased to 32 IU mL 1 at 2.0 h 1. Aerobic conditions caused an increase in both soluble and cell-bound activities, by 1.5–2-fold and 4–6-fold,

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P. Desjardins et al. / International Dairy Journal 11 (2001) 943–951

Table 3 Effect of dilution rate and aeration on nisin Z production during continuous free cell culturesa Dilution rate (h 1)

0.15

0.20

0.25

0.30

0.35

0.40

0.60

0.80

1.00

No aeration Soluble activity (IU mL 1) Cell-bound activity (IU mL 1) Total activity (IU mL 1) Specific production (IU (107 cfu) 1)

1325 128 1493 1.19

1024 128 1152 0.93

1024 128 1152 0.94

1024 128 1152 0.95

1024 128 1152 0.95

1024 109 1133 1.03

512 32 544 0.99

366 32 398 1.31

256 18 274 1.31

Aeration Soluble activity (IU mL 1) Cell-bound activity (IU mL 1) Total activity (IU mL 1) Specific production (IU (107 cfu) 1)

2048 512 2560 2.23

1024 512 1536 1.49

1024 128 1152 4.06

128 8 136 3.89

a

Reported data are means of duplicate cultures, except for D of 0.35 or 0.40 h 1 and 0.15 or 0.60 h 1 which were tested with 3 and 4 replications, respectively, for non-aerated cultures. Bacteriocin titres differed by no more than one well for the repeated cultures with the microtitre assay.

Fig. 3. Effect of dilution rate and aeration on nisin productivity during continuous free cell (’, &) and immobilized cell (m, n) culture. Filled and open symbols correspond to non-aerated and aerated cultures, respectively.

respectively, compared with non-aerated IC cultures (Table 4). Gel bead activity did not change with D for non-aerated cultures, averaging 2300 IU g 1, but increased from 8000 to 22 200 IU 1 g 1 with D in the tested range from 0.25 to 2.0 h 1 for aerated cultures. Nisin productivity continuously increased with D in the tested range for both aerated and non-aerated cultures, and aeration had a positive effect (Fig. 3).

4. Discussion Bacteriocins are traditionally produced in batch cultures. The production of bacteriocins, such as nisin, from lactic acid bacteria is associated with the growth of

the producing strain, and generally, maximum production corresponds to maximum cell concentration (Parente, Ricciardi, & Addario, 1994; De Vuyst & Vandamme, 1994; Matsusaki et al., 1996; Amiali et al., 1998). Therefore increased cell concentrations in a high cell-density reactor is expected to increase bacteriocin production. In this study, nisin production during FC and IC continuous cultures in supplemented whey permeate medium decreased when D increased in the tested range from 0.15 to 1.0 h 1 and from 0.25 to 2.0 h 1, respectively (Tables 3 and 4). Maximum soluble activity was similar for both systems, considering the low accuracy of the serial 2-fold dilution micromethod used for activity determination, with 1325 and 1024 IU mL 1 for FC and IC, respectively. Volumetric productivity was also very similar for FC and IC cultures when D was lower than 0.5 h 1, but increased continuously with D for non-aerated and aerated IC cultures, to a maximum of 1090 and 1760 IU mL 1 h 1, while it decreased during FC cultures for D higher than 0.5 h 1 (Fig. 3). Maximum total activity obtained during non-aerated and aerated FC batch cultures (4230 IU mL 1 after 8 h and 20 480 IU mL 1 after 24 h fermentation, respectively) with the same strain and culture conditions (Amiali et al., 1998) was much higher than that measured for continuous FC and IC cultures in our study (Tables 3 and 4). However, volumetric productivity during non-aerated and aerated batch cultures (530 and 850 IU mL 1 h 1, respectively) was lower than maximum productivity during non-aerated and aerated continuous IC cultures (1090 and 1760 IU mL 1 h 1 for D of 2.0 h 1, respectively), but slightly higher than that during continuous FC cultures (450 IU mL 1 h 1 for both aerated and non-aerated cultures for D of 0.4 h 1, Fig. 3). Therefore, when considering the cleaning-preparation periods between batches, the maximum productivity of continuous IC cultures is more than twice that for FC batch cultures.

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P. Desjardins et al. / International Dairy Journal 11 (2001) 943–951 Table 4 Effect of dilution rate and aeration on nisin Z production during continuous immobilized cell culturesa Dilution rate (h 1)

0.25

No aeration Soluble activity (IU mL 1) Cell-bound activity (IU mL 1) Total activity (IU mL 1) Specific production (IU (107 released cells) 1) Total specific productionb (IU (109 cfu) 1) Gel bead activity(IU g 1)

1024 64 1088 1.19 0.21 1778

1024 96 1120 1.49 0.09 2656

768 64 832 1.41 0.06 1917

512 32 544 1.36 0.03 2856

Aeration Soluble activity (IU mL 1) Cell-bound activity (IU mL 1) Total activity (IU mL 1) Specific production (IU (107 released cells) 1) Total specific productionb (IU (109 cfu) 1) Gel bead activity (IU g 1)

2048 384 2432 4.07 0.66 7968

2048 384 2432 4.34 0.35 13 280

1024 256 1280 4.96 0.12 18554

768 112 880 4.84 0.08 22 194

0.50

1.25

2.00

a

Reported data are means of duplicate cultures. Bacteriocin titres differed by no more than one well for the repeated cultures with the microtitre assay. b Total specific activity was calculated with total (immobilized and released) viable cell counts.

Specific nisin production of non-aerated continuous FC and IC cultures did not change with D; it averaged 1.07 and 1.36 IU (107 cfu) 1, respectively, indicating that for both systems, nisin production was associated with cell growth (Tables 3 and 4). The continuous supply of fresh medium allows nutrient feeding and also removal of metabolites, such as lactic acid and nisin, which are potential inhibitors for cell growth. Increasing D during continuous FC and IC cultures resulted in a decrease in lactic acid yield from lactose (Tables 1 and 2), particularly for high D; which can be explained by an increased use of lactose for biomass production when nutrients or products are not limiting or inhibitory, respectively. Aeration demonstrated a stimulatory effect on nisin Z production by L. diacetylactis UL719. Soluble activity increased by 1.5–2-fold with aeration at a low D of 0.15 h 1 and over the tested range, respectively, while cell-bound activity increased 4–6-fold (Tables 3 and 4). However, aeration decreased nisin production of FC cultures at DX0.4 h 1 which can be explained by the large negative effect of aeration on cell growth observed during FC cultures at high D (Table 1). A significant but smaller effect of aeration was also noticed on released cell concentration for IC cultures (Table 2). These data can be explained by the establishment of sharp oxygen concentration gradients within gel beads which protected immobilized cells from this toxic compounds (Hooijmans et al., 1990). Few studies have been reported on bacteriocin production in non-optimal stressing conditions, such as aeration, for bacterial growth and reported effects are strain-dependent. Previous research with the same strain and fermentation conditions as in this study showed that aeration, when started after 2.5 h incubation, had a

positive effect on nisin production during FC batch cultures, and largely increased cell-bound activity (Amiali et al., 1998). On the other hand, aeration has been reported to be antagonistic to the production of nisin A (Hurst, 1981), lactocin S (Mrtved-Abildgoard et al., 1995), and LIQ-4 bacteriocin (Kuhnen, . Sahl, & Brandis, 1985), while it resulted in an increase in nisin Z production by L. lactis IO-1 (Chinachoti et al., 1997), and an increase in specific production rate of amylovorin by Lb. amylovorus (De Vuyst, Callewaert, & Crabbe, 1996). The stimulation effect of aeration on nisin production during continuous FC and IC cultures was shown by the 1.5–4-fold increase of specific nisin production for aerated cultures compared to non-aerated cultures (Tables 3 and 4). Specific nisin production in nonaerated continuous FC and IC cultures and FC batch cultures (Amiali et al., 1998) was very close, averaging 1.07, 1.36 and 1.55 IU (107 cfu) 1 and aeration increased the mean specific production to 2.92, 4.55, and 11.7 IU (107 cfu) 1, respectively, corresponding to a 13-, 3- and 7.5-fold increase. Specific nisin production and lactic acid yield from lactose in non-aerated continuous FC cultures increased and decreased, respectively, for high D while they did not change with D in continuous IC cultures (Tables 3 and 4). These data may indicate that regulation for both productions is related. Total specific production calculated for total biomass (released and immobilized cell populations) for continuous IC cultures was very low compared with FC batch and continuous cultures, and decreased significantly with D; from 0.21 to 0.03 IU (109 cfu) 1, and 0.66 to 0.08 IU (109 cfu) 1 during non-aerated and aerated cultures, respectively (Table 4). Thus, the nisin production by immobilized cells was low, which agrees with data reported by

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Huang et al. (1996) for pediocin. Different factors were proposed to explain this observation: (a) limitation of diffusion of bacteriocin into the bulk medium; (b) increased adsorption of bacteriocin to immobilized cell surfaces; (c) inhibition of prebacteriocin production, transport or maturation in the local environment of immobilized cells (low pH and substrate concentration, high lactic acid concentration). In this study, nisin activity in gel beads was high and increased with D from 8000 to 22 200 IU g 1 for continuous IC cultures with aeration, but did not change with D during non-aerated cultures, averaging 2300 IU g 1 (Table 4). A theoretical gel bead activity can be calculated for different D; using cell-bound activity for released cells, released and immobilized cell concentrations and soluble activity in the bulk medium. The theoretical gel bead activity represented close to 100% (from 60% to 120%) of the measured activity for nonaerated IC cultures, taking into account the low accuracy of the activity test, but only from 30% to 50% for aerated cultures. Therefore, aeration apparently increased cell-bound activity for immobilized cells, but diffusion of bacteriocin was not apparently a limiting factor as shown by the low activity observed in gel beads of non-aerated continuous IC cultures. Maximum bacteriocin productions and specific activity for continuous FC and IC cultures obtained in this study with nisin and in previous work with pediocin (Huang et al., 1996) were low compared with those measured during FC batch cultures (Amiali et al., 1998; Goulhen et al., 1999; Huang et al., 1996). This suggests that during continuous cultures at steady or pseudosteady state, all the steps involved in the production of the mature peptide are not expressed at their maximum potential, and some processes (post-translational reactions, transport, or maturation) may be limiting (Engelke, Gutowski-Eckel, Kiesau, Hammelmann, & Entian, 1994; Parente & Ricciardi, 1999). The maximum total nisin production obtained during continuous FC and IC cultures with aeration (2560 and 2432 IU mL 1 for D equal to 0.15 and 0.25 h 1, respectively) are comparable to that generally obtained under the optimal conditions for nisin A or Z production, which correspond to 1600–4000 IU mL 1 (De Vuyst & Vandamme, 1992; Matsusaki et al., 1996; Van’t Hul & Gibbons, 1997; Chinachoti et al., 1997). However, continuous IC cultures lead to a large increase in volumetric productivity, particularly at high D; compared with batch and continuous FC cultures (Fig. 3).

5. Conclusions Continuous FC and IC cultures in supplemented whey permeate medium exhibited lower nisin produc-

tion than during pH-controlled batch cultures with the same culture conditions. For all tested fermentation types, nisin production was growth-associated, and aeration exhibited a stimulatory effect on production. However, the high cell densities obtained in the continuous IC bioreactor did not result in increased nisin production compared with continuous FC cultures for which viable cell concentration was about 10 times lower. Data from this study may indicate that some biosynthesis steps of the mature peptide are limiting due to steady-state conditions maintained during continuous cultures.

Acknowledgements This research was carried out within the program of the Canadian Research Network on Lactic Acid Bacteria, supported by the National Sciences and Engineering Research Council of Canada, Agriculture and Agri-Food Canada, Novalait inc., The Dairy Farmers of Canada and Rosell-Lallemand inc., and was also supported by the Fond pour les Chercheurs et l’Avancement de la Recherche from the Province of Quebec.

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