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- Materials and methods
The conditions for high production of nisin Z and pediocin during pH-controlled, mixed-strain batch cultures in a supplemented whey permeate medium with Lactococcus lactis subsp. lactis biovar. diacetylactis UL719, a nisin Z producer strain, and variant T5 of Pediococcus acidilactici UL5, a pediocin-producing strain resistant to high concentrations of nisin, were studied. Mixed cultures were performed at 37 °C and pH 5·5 by first inoculating with variant T5 and then with L. diacetylactis UL719 after 8 h incubation, and were compared with single-strain batch cultures. High productions of both nisin Z and pediocin were obtained after 18 or 16 h incubation during mixed cultures, with titres of 3000 and 730 AU ml−1, or 1060 and 1360 AU ml−1, respectively, corresponding to approximately 75 and 55, or 25 and 100 mg l−1 of pure nisin Z and pediocin, respectively. In pure cultures, nisin Z and pediocin productions were higher than in mixed cultures, and maximum activities were obtained after 10 h incubation, with approximately 10 000 AU ml−1 (250 mg l−1 pure nisin Z) and 2500 AU ml−1 (190 mg l−1 pure pediocin). During mixed cultures, significant pediocin degradation was observed in the culture supernatant fluid after 16 h incubation, together with a sharp drop in Ped. acidilactici UL5 cell viability. In the test conditions, the feasibility of producing a nisin/pediocin mixture by mixed-strain fermentation was demonstrated. The bacteriocin mixture produced in a supplemented whey permeate medium could be used as a natural food-grade biopreservative with a broad activity spectrum.
Bacteriocins are bacterial antimicrobial proteins that are active against bacteria closely related to the producing organism and, depending on the bacteriocin, against a wide range of Gram-positive bacteria ( Klaenhammer 1993; Jack et al. 1995 ). Bacteriocinogenic lactic acid bacteria (LAB) may play an important role in fermented food products because they have been shown to inhibit the growth of food spoilage or pathogenic micro-organisms ( Jack et al. 1995 ). Among bacteriocins produced by LAB, pediocin and nisin are the most studied, not only because they exhibit a broad spectrum of activity, but also because they are bactericidal at low concentrations and exhibit thermal and pH stability in foods ( Ray & Hoover 1993; De Vuyst & Vandamme 1994). Nisin, produced by Lactococcus lactis subsp. lactis, is the only bacteriocin currently approved by the Food and Drug Administration as GRAS (Generally Recognized As Safe) ( Federal Register 1988). Nisin is currently used in more than 40 countries for specific food applications ( Delves-Broughton 1990). Recently, much attention has been focused on pediocin because of its high level of activity against Listeria species ( Muriana 1996).
To inhibit pathogenic or spoilage micro-organisms, bacteriocinogenic strains or partially purified bacteriocins can be added to foods ( Muriana 1996). However, the effectiveness of bacteriocins in foods may be reduced by different factors ( Hanlin et al. 1993 ; Muriana 1996). First, the minimum inhibitory concentration (MIC) differs widely among bacteriocins and sensitive strains ( Muriana 1996). Secondly, the activity spectrum of bacteriocins produced by Gram-positive bacteria is usually limited and does not include Gram-negative bacteria. Harris et al. (1992a) also demonstrated that bacteriocin-resistant variants may appear and grow in the presence of a bacteriocin, and therefore limit its efficacy. Despite the large number of publications on bacteriocin resistance mechanisms, they are still not entirely understood ( Ming & Daeschel 1995; Maisnier-Patin & Richard 1996; Goulhen et al. 1998 ).
The use of a combination of bacteriocins has been proposed to reduce the probability of selection and the development of bacteriocin-resistant variants ( Klaenhammer 1993). Hanlin et al. (1993) demonstrated an increased bactericidal activity against undesirable micro-organisms when using a mixture of pediocin and nisin compared with a single bacteriocin. The synergistic action of mixed bacteriocins could be attributed to two effects. First, different modes of bacteriocin action might explain the different levels of sensitivity of micro-organisms to these components ( Hanlin et al. 1993 ). Thus, a variant that is resistant to the first bacteriocin in a sensitive culture could eventually be eliminated by the other bacteriocin in the mixture ( Muriana 1996). Secondly, the use of a mixture of two bacteriocins might broaden their activity spectrum as a strain that is resistant to one bacteriocin could be weakened by the other bacteriocin and then become sensitive to the first bacteriocin.
Mixed cultures of a nisin producer and a non-bacteriocinogenic, nisin-resistant strain have been used for sauerkraut production ( Harris et al. 1992b ). However, there are no published data on the production of bacteriocin mixtures by mixed-strain fermentation with two bacteriocinogenic cultures. This production would require the selection of efficient bacteriocin-producing strains that are resistant to the bacteriocin produced by the other strain of the mixed culture. Another difficulty is the development of accurate methods to quantify selectively each bacteriocin in the mixture.
Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 produces a nisin derivative (nisin Z) that differs from nisin A by a single amino acid at position 27 ( Mulders et al. 1991 ; Meghrous et al. 1997 ). Although nisins A and Z have similar antimicrobial activities, nisin Z exhibits improved solubility and diffusion characteristics at pH > 6·0, which are important for foods ( De Vos et al. 1993 ). Recently, Amiali et al. (1998) reported high nisin Z production in a supplemented whey permeate medium during pH-controlled batch fermentation with this strain, with a maximum total activity of 20 500 and 4500 IU ml−1 with or without aeration, respectively. Goulhen et al. (1998) recently isolated and characterized variants of Pediococcus acidilactici UL5, a pediocin producer, which were resistant to various concentrations of nisin. The purpose of this study was therefore to demonstrate the feasibility and identify the optimum conditions for the production of a mixture of nisin Z and pediocin by batch fermentation of a supplemented whey permeate medium, using L. diacetylactis UL719 and the nisin-resistant variant T5 from Ped. acidilactici UL5.
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- Materials and methods
The production of a mixture of two broad-spectrum bacteriocins, nisin Z and pediocin, by pH-controlled batch fermentation of SWPM required the elucidation of strain compatibilities and their specific demands for growth and bacteriocin production. Pure cultures of L. diacetylactis UL719 and variant T5 of Ped. acidilactici UL5 were first studied using conditions from previous experiments that gave high cell growth and bacteriocin production by both strains, i.e. a pH of 5·5, a temperature of 37 °C, and a whey-based medium supplemented with 2% yeast extract, 0·5% glucose and 0·1% Tween-80. The maximum cell count for L. diacetylactis UL719 obtained after 8 h incubation under these conditions (1·2 × 1010 cfu ml−1) was about two-thirds that reported by Amiali et al. (1998) at pH 5·5 and 30 °C in a whey-based medium containing 1% yeast extract, 0·1% Tween-80 and no glucose. Moreover, Amiali et al. (1998) obtained a maximum total nisin Z titre of 4100 IU ml−1 after 8 h incubation, which remained constant until the end of the 24 h fermentation. In this study, a higher maximum for total nisin Z activity of 9216 AU ml−1 (1 IU = 1 AU) was obtained after 10 h incubation, but this titre decreased to 5461 AU ml−1 at the end of the culture, suggesting that nisin Z degradation occurred at 37 but not at 30 °C. This effect could also be partially attributed to readsorption of soluble nisin Z to the cell wall of the producer strain. The cell-bound nisin Z titre increased from 0 to 2560 AU ml−1 between 8 and 24 h of incubation ( Fig. 1); this titre was approximately fivefold higher than that reported by Amiali et al. (1998) for the same period of incubation. As shown in Fig. 1, soluble nisin Z production parallels that of biomass and thus, shows primary metabolite kinetics ( De Vuyst & Vandamme 1994; Amiali et al. 1998 ). The effect of temperature in the suboptimal range from 30 to 37 °C on cell growth and nisin Z production may be strain-specific. Matsusaki et al. (1996) reported that nisin Z production by L. lactis IO-1 at 37 °C, which is the optimal temperature for growth and lactic acid production, was only 75% of the activity obtained at 30 °C.
For mixed cultures, we used a nisin-resistant variant, T5, of Ped. acidilactici UL5, which was isolated on a nisingradient and shown to resist high concentrations of nisin A ( Goulhen et al. 1998 ). The effect of culture medium and incubation time during batch culture with Ped. acidilactici UL5, at 30 °C and without pH control, was studied by Daba et al. (1993) . Maximum biomass and pediocin production was observed after 9 h incubation in MRS broth supplemented with 2% yeast extract, giving 6·9 × 109 cfu ml−1 and 8192 AU ml−1, respectively. High biomass and pediocin production was also obtained in whey permeate supplemented with 2% yeast extract and 0·1% Tween-80, giving 7·6 × 108 cfu ml−1 and 2048 AU ml−1, respectively ( Daba et al. 1993 ). These values are very similar to those obtained in the present study of pH-controlled batch culture at 37 °C and variant T5 of Ped. acidilactici UL5; maximum biomass production and soluble pediocin titre were obtained after 10–12 h incubation and remained constant thereafter at 6 × 108 cfu ml−1 and 1900 AU ml−1, respectively ( Fig. 2). External control of pH did not appear to affect pediocin production in SWPM, which supports earlier reported data using MRS medium ( Daba et al. 1993 ). Finally, the maximum soluble titre (1900 AU ml−1) obtained between 10 and 24 h incubation in this study was very similar to the 2048 AU ml−1 reported by Daba et al. (1993) after 24 h incubation in MRS broth at the same pH of 5·5. Therefore, the conditions of batch fermentations selected in this study resulted in high pediocin production (estimated at 150 mg l−1) in SWPM.
Production of a mixture containing two broad-spectrum bacteriocins has never been reported, possibly because of the incompatibility or sensitivity of strains producing different bacteriocins, and to other problems related to data collection and interpretation. Although variant T5 was resistant to high concentrations of nisin, its competitiveness in mixed culture with L. diacetylactis UL719 was limited. These data might be explained in part by the low growth rate of variant T5 compared with L. diacetylactis UL719 during pH-controlled batch cultures (0·42 ± 0·03 h−1 and 1·23 ± 0·09 h−1, respectively), and the competition for the carbon source (glucose) in mixed cultures. Pediococcus acidilactici UL5 does not utilize lactose ( Daba et al. 1993 ) and therefore relies exclusively on glucose added to SWPM. Lactococcus diacetylactis UL719 consumed both glucose and lactose in the SWPM, leading to rapid utilization of residual glucose when this culture was inoculated after 8 h of mixed fermentation ( Fig. 3). Exhaustion of glucose was observed after 14 and 18 h incubation in mixed and pure cultures of variant T5, respectively. In order to promote cell growth and pediocin production by variant T5 in mixed culture, L. diacetylactis UL719 was inoculated 8 h after the inoculation of variant T5; this corresponded to the end of the exponential growth phase of T5 in pure culture.
In the test conditions, the feasibility of producing a nisin/ pediocin mixture was demonstrated by mixed-strain fermentations. High bacteriocin titres for both bacteriocins were obtained by a two-step fermentation with successive inoculations with variant T5 followed by, after 8 h incubation, L. diacetylactis UL719. However, the maximum total nisin Z production in mixed culture was significanty lower than in pure cultures; these maxima were 2976 and 10 176 AU ml−1, respectively, obtained 10 h after inoculation with L. diacetylactis UL719. This difference might be explained by the corresponding lower cell counts for mixed (4·2 × 109 cfu ml−1) rather than pure cultures (1·2 × 1010 cfu ml−1). In order to avoid pediocin degradation, which occurred during long incubation periods, and to produce high nisin Z titres, the fermented broth should be optimally harvested after approximately 16 h incubation ( Figs 1 and 2). Increasing the inoculation rate of L. diacetylactis UL719 might lead to a reduction in incubation time which would then enable the maximum nisin Z titre to be achieved and the fermented medium to be harvested after only 16 h fermentation ( Fig. 2). Finally, bacteriocin production did not appear to be stimulated in mixed culture as indicated by the similar mean specific pediocin (4·5 and 6·3 × 10−6 AU cfu−1 ml−1) or nisin (8·4 and 7·4 × 10−7 AU cfu−1 ml−1) production for pure and mixed cultures at incubation times corresponding to maximum titres, and considering the low accuracy of the serial twofold dilution activity test.
Maximum nisin production during pH-controlled batch cultures, obtained under the presumed optimal conditions, has been reported to be in the range 2000–3800 IU ml−1 ( De Vuyst & Vandamme 1994; Matsusaki et al. 1996 ). Recently, Amiali et al. (1998) reported a very high nisin Z production (20 500 IU ml−1 corresponding to approximately 500 mg l−1 of pure nisin) during pH-controlled, aerated batch fermentation of SWPM with L. diacetylactis UL719, with high cell-bound activity accounting for 80% of the total titre. Total nisin Z and pediocin production for mixed cultures was about 3000 AU ml−1 and 730 AU ml−1 (corresponding to 75 and 55 mg l−1 pure nisin and pediocin, respectively) after 18 h incubation, and 1063 and 1359 AU ml−1 (25 and 100 mg l−1, respectively) after 16 h incubation. Therefore, the ratio of nisin to pediocin in fermented broth was very dependent on incubation time and increased from 0·25 to 1·34 during the 16–18 h incubation period, although total bacteriocin production remained stable at approximately 125 mg l−1.
The estimation of both nisin Z and pediocin titres in broth samples from mixed cultures is a complex issue; it requires either the selection of an indicator strain that is sensitive to one of the bacteriocins in the mixture, or the specific inactivation of either bacteriocin. Both nisin Z and pediocin are small, heat-stable peptides which are relatively stable over a large pH range. Our first approach consisted of testing different enzymes at a concentration of 1 mg ml−1 for specific inactivation of one bacteriocin. Trypsin was shown to inactivate pediocin but not nisin Z after 1 h incubation at 37 °C. However, nisin Z titre could be directly estimated in culture samples by the microtitre assay using indicator strain Ped. acidilactici UL5, which is nisin-sensitive but immune to pediocin. However, conditions for specifically inactivating nisin Z by enzymes were not found. In addition, a strain that was sensitive to pediocin but resistant to nisin was not found. Listeria ivanovii ATCC 19119 and Ped. acidilactici UL5 were then selected for determining total bacteriocin and nisin Z titres, respectively, and the pediocin titre was estimated indirectly, as described in the Materials and Methods. However, because of the low sensitivity of Listeria strains to nisin compared with pediocin, the nisin Z contribution to total titres, as estimated using L. ivanovii ATCC 19119, was always low, and represented a maximum of 10% of the total titre in mixed culture. Therefore, when considering the low accuracy of the twofold dilution micro-method, the nisin Z contribution to total bacteriocin titres, as determined with L. ivanovii ATCC 19119, was within the experimental error of the method.
In conclusion, this study demonstrated the feasibility of, and identified conditions for, the production of a bacteriocin mixture in mixed-strain fermentation of SWPM by successive inoculations with variant T5 of Ped. acidilactici UL5, a nisin-resistant and pediocin-producing strain, and L. diacetylactis UL719, a nisin-producer strain. In the test conditions, high production of both pediocin and nisin Z was obtained after 16–18 h fermentation. Additional research might be required to optimize the inoculation rate of L. diacetylactis UL719 to increase the production of nisin Z while avoiding pediocin degradation, which occurs during long incubation periods exceeding 16 h. The bacteriocin mixture produced in SWPM could be used as a natural food biopreservative with a broad spectrum of activity resulting from the synergistic action of nisin Z and pediocin ( Hanlin et al. 1993 ).