Effect of the extremely low frequency magnetic field on nisin production by L lactis using cheese whey permeate

Effect of the extremely low frequency magnetic field on nisin production by L...

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Effect of the extremely low frequency magnetic field on nisin production by Lactococcus lactis subsp. lactis using cheese whey permeate

David Chacon Alvarez, Victor Haber Perez*, Oselys Rodrıguez Justo, Ranulfo Monte Alegre

Department of Food Engineering, School of Food Engineering, State University of Campinas, SP, Brazil Received 13 December 2005; received in revised form 2 March 2006; accepted 10 April 2006

Abstract

The effect of the extremely low frequency (ELF) magnetic field on nisin production by Lactococcus lactis subsp. lactis using cheese whey permeate was studied during batch fermentation. The cellular suspension from the fermentor was externally recycled through a stainless steel tube inserted between the magnetic bars. The exposure time, recycle velocity and intensity of the magnetic field varied in a range of 4–12 h, 0.85– 1.50 m s 1 and 5–20 mT, respectively, according to a 23 complete factorial design. In all experiments, the produced biomass was almost constant. However, under the best conditions with the magnetic field treatment (4 h, 1.50 m s 1 and 5 mT) the nisin yield with respect to the substrate consumption and formed biomass were three and five times higher than the values of the control experiment. The application of the magnetic field allowed to deviate the metabolic pathway in order to intensify or to inhibit the nisin production. # 2006 Elsevier Ltd. All rights reserved.

Keywords: Nisin; Lactococcus lactis; ELF; Magnetic field; Biological effects

1. Introduction

Nisin is a lantibiotic used as a food preservative in several countries [1] and researches in order to increase its production have been carried out in batch and continuous fermentation systems [2–6]. The lantibiotics are polycyclic peptides containing the uncommon lanthionine amino acids and unsaturated amino acids such as dehydroalanine, which are produced by Gram positive bacteria [1]. In addition, several works about the properties, action mode and nisin applications have been published [1,7,8].

The biological effects of the weak low frequency magnetic field have attracted attention of many researchers, not only to establish the basic mechanisms of this interaction but also its practical applications potential. However, the mechanisms through which these fields might interact with the biological system are unclear. The biological effects of electromagnetic fields have been studied, particularly on cells (mammalian,

T-lymphocytes, tissue, tumours), bio-molecules, chemical reactions (influencing the electronic spin states of reaction intermediates) and microorganisms, as well as on other animal and human cells [9] and more recently, the interaction with cell membranes [10].

Moore [1] reported that the stimulation or inhibition of the growth of five bacteria species and yeasts was dependent on the field strength, frequency and bacterium kind. The stimulation or inhibition of microbial growth was also reported by other authors [12–16].

Several magnetic field generator devices [10,12,16–20] have been developed to accomplish researches about the biological effects of electromagnetic field on biological materials. These apparatus allow to expose the cell cultures at the magnetic field irradiation in small systems, such as Petri plates, slant tubes or small vials and reservoirs for cellular suspensions. At the same time, other applications of magnetic and electromagnetic fields in fermentation and enzymatic processes have been published [13–15]. Although the mentioned cases, the application on industrial scale is very difficult, due to the magnetic field devices generator be designed to cover all or most part of the fermentor and certainly, this is technically and economically unviable for application on larger volumes.

Few studies on microorganisms growth with metabolites production under electromagnetic field have been published.

w.elsevier.com/locate/procbio Process Biochemistry 41 (2006) 1967–1973

* Correspondence to: Universidade Estadual de Campinas, Cidade Universitaria ‘‘Zeferino 7 Vaz’’ s/n, Distrito de Barao Geraldo, DEA/FEA, Caixa Postal 6121, Campinas, CEP 13083-970, SP, Brazil. Tel.: +5 19 37884029; fax: +5 19 37884027. E-mail address: victorh@fea.unicamp.br (V.H. Perez).

1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.04.009

The lactic acid production can be stimulated by alternating magnetic field from 1 to 10 Hz [21]. According to Kropinski et al. [19] the protein synthesis by Escherichia coli can be stimulated under electromagnetic field of 3 mT and 60 Hz together with the temperature rise (3 8C for 70 min of exposure), with an increase of six-fold in the protein concentration. These authors also related that the synthesis of new proteins occurred in the exposed cells. Recently, the porphyrin production has been studied using Rhodobacter sphaeroides under anaerobic-light conditions and an external magnetic field of 0.13–0.3 T [2]. The microorganism growth was suppressed and the production of extra cellular porphyrin was activated up to 5.3-fold higher than in the control experiment (without magnetic field application) when the field induction was 0.3 T. Grosse et al. [23] studied the influence of pulsative electromagnetically induced currents (PEMIC) on the antibiotic nourseothricin production using Streptomyces noursei. Nevertheless, the effect of the magnetic field has been also observed on metabolite production by plant cell culture [24].

The nisin metabolite production by Lactococcus lactis subsp. lactis in submerse fermentation was investigated under extremely low frequency magnetic field. The medium of fermentation containing the cells (cellular suspension) was recycled through stainless steel tube inserted between magnet prisms (magnetic field generator), which is an apparatus that can be easily implemented on industrial scale. In addition, using the nisin production as a model, this paper intends to contribute to the knowledge and applications of the magnetic field biological effects on fermentative processes.

2. Material and methods

2.1. Microorganisms

L. lactis subsp. lactis was obtained from the American Type Culture

Collection, USA (ATCC No. 7962) and used for nisin production. It was maintained in a medium containing (g L 1): sucrose (10), yeast extract (10), peptone (10), K2HPO4 (10), NaCl (2) and MgSO4 (0.4) with the pH adjusted to 7.0 with NaOH (2 mol L 1). Lactococcus lactis subsp. cremoris from the

Tropical Culture Collection of Andre Tosello Foundation, Brazil (CCT No. 361) was used as the test microorganism to determine nisin mass. All chemical reagents and bacteriological media used in this work were purchased from Merck and SIGMA, respectively.

2.2. Inoculum preparation

L. lactis subsp. lactis (ATCC No. 7962) was maintained in a medium containing (g L 1): glucose (1), yeast extract (1), meat extract (1), tryptone

(1), Na2HPO4 (0.2) and NaCl (0.5). The pH was adjusted to 7.0 with NaOH (2 mol L 1).Thefermentedbrothwasdistributedintesttubescontainingglycerol solution,mixedandfrozenat 20 8C.Thefinalglycerolconcentrationinthetubes was 40%, which were used to inoculate the pre-culture in the Erlenmeyer flasks. Therefore,theinoculumwaspreparedin500 mlErlenmeyerflaskswith300 mlof the medium described above at 30 8C for 24 h and 50 rpm on shaker.

2.3. Fermentation procedure

The fermentations were carried out in a BioFlo-I fermentor (New Brunswick Scientific, USA) with a working volume of 4 L, and inoculated with 10% (v/v) of the pre-culture at 30 8C for 24 h and 50 rpm. The medium used in the pre-culture and in the fermentor contained (g L 1): cheesewhey permeate (final lactose concentration—10), K2HPO4 (10), NaCl (1), MgSO4 (0.2) and 1.3 g L 1 of each amino acid: alanine, arginine, glutamic acid, histidine, valine, threonine and methionine. All media were sterilised at 121 8C for 15 min and the pH was adjusted to 7.0 with NaOH or HCl 2 M 1 prior to sterilization.

2.4. Experimental setup

The fermentation medium was recycled through magnetic field generator (a stainless steel tube of 5.0 m of internal diameter inserted between three pars of magnetic primes with opposite poles) from Metalmag (Brazil), which sketch is shown in Fig. 1. Each magnet was a square prism with two faces of 4.8 cm 4.8 cm and thickness of 2.4 cm. They were inserted in a stainless steel box separated by iron bar of 2.4 cm 4.8 cm and thickness of 0.2 cm. The distances of the two boxes from the recycle tube were adjusted in three positions: 5.0, 15 and 35 m supplying magnetic inductions of 20 0.001, 12.5 0.001 and 5 0.001 mT, respectively. This apparatus permits to obtain uniform magnetic fields in the space among the magnets, generating a square wave with perpendicular field lines for fluid movement. A MG-3D Gauss-meter (Walker Scientific Inc., USA) was used to determine the field strength.

The velocity input on the fluid in the recycle ranged from 0.85 to 1.5 m s 1 allowing to work with lower frequencies than 20 Hz, that are known as extremely low frequency (ELF, ranging below 300 Hz). After the treatment time (4, 8 and 12 h) with the magnetic field, the generator was removed from the recycling loop. However, the medium (cellular suspension) recycling was kept until the end of the experiment. Previously, an experiment was carried out for 24 h with magnetic field exposure.

2.5. Analytical methods

2.5.1. Dry mass 10 ml medium samples were centrifuged at 2100 g (Exelsa Baby centrifuge 206-R, Fanen, Brazil). The harvested cells were washed twice with distilled water and dried at 65 8C and 8 kPa until constant weight in a vacuum oven GST-920 model (SUPRILAB, Brazil).

2.5.2. Total reducing sugars

The total remaining lactose in the fermentation medium was determined through the Somogyi–Nelson method [25].

2.5.3. Extraction and measurement of produced nisin mass

The nisin mass was determined through the microorganism inhibition test (L. lactis subsp. cremoris). One test tube containing 5 mL of medium, 1 mL of

D.C. Alvarez et al./Process Biochemistry 41 (2006) 1967–19731968

Fig. 1. Fermentor setup for the magnetic field treatment of the Lactococcus lactis cell suspensions. Drawing not to scale. (1) Fermentor, (2) cellular suspension, (3) three-way valve, (4) peristaltic pump, (5) stainless steel tube and (6) magnetic field generator.

the test microorganism culture and 2 ml of the L. lactis subsp. lactis culture supernatant was incubated at 37 8C for 6 h, according to Berridge and Barret [26] modified method [27]. After this time the microorganism growth was stopped by the addition of 1 mL of thiomersalate solution (0.0004%). The absorbance was measured at 600 nm and converted in to nisin mass through a standard curve that was drawn replacing the L. lactis subsp. lactis supernatant by 2 ml of nisin (SIGMA, powder with 2.5% of nisin) solution at different concentration.

To extract nisin from the cells, the cells harvested from each sample were suspended with 5 mL HCl solution (pH between 1.8 and 2.0) and boiled for 5 min. After centrifugation in an EPPENDORF refrigerated centrifuge (model 5804R) the nisin mass in the supernatant was measured as described above. The total nisin mass was the sum of the nisin mass from the medium supernatant and the nisin mass extracted from bacterium cells.

2.6. Statistical analysis

The experimental design with triplicate central points was used to evaluate the biological effects of the magnetic field on bacterial growth and nisin production. The magnetic induction (B), the exposure time (t) and the flow rate through the recycle tube (v) were combinedto obtain a 23 completefactorial design with central point. The experimental conditions and decoded values for the independent variables were: level 1 (0.005 T, 4 h and 0.85 m s 1); level +1 (0.020 T, 12 h and 1.50 m s 1) and central point (0.0125 T, 8 h and 1.175 m s 1), respectively. The central point is repeated three times for the experimental error estimate. The responses were expressed as yield coefficients: Growth (Yx/s) and nisin (Yp/s) yields, both with respect to the consumed substrate and nisin yield (Yp/x) with respect to the formed cell mass, which were determined from the growth, substrate consumption and product formation kinetic curves.

Analysis of variance (ANOVA) of the obtained data was performed through

The Statistical Software version 5.0 (Stat Soft Co.). Significance levels of P < 0.05 were assumed as the effect of the magnetic field on the variables statistically significant in this study. Besides, the F-test was used to verify the mathematical models quality, in which the calculated F-test (Fcalc) was compared with the listed F (Flisted).

3. Results

Several assays of nisin production were carried out without magnetic field application which were split into two groups, with and without recycling, to verify the effect of recycling on microbial growth and nisin production. Thus, the flow rate through the recycle tube was adjusted to a mean velocity of 0.85 m s 1 during all fermentation courses. The results (Table 1) showed that the nisin yield from the biomass (Yp/ x) decreased 62% and the nisin yield from the substrate (Yp/s) decreased 53%, when the recycle was introduced in the system, although the biomass yield from the substrate (Yx/s) has not presented a considerable variation. In both cases, the sugar consumption was up to 50%.

Fig. 2 shows the effect of the magnetic field on the process kinetics when the exposure time and recycling velocity were fixed in 24 h and0.85 ms 1, respectively, with twodifferent field intensities (5 and 20 mT), during all fermentation courses (24 h). There were stimulus of the substrate consumption and biomass

D.C. Alvarez et al./Process Biochemistry 41 (2006) 1967–1973 1969

Table 1 Fermentation of cheese whey permeate by L. lactis subsp. Lactis for nisin production, after 24 h of fermentation, with and without medium recycling and without exposure to a magnetic field (control experiment), at 30 8C, 50 rpm in a bioreactor, with 0.85 m s 1 flow rate of the fermentation medium (cellular suspensions)

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