food and bioproducts processing 9 3 ( 2 0 1 5 ) 115–121 Contents lists available at ScienceDirect Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp Short communication Low-cost puriﬁcation of nisin from milk whey to a highly active product Angela Faustino Jozala a,∗ , Letícia Celia de Lencastre Novaes a , Priscila Gava Mazzola b , Laura Oliveira-Nascimento a , Thereza Christina Vessoni Penna a , José António Teixeira c , Luis António Passarinha d , João António Queiroz d , Adalberto Pessoa Júnior a a Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, Universidade de São Paulo (USP), São Paulo, Brazil b Department of Clinical Pathology, School of Medical Sciences, Universidade de Campinas (Unicamp), Brazil c Department of Biological Engineering, Universidade do Minho, Braga, Portugal d CICS – Centro de Investigac¸ão em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal a b s t r a c t Nisin is a natural peptide used as a preservative in a variety of food products, in which it inhibits mainly Grampositive bacterial growth, including multidrug-resistant pathogens. However, its application range depends on the cost-effective production and puriﬁcation of this molecule. Our group has previously produced nisin by Lactococcus lactis cultivation in milk whey, which is an industrial residue from dairy production. To our knowledge, no report used milk whey as a culture medium, although several investigators have puriﬁed nisin using different techniques. We thus aimed to establish a low-cost puriﬁcation of nisin obtained by this process. Samples were diluted in ammonium sulphate, applied onto HIC columns (butyl sepharose CL 4B matrix), and eluted with Milli-Q water or PBS. Elution fractions were monitored for protein content and nisin antibacterial activity. Water elution resulted in puriﬁcation factor values (270, commercial nisin; 775, nisin produced in-house) higher than those obtained with PBS elution. We concluded that puriﬁcation of nisin does not require precipitation with ammonium sulphate, therefore allowing step/cost reduction. Moreover, puriﬁcation from milk whey using HIC provides nisin with high activity and low salt content, which can further be applied to a variety of areas. © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Nisin; Hydrophobic interaction chromatography (HIC); Milk whey; Puriﬁcation; Lactococcus lactis; Antimicrobial activity 1. Introduction Certain Lactococcus lactis strains produce the antimicrobial peptide nisin as a response to the presence of competitive bacteria, including their spores (Delves-Broughton et al., 1996). Nisin has structure variants due to point mutation that changes its 34 amino acid chain (Field et al., 2008). Regardless of the variant, it can conserve biological products by preventing contamination without toxicity. This property has guaranteed its use in food industry as an established preservative (De Arauz et al., 2009). ∗ Corresponding author at: Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas da USP, Av. Prof. Lineu Prestes, 580, B16, 05508-000 São Paulo, SP, Brazil. Tel.: +55 11 3091 2376; fax: +55 11 3815 6386. E-mail address: [email protected] (A.F. Jozala). Received 27 February 2013; Received in revised form 4 November 2013; Accepted 7 December 2013 Available online 15 December 2013 0960-3085/$ – see front matter © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fbp.2013.12.003 116 food and bioproducts processing 9 3 ( 2 0 1 5 ) 115–121 Nisin advantages reached both experimental and commercial frontlines of pharmaceutical, veterinary, and health-care products (De Arauz et al., 2009; Delves-Broughton et al., 1996; Liu et al., 2004; Ukuku and Fett, 2004; Sakamoto et al., 2001; Turner et al., 2004; Aranha et al., 2004; Von Staszewski and Jagus, 2008). The extent of applications requires different levels of product purity, which are achieved by several methods including ion exchange, immunoafﬁnity chromatography, capillary electrophoresis, ammonium sulfate precipitation, and liquid-liquid and organic solvent extraction (Cheigh et al., 2004; Prioult et al., 2000; Suarez et al., 1997; Yang et al., 1992; Taylor et al., 2007; Jozala et al., 2008; Abts et al., 2011; Espitia et al., 2012). Hydrophobic interaction chromatography (HIC) is another widespread technique for puriﬁcation of biomolecules, including nisin (Mahn et al., 2005; Passarinha et al., 2007; Josic et al., 2012). The advantage of HIC for protein puriﬁcation comes from its ability to separate closely related variants (Zolodz et al., 2010). In addition, columns can be packed with a variety of wide range of media by varying the base matrices and the chemical nature of the ligands, which allows variation in hydrophobicity and consequent protein selectivity (Perkins et al., 1997). Our research group developed an efﬁcient protocol to produce nisin from L. lactis, with whey milk as culture medium (Jozala et al., 2007). This by-product from dairy industry is a low-cost alternative for industrial production, which can provide high yields of this antimicrobial agent. To our knowledge, there are no reports on nisin puriﬁcation from this medium. In fact, all methods described for nisin puriﬁcation used complex formulated media, mostly Man, Rogosa, and Sharpe (MRS) medium. The aim of the present work was to purify produced nisin through hydrophobic interaction chromatography (HIC), based on puriﬁcation proﬁles of commercial nisin. 2. Materials and methods 2.1. Bacterial strains and media L. lactis (ATCC 11454) and Lactobacillus sakei (ATCC 15521) strains were stored in MRS broth (Difco, Detroit, MI, USA), and supplemented with 40% glycerol at −80 ◦ C (Jozala et al. 2007). L. sakei, the nisin-sensitive strain, was grown in MRS broth or agar (Difco, Detroit, MI, USA). L. lactis, the nisin-producing strain, was cultivated in milk whey (pH 6.8, kindly provided by a local dairy plant, Brazil). 2.2. Standard nisin Commercial nisin (2.5%, with sodium chloride and denatured milk solids, Sigma) was used as a standard during puriﬁcation. 2.3. Nisin production Initially, a frozen aliquot of L. lactis (108 CFU) was incubated in MRS broth (50 mL; shaker, 100 rpm, 36 h, 30 ◦ C). An aliquot (5 mL) from this pre-culture was transferred to milk whey (50 mL) and incubated (shaker, 100 rpm, 36 h, 30 ◦ C). This culture was sub-cultivated ﬁve times, always inoculating aliquots (5 mL) from the previous culture in fresh milk whey (50 mL), and incubated in the same conditions (100 rpm, 36 h, 30 ◦ C). The process was monitored for microorganism contamination (colony morphology and Gram staining) and nisin activity. The last sub-culture was centrifuged (13,200 × g, 10 min, and 10 ◦ C), and the supernatant collected and sterile ﬁltered (22-m pore diameter, Millipore). The sterile solution was called “nisin produced in-house” and stored (4 ◦ C) for further puriﬁcation. 2.4. Agar diffusion assay for determination of nisin activity Nisin activity was determined by agar diffusion assay, using L. sakei as nisin-sensitive microorganism. Serial dilutions of commercial nisin were utilized to construct the standard curve (10–105 Arbitrary Units (AU/mL)), as previously described elsewhere (Arauz et al., 2011). Brieﬂy, L. sakei was grown in MRS broth (shaker, 100 rpm, 30 ◦ C, 24 h); an aliquot from this culture was diluted in MRS agar and plated in Petri dishes (106 UFC/dish). After agar solidiﬁcation, 3-mm wells were caved. Samples and standard were independently transferred to wells (50 L/well) and incubated (4 ◦ C, 12 h; 30 ◦ C, 24 h). After incubation, diameter of growth inhibition zones was determined as the average of four independent measurements. Diameters (mm) determined for commercial nisin were related to the standard curve (AU/mL) (Fig. 1). 2.5. Protein quantiﬁcation After elution, the samples were spectrophotometrically monitored (280 nm, Molecular Devices, LLC, USA) for protein content. Samples with nisin activity were quantiﬁed by the bicinchoninic acid assay (QuantiProTM BCA Assay Kit, Sigma) according to the manufacturer’s protocol. 2.6. Puriﬁcation of nisin by hydrophobic interaction chromatography Commercial nisin was previously diluted in phosphatebuffered saline (0.1 M, pH 7.2, PBS) or Milli-Q water (100 mg/mL, 4 log10 AU/mL), whereas nisin produced in-house (4982 g/mL of total protein; 4 log10 AU/mL) was not diluted. To guarantee the hydrophobic interaction, ammonium sulphate was added to the samples in sufﬁcient amount to achieve 2 M of ﬁnal concentration. From these solutions, a sample (3 mL) was loaded onto the column. A column (10-mm diameter, 10-cm length) was packed with butyl sepharose CL 4B matrix (5 mL; GE Healthcare, Uppsala, Sweden). It was equilibrated using 3–4 column volumes (1 mL/min) of ammonium sulphate (2 M) in PBS (ﬁrst proﬁle) or water (second proﬁle). Diluted samples were sequentially eluted with 3 column volumes of (a) 2 M ammonium sulphate, (b) 1 M ammonium sulphate and (c) solvent (PBS or water). Elution was performed at a 1 mL/min ﬂow rate and each collected fraction contained 1 mL. Fractions were monitored by absorbance (280 nm), protein content, and nisin activity. 2.7. Electrophoresis Eluted samples that exhibited nisin activity were analyzed by SDS-PAGE (gradient precast gel 4–20%, Bio-Rad, USA) and compared with non-puriﬁed commercial nisin. The gel was stained using the silver stain kit (Bio-Rad, USA). Kaleidoscope polypeptide standard (Bio-Rad, USA) was used as molecular weight standard. food and bioproducts processing 9 3 ( 2 0 1 5 ) 115–121 3. Results and discussion 3.1. Nisin production/analysis 117 We produced nisin from sweet whey, as described above, and labeled it as “nisin produced in-house”. As expected, no cross-contamination was detected. Nisin produced inhouse (4982 g/mL total protein; 4 log10 AU/mL) was mixed with ammonium sulphate (1 or 2 M) and tested for antimicrobial activity and protein content after 24 h. Addition of ammonium sulphate did not change the parameters evaluated over time (p < 0.05, T-test). Nisin activity is generally measured by the method used in this paper, while protein content determination of peptides varied greatly. Protein content was ﬁrst measured using the Bradford method, but we did not obtain reproducible results when testing samples with low protein/high nisin content (data not shown). The variability might be due to nisin size (3.4 kDa), which is close to Bradford lower limit (3 kDa), or/and nisin amino acid sequence, which has only ﬁve basic groups to react with the dye. On the contrary, BCA has an expanded lower limit (2 kDa) and reacts with peptide bonds at higher temperatures. Use of BCA in our experiments improved nisin detection and allowed reproducible results. 3.2. Fig. 1 – Agar diffusion assay of the nisin samples. (A) Commercial nisin activity puriﬁed fraction and (B) commercial nisin activity in crude. The diameter of growth inhibition zones was determined as the average of four independent measurements 2.8. Statistical and mathematical analysis Standard curves (log AU × halo diameter) were analyzed by linear regression analysis (R2 > 0.9). Experimental AU values were calculated using the following equation: AU/mL = 10[(0.1423×halo diameter)+0.1035] (1) Yield (Y) and puriﬁcation factor (PF) were calculated using the following equations: PF = AUﬁnal /mg total proteinﬁnal AUinitial /mg total proteininitial Y = 100 × AUﬁnal × Vﬁnal AUinitial × Vinitial Nisin elution proﬁles Commercial nisin was diluted in water and loaded onto a HIC column (Fig. 2A). In the ﬁrst step, the eluate (2 M ammonium sulphate) contained the highest protein content, although without nisin activity. In the following step, the eluate (1 M ammonium sulphate) contained a low amount of protein. Nisin activity was detected only in the water elution step (Fig. 2A, samples 35–40), in which protein content was lower than in the previous steps. Although elution of commercial nisin with PBS showed a similar proﬁle (Fig. 2B), a decrease was observed in the number of samples with detectable activity (Fig. 2B, samples 36–39). Regardless of the eluent (PBS or water), nisin was selectively eluted in the step without ammonium sulphate. The second step (elution with 1 M (NH4 )2 SO4 ) of commercial nisin puriﬁcation yielded low protein content without nisin activity. Therefore, we decided to suppress this step and perform a 2-step protocol to purify the nisin produced in-house (elution with 2 M (NH4 )2 SO4 and water or PBS). Elution of nisin produced in-house exhibited the same behavior observed with the commercial one; nisin was selectively eluted in the step without ammonium sulphate. A peak with high protein content was recovered in the ﬁrst step (2 M ammonium sulphate, with water or PBS), but no nisin activity was found. Most samples collected in the last step were highly active (Fig. 3A and B), and some of them even showed increased activity around 1 log after puriﬁcation. Nisin was previously puriﬁed (produced in MRS) by HIC, in which it was also recovered in an eluent without ammonium sulphate (Gujarathi et al., 2008). However, conditions used in this work are different from the published literature. (2) 3.3. (3) Puriﬁcation factor and yield Nisin produced in-house showed the highest values for puriﬁcation factor (PF) 774 for water elution and 384 for PBS elution, 118 food and bioproducts processing 9 3 ( 2 0 1 5 ) 115–121 Fig. 2 – Chromatograms of commercial nisin puriﬁcation by HIC. Black line represents the chromatogram proﬁle recorded at 280 nm (non-bound and bound proteins). Gray solid bars represent nisin activity (Log AU). (A) Commercial nisin, water elution. (2) Commercial nisin, PBS elution. 3 times higher than PF from commercial nisin it was 268 for water elution and 135 for PBS elution (Table 1). Differences between nisin formulations might explain the discrepancy between yield values for the commercial and in-house samples: the commercial one contains sodium chloride, whereas nisin produced in-house was not supplemented with salt. Sodium chloride contributes to lipid oxidation (Ladikos and Lougovois, 1990) and might increase Table 1 – Speciﬁc activity, yield and puriﬁcation factor of nisin after HIC puriﬁcation. Samplesa Nisin activity (AU/mL) Commercial nisin Initial Water elution PBS elution Protein content (g/mL) Speciﬁc activity (AU/mg) Yield (%) Puriﬁcation factor (PF) 51,767.18 3309.97 926.57 10, 000 2.39 1.33 5.18 1384.92 697.19 100 6.39 1.79 1 267.53 134.68 Nisin produced in-house 3837.16 Initial 10,931.35 Water elution 5833.14 PBS elution 4982.43 18.35 19.73 0.77 595.71 295.72 100 284.88 152.02 1 773.52 383.99 a Samples were analyzed before (initial) and after puriﬁcation with water or PBS as the eluent. food and bioproducts processing 9 3 ( 2 0 1 5 ) 115–121 119 Fig. 3 – Chromatograms of nisin produced in-house puriﬁcation by HIC. Black line represents the chromatogram proﬁle recorded at 280 nm (non-bound and bound proteins). Gray solid bars represent nisin activity (Log AU). (A) Nisin produced in-house, water elution. (B) Nisin produced in-house, PBS elution. nisin availability, since nisin-fat clusters were reported to reduce antimicrobial activity (Jung et al., 1992). In addition, this salt prevents formation of insoluble protein aggregates (Costantino et al., 1995; Middelberg, 2002) that may entrap active nisin. Despite sodium chloride advantages in this case, it is likely that the salt interferes with nisin sporicidal activity (Bell and Lacy, 1985) and may reduce HIC efﬁciency (Roettger et al., 1989). High salt concentration can also induce hyperosmotic stress and kill cells (Burg et al., 2007), which is an undesired outcome for in vitro cytotoxicity tests. We believe that HIC puriﬁcation concentrated the active peptide in commercial samples, resulting in high values for PF. A selection to obtain the most puriﬁed fractions can signiﬁcantly reduce their yield (Ward and Swiatek, 2009), as occurred in this puriﬁcation. On the contrary, nisin produced in-house has demonstrated better activity than commercial nisin. In this case, puriﬁcation probably separated impurities that were interacting with nisin in the crude extract. The increase in activity resulted in high values for PF and yield. Regarding the eluent effect, the values for yield and PF in all preparations eluted with water were higher than those eluted with PBS (Table 1). This might be explained by the pH factor: nisin is more active in acidic solutions, whereas PBS maintains a neutral pH. As a consequence, the antimicrobial activity decreases, which reﬂects on the yield/PF values. Fig. 4 – SDS-PAGE (gradient gel 4–20%, silver stain). SDS-PAGE (gradient precast gel 4–20%, Bio-Rad, USA). The gel was stained using the silver stain kit (Bio-Rad, USA). Kaleidoscope polypeptide standard (Bio-Rad, USA) was used as molecular weight standard. At 150 V, 200 mA for 90 min. (A) Molecular weight standard; (B) puriﬁed form of commercial nisin; (C) puriﬁed fraction of nisin produced in-house; (D) commercial nisin in crude. 120 food and bioproducts processing 9 3 ( 2 0 1 5 ) 115–121 Researchers Gujarathi et al. (2008), who puriﬁed MRSderived nisin using HIC, obtained 50% recovery and a PF of 10.87. As stated above, most conditions used by this group were different from ours: their protocol involved several puriﬁcation steps; the sample was previously concentrated by ammonium sulphate precipitation; the column was packed with phenyl Sepharose instead of butyl Sepharose, among other details (Gujarathi et al., 2008). To our knowledge, there are no articles describing puriﬁcation of whey milk-derived nisin, thus precluding any comparisons. 3.4. Electrophoresis Puriﬁed and crude samples were subjected to SDS-PAGE (4–20%) and revealed with silver (Fig. 4). The gels showed the “smiley effect”, probably due to high salt content in some samples. Puriﬁed samples showed no bands but nisin, in agreement with nisin activity (high) and protein content (low) assays. As expected, commercial nisin (not puriﬁed, Fig. 3B) showed multiple bands and contained high protein content. 4. Conclusion The present study established a low-cost puriﬁcation of milk whey-derived nisin by HIC. We obtained a high degree puriﬁcation of nisin without protein precipitation, which allows step/cost reduction. It was possible to recover nisin with water or PBS, which allows more possible applications of nisin. Water was the best eluent, with an increase of up to 775fold in the antimicrobial activity. As per the outcomes of this work, milk whey probably contains factors, as remains of precipitated proteins, derived from the nisin production process, that decrease its activity, although high activity values were obtained in the crude extract. These factors were easily removed by HIC puriﬁcation, evidenced by an even higher antimicrobial activity. We conclude that production of nisin using milk whey as culture medium and HIC in the downstream process is costeffective and results in a highly puriﬁed nisin product. 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