Nelson Pérez Guerra

Nutritional factors affecting the production of two bacteriocins from lactic acid bacteria on whey

The ability of Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 to produce bacteriocins on both diluted and concentrated whey was investigated in batch fermentations. Both strains produced the higher amounts of biomass and bacteriocin titres on diluted whey. Luedeking and Piret expression was able to model the production of nisin, which was produced as a primary metabolite on both culture media. However, the pediocin production could not be typified in any case due to the negligible growth of P. acidilactici. Although the whey supported the growth and bacteriocin production by the two strains, both biomass and bacteriocin productions were lower than those obtained on MRS broth. The effect of total sugar, nitrogen, phosphorous and buffer concentrations on the production of nisin and pediocin was studied in diluted whey using factorial experiments and empirical modelling. The production of nisin was greatly inhibited by the increase in nitrogen, buffer, and to a lesser extent, sugar concentration in the medium, nevertheless, the used phosphorous source produced a light stimulatory effect on bacteriocin synthesis. In addition, the growth of Lc 1.04 was mainly affected by the nitrogen source used. On the other hand, pediocin was inhibited by the increase in buffer, phosphorous, and to a lesser degree, by the sugar and nitrogen concentration. The inhibitory activity of pediocin disappeared almost totally after 15 min of treatment with trypsin, papain, subtilisin and pepsin. The activity of nisin was drastically reduced by treatment with trypsin, subtilisin and pepsin. Nevertheless, 50% of the initial activity was retained when nisin was treated with papain. Both bacteriocins showed the highest heat stability at acidic pH and short incubation times.

Development of a bioactive packaging cellophane using Nisaplin as biopreservative agent.

Aims:  Production of a nisin‐containing cellophane‐based coating to be used in the packaging of chopped meat.

Modelling the influence of pH on the kinetics of both nisin and pediocin production and characterization of their functional properties

Abstract A kinetic model of the production of bacteriocins on MRS medium by Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 has been developed. The model is a modification of the Luedeking and Piret expression, including a term for the influence of pH on both nisin and pediocin production. With this model it was demonstrated that these bacteriocins are produced as primary metabolites depending on the pH. By modeling the effects of temperature and pH on their activities, the high heat stability of both bacteriocins was demonstrated, with optimum values at acidic pH and short incubation times. The initial activity of both nisin and nisaplin was drastically reduced by treatment with trypsin, subtilisin and pepsin, but 50% activity was retained when both antibacterial substances were treated with papain. The inhibitory activity of pediocin almost totally disappeared after 15 min of treatment with these four proteases.

Influence of pH drop on both nisin and pediocin production by Lactococcus lactis and Pediococcus acidilactici.

Aims: To develop a kinetic model for describing the specific effect of pH drop on nisin and pediocin production in whey.

Nisin and pediocin production on mussel-processing waste supplemented with glucose and five nitrogen sources

Aims: Optimization of bacteriocin production by L. lactis subsp. lactis CECT 539 and Ped. acidilactici NRRL B‐5627 on mussel‐processing wastes.

Production of bacteriocins from Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B‐5627 using mussel‐processing wastes

The growth and bacteriocin production by Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B‐5627 were investigated on mussel‐processing wastes. Both bacteriocin productions were satisfactorily modelled using a modified form of the Luedeking and Piret expression, which includes a term for the influence of the pH reduction rate. Experimental data from cultures buffered at different initial concentrations (0, 0.03, 0.10 and 0.25 M) of both bacteria were used to fit and verify the model. The influence of total sugars, nitrogen, phosphorus and buffer concentration on nisin and pediocin production was also studied using response‐surface methodology and empirical modelling. Enhanced nisin production (33 BU/ml) was achieved in media buffered with 0.10 M potassium hydrogen phthalate/NaOH. However, the highest levels of pediocin (368 BU/ml) were obtained in the non‐buffered media.

Enhancement of nisin production by Lactococcus lactis in periodically re-alkalized cultures

Synthesis of nisin as well as biomass production by Lactococcus lactis subsp. lactis CECT (Colección Española de Cultivos Tipo) 539 on both hydrolysed mussel‐processing waste and whey medium were followed in three fixed volume fed‐batch fermentations, with re‐alkalization cycles. The two cultures on mussel‐processing waste (MPW) were fed with a 240 g/l concentrated glucose and with a concentrated MPW (about 100 g of glucose/l). The culture on whey was fed with a mixture of concentrated whey (48 g of total sugars/l) and a 400 g/l concentrated lactose. The three cultures were mainly characterized with higher nisin titres [49.7, 109.6 and 124.7 bacteriocin activity units (AU)/ml respectively] compared with the batch process on de Man, Rogosa and Sharpe [(1960) J. Appl. Bacteriol. 23, 130–135] medium (49.6 AU/ml), MPW (9.5 AU/ml) and whey (22.5 AU/ml) [1 AU/ml is the amount of antibacterial compound needed to obtain 50% growth inhibition (LD50) compared with control tubes]. In the three fed‐batch cultures a shift from homolactic to mixed‐acid fermentation was observed, and other products (acetic acid, butane‐2,3‐diol or ethanol) in addition to lactic acid were detectable in the medium. However, their contributions to the total antibacterial activity of the post‐incubates (the cell‐free culture supernatant obtained at the end of the fermentation process) of L. lactis CECT 539 against Carnobacterium piscicola CECT 4020 were very low.

Enhanced nisin and pediocin production on whey supplemented with different nitrogen sources

Production of nisin and pediocin were followed, respectively, in Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 grown with lactose and four different nitrogen sources. Neither NH4Cl nor glycine improved production of the bacteriocins. Both yeast extract and Casitone increased pediocin production from 55 BU ml−1 to 195 BU ml−1 and 185 BU ml−1, respectively. Nisin increased from 21 BU ml−1 to 74 BU ml−1 and 59 BU ml−1 with these nitrogen sources.

Antimicrobial activity of nisin adsorbed to surfaces commonly used in the food industry.

The adsorption isotherms of nisin to three food contact surfaces, stainless steel, polyethyleneterephthalate (PET), and rubber at 8, 25, 40, and 60 degrees C, were calculated. For all surfaces, the increase in temperature led to a decrease in the affinity between nisin and the surface. The rubber adsorbed a higher amount of nisin (0.697 microg/cm2) in comparison with PET (0.665 microg/cm2) and stainless steel (0.396 microg/cm2). Adsorption of nisin to the stainless steel surface described L-2 type curves for all temperatures assayed. However, for PET and rubber surfaces, the isotherms were L-2 type (at 40 and 60 degrees C) and L-4 type curves (at 8 and 25 degrees C). Nisin retained its antibacterial activity once adsorbed to the food contact surfaces and was able to inhibit the growth of Enterococcus hirae CECT 279 on Rothe agar medium. The attachment of three Listeria monocytogenes strains to the three surfaces was found to be dependent on the surface, the strain, and the initial bacterial suspension in contact with the surface. The adsorption of Nisaplin on surfaces reduced the attachment of all L. monocytogenes strains tested. The effect of PET-based bioactive packaging in food was very encouraging. When applied to a food system, nisin-adsorbed PET bottles reduced significantly (P < 0.05) the levels of the total aerobic plate counts in skim milk by approximately 1.4 log units after 24 days of refrigerated storage (4 degrees C), thus extending its shelf life.

Fed‐batch pediocin production by Pediococcus acidilactici NRRL B‐5627 on whey

Cell growth and pediocin production by Pediococcus acidilactici NRRL B‐5627 on whey were compared by using batch fermentation and re‐alkalized fed‐batch fermentation. The batch fermentations were performed on DWG [DW (diluted whey) supplemented with 1% (w/v) glucose], DWYE [DW supplemented with 2% (w/v) yeast extract] and DWGYE (DW supplemented with 1% glucose plus 2% yeast extract) media. The fed‐batch culture on DWYE medium was fed with a mixture of concentrated whey (48 g of total sugars/l) supplemented with 2% yeast extract and 400 g/l concentrated glucose. The re‐alkalized fed‐batch culture was characterized by higher biomass (6.57 g/l) and pediocin [517.6 BU (bacteriocin activity units)/ml] concentrations compared with the batch processes on MRS (de Man, Rogosa and Sharpe) broth (1.76 g/l and 493.2 BU/ml), DW (0.17 g/l and 57.7 BU/ml), DWG (0.14 g/l and 53.6 BU/ml), DWYE (1.43 g/l and 187.6 BU/ml) and DWGYE (1.28 g/l and 167.3 BU/ml) media. A mixed acid fermentation was observed during the growth of P. acidilactici NRRL B‐5627 in the fed‐batch culture on DWYE medium, and other products (acetic acid and ethanol) in addition to lactic acid accumulated in the medium. Mathematical models were set up to describe fed‐batch production of biomass and pediocin by P. acidilactici. The models developed offer a better fit and a more realistic description of the experimental biomass and pediocin production data when compared with the logistic and Luedeking and Piret [(1959) J. Biochem. Microbiol. Technol. Eng. 1, 431–459] model.